Composite matrices used to promote innervation, osteogenesis and angiogenesis

Abstract

Composite materials are provided that can be used to promote innervation, osteogenesis, and angiogenesis.

Description

[Technical field]

[0001] The present invention relates to composite matrices useful for promoting innervation, osteogenesis and angiogenesis. [Background technology]

[0002] Our team (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) used a co-culture model of sensory neurons, mesenchymal cells and endothelial cells to demonstrate the impact of the communication between these three cell types on bone formation and the importance of the neurovascular dialogue. Based on these observations, the team developed an elastin peptide-based hydrogel that can stimulate the recruitment of nerve fibers (Paiva dos Santos et al., Acta Biomaterialia, 2019 Nov;99:154-167; PCT / EP2019 / 055075), in particular sensory neurons, and can also accommodate other cell types. Considering this, the inventors seek to improve various properties of said hydrogels, in particular in terms of increasing cell colonization uniformity and biodegradability of the material, and propose a biocompatible injectable composite matrix capable of stimulating innervation, tissue vascularization and exhibiting osteogenic properties for applications in bone tissue engineering. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] PCT / EP2019 / 055075 [Patent Document 2] International application WO2017021334 [Non-patent literature]

[0004] [Non-Patent Document 1] Silvaら, Cell Death and Disease, 2017 Dec 13;8(12):3209 pages

Non-patent document 2

Non-patent document 3

Non-patent document 4

Non-patent document 5

Non-patent document 6

Non-patent document 7

Non-patent document 8

Non-patent document 9

Non-patent document 10

Non-patent document 11

Non-patent document 12

[0005] The inventors have developed a novel composite material comprising an organic phase and an inorganic phase. This material is a biocompatible injectable material capable of stimulating innervation and tissue vascularization and exhibiting osteogenic properties for use in bone tissue engineering. The invention more particularly relates to a composite matrix combining an organic phase functionalized with a bioactive peptide and an inorganic phase comprising calcium phosphate. The composite matrix according to the invention is capable of recruiting sensory neurons and of hosting osteoforming and endothelial cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0006] The composite matrix according to the invention is characterized in that it comprises an organic phase comprising an elastin-like peptide, at least one bioactive peptide, and an inorganic phase comprising calcium phosphate.

[0007] 1. Elastin-like peptides The first component of the composite matrix according to the invention is an elastin-like peptide (or ELP for elastin-like polypeptide) comprising at least one methionine residue that is alkenylated prior to the formation of the composite matrix. This type of peptide, its production by genetic engineering and its purification are known to the skilled person and reference can be made in particular to the 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). The skilled person can also refer to the examples presented below with reference to the generation of an ELP called ELPM80.

[0008] 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 moiety that contains an alkene group, i.e., that contains 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 moiety linked to the methionine residue. According to a particular embodiment, the methionine moiety has the following formula (I):

[0009] [ka]

[0010] is connected to the part.

[0011] According to one embodiment, the synthesis of alkenylated ELPs according to the moiety of formula (I) can be carried out by chemoselective thioalkylation of the methionine side chain using allyl glycidyl ether according to the procedure described in Petitdemange et al. (Bioconjug Chem. 2017 May 17;28(5):1403-1412). The skilled person can in particular carry out the thioalkylation of the ELP peptide called ELPM80, with reference to the examples of the present application.

[0012] According to one embodiment, the alkenylated ELP embodied in the present invention comprises at least one occurrence of the amino acid sequence VPGMG in which the methionine residue is alkenylated.

[0013] According to a particular embodiment, the alkenylated ELP used to produce the composite matrix according to the invention has a high molecular weight, in particular an ELP greater than 20 kDa.

[0014] In one embodiment, the alkenylated ELP has the formula (II): Z - [VPGXG] n (II) The structure is During the ceremony: Z is a peptide containing 1 to 20 amino acids, X is a glycine residue, a valine residue or an alkenylated methionine residue, in particular a residue of formula (III):

[0015] [ka]

[0016] represents an alkenylated methionine residue of n is an integer comprised between 40 and 160, more particularly between 60 and 100, in particular between 70 and 90, The molar ratio of valine / methionine alkenylated at position X is comprised 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.

[0017] According to one embodiment, X represents a glycine residue or an alkenyl methionine residue, in particular an alkenyl methionine residue of formula (III). According to another preferred embodiment, X represents a valine residue or an alkenyl methionine residue, in particular an alkenyl methionine residue of formula (III).

[0018] According to one embodiment, n is an integer comprised between 60 and 100, in particular between 70 and 90, more particularly between 76 and 84, more particularly n is equal to 76, 77, 78, 79, 80, 81, 82, 83 or 84, more particularly n is equal to 80.

[0019] According to a particular embodiment, Z is a peptide whose amino terminal amino acid residue is methionine. According to another embodiment, [VPGXG] n The amino acids contained in the Z immediately upstream of the unit correspond to a MW dipeptide. In particular, Z may consist of or comprise a MW dipeptide. In certain embodiments, Z consists of a MW dipeptide.

[0020] According to one embodiment, the ELP used has the formula Z-[VPGXG] nwherein the molar ratio of valine / methionine alkenylated in position X is comprised between 1:1 and 5:1, in particular between 2:1 and 4:1, said ratio being more particularly 3:1.

[0021] According to another embodiment, the ELP used has the formula Z-[(VPGVG)(VPGMG)(VPGVG)] x , especially MW[(VPGVG)(VPGMG)(VPGVG)2] x where x is an integer comprised between 15 and 25, in particular between 19 and 21, and x is more particularly equal to 20.

[0022] According to one embodiment, the ELP used is the peptide MW [(VPGVG)(VPGMG)(VPGVG)2] described in the Examples. 20 (ELPM80), said peptide containing at least one alkenylated methionine residue. According to another particular embodiment, the ELP used has the formula MW[(VPGVG)(VPGMaG)(VPGVG)2] as described in the examples. 20 (ELPM(alkene)-80), where Ma represents an alkenylated methionine residue of formula (III) above.

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

[0024] 2. Bioactive peptides The composite matrix may also contain a bioactive peptide, which is capable of exerting a biological function when the composite matrix is ​​implanted in a tissue or organ of a subject. Such a bioactive peptide may be an adhesion peptide or a calcium-binding peptide, which is capable of binding to cells of interest in particular.

[0025] These bioactive peptides contained in the composite matrix of the present invention may contain a cysteine ​​residue at each of their termini. The cysteine ​​residue may be covalently linked to the amino acid sequence of the peptide directly or via a spacer, in particular a peptide or pseudopeptide spacer. In particular, the spacer may 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.

[0026] According to a particular embodiment, the amount of bioactive peptide in the complex matrix according to the invention is comprised between 99.9 and 0.1% (w / v). According to another particular embodiment, for a complex matrix having a concentration comprised between 3 and 5% (w / v) and for a thiol:alkene ratio of 1:1, said matrix comprises 0.1-3% (w / v) of at least one bioactive peptide.

[0027] 2.1. Adhesion peptides The first category of bioactive peptides that can be used in the composite matrix of the present invention are adhesion peptides that can recruit nerve cells, mesenchymal cells and / or endothelial cells. Peptides that can recruit endothelial cells can be specifically peptides derived from laminin, fibronectin or type I collagen, more specifically peptides derived from laminin. Thus, adhesion peptides can be selected from fibronectin-derived peptides REDV, RGD and GRGDSP, laminin-derived peptides IKLLI, IKVAV, PDSGR and YIGSR, and collagen type I-derived peptides DGEA. Peptides that can recruit nerve cells can be specifically peptides derived from laminin YIGSR, RNIAEIIKDI and IKVAV.

[0028] In a preferred embodiment, the composite matrix comprises at least one adhesion peptide selected from IKVAV and YIGSR.

[0029] According to a particular embodiment, the adhesion peptide is an IKVAV peptide, which may in particular be an IKVAV peptide of the formula Cys-{spacer}-Ile-Lys-Val-Ala-Val-{spacer}-Cys, in particular a peptide of the formula Cys-{Beta-Ala}-Ile-Lys-Val-Ala-Val-{Beta-Ala}-Cys.

[0030] According to another particular embodiment, the adhesion peptide is a YIGSR peptide, which may in particular be a YIGSR peptide of the formula Cys-{spacer}-Tyr-Ile-Gly-Ser-Arg-{spacer}-Cys, in particular a peptide of the formula Cys-{Beta-Ala}-Tyr-Ile-Gly-Ser-Arg-{Beta-Ala}-Cys.

[0031] According to an even more preferred embodiment, the composite matrix comprises the IKVAV peptide and the YIGSR peptide.

[0032] According to a preferred alternative embodiment, in order to optimize and control the cross-linking of the composite matrix, the latter comprises a single adhesive peptide comprising at least two adhesive peptides, in particular a biomimetic peptide comprising the adhesive peptides IKVAV and YIGSR. The peptides constituting the single adhesive peptide can be included in any order. According to an alternative embodiment, the single adhesive peptide comprises, from the amino-terminal end towards the carboxy-terminal end, the peptides YIGSR and IKVAV. In a preferred alternative embodiment, the single adhesive peptide comprises the peptides IKVAV and YIGSR, from the amino-terminal end towards the carboxy-terminal end. The single adhesive peptide can comprise a cysteine ​​residue at each of its ends. The cysteine ​​residue can be covalently linked to the amino acid sequence of the single adhesive peptide directly or via a spacer, in particular a peptide or pseudopeptide spacer. The spacer can 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 constituting the single adhesive peptide can be covalently linked to each other directly or via a spacer. The spacer between the peptides constituting the single adhesive peptide may in particular be a peptide or pseudopeptide spacer, in particular a glycine amino acid, diglycine or triglycine.

[0033] Furthermore, a single adhesion peptide comprising at least two adhesion peptides may also advantageously comprise one or more target sequences of one or more metalloproteases, in particular metalloproteases 2 and 9. By way of example, the peptide: - PVGLIG, - the peptides QPQGLAK, GPLGLSLGK and GPLGMHGK described in Jha et al., Biomaterials, May 2016; 89; pp. 136-47; and - The peptides GPQGIAGQ, GPQGIWGQ and VPMSMRGG described in Lin et al., Acta Biomaterialia, 10(12), December 2014, pp. 5106-5115 can be mentioned.

[0034] Such metalloprotease target peptides can be introduced to allow cleavage between adhesive peptides that remain bound to the matrix of the present invention but are available for cell adhesion. Furthermore, metalloprotease target peptides can be useful for triggering matrix degradation and thereby promoting cell adhesion. Metalloprotease target peptides can be included in a single adhesive peptide at any position, particularly at its amino-terminal end, between two adhesive peptides contained in the single adhesive peptide, or at its carboxy-terminal end. Preferably, metalloprotease target peptides are located between two adhesive peptides in the single adhesive peptide.

[0035] According to a particular embodiment, the single adhesion peptide comprises at least one occurrence, in particular a single occurrence, of the target sequence for metalloproteases 2 and 9, PVGLIG.

[0036] 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 has the formula: IKVAV (spacer) PVGLIG (spacer) YIGSR, YIGSR (spacer) PVGLIG (spacer) IKVAV, PVGLIG(spacer)YIGSR(spacer)IKVAV, PVGLIG(spacer)IKVAV(spacer)YIGSR, IKVAV(spacer) YIGSR(spacer) PVGLIG, or YIGSR(spacer)IKVAV(spacer)PVGLIG.

[0037] Preferably, the single adhesion peptide has the formula IKVAV(spacer)PVGLIG(spacer)YIGSR.

[0038] The spacers designated as "(spacer)" in the above unique sequences may be identical or different sequences, in particular identical. In particular, the spacers may be selected from glycine residues, diglycine dipeptides or triglycine tripeptides.

[0039] According to one alternative embodiment, the single adhesion peptide has the formula: IKVAV-GGG-PVGLIG-GGG-YIGSR, YIGSR-GGG-PVGLIG-GGG-IKVAV, PVGLIG-GGG-YIGSR-GGG-IKVAV, PVGLIG-GGG-IKVAV-GGG-YIGSR, IKVAV-GGG-YIGSR-GGG-PVGLIG, or YIGSR-GGG-IKVAV-GGG-PVGLI.

[0040] Preferably, said single adhesion peptide has the formula IKVAV-GGG-PVGLIG-GGG-YIGSR.

[0041] The peptides that allow the incorporation of a single peptide into the composite matrix prior to crosslinking and formation of said composite matrix may have the formula: CβA-IKVAV(spacer)PVGLIG(spacer)YIGSR-βAC, CβA-YIGSR (spacer) PVGLIG (spacer) IKVAV-βAC, CβA-PVGLIG(spacer)YIGSR(spacer)IKVAV-βAC, CβA-PVGLIG(spacer)IKVAV(spacer)YIGSR-βAC, CβA-IKVAV(spacer)YIGSR(spacer)PVGLIG-βAC, or CβA-YIGSR (spacer) IKVAV (spacer) PVGLIG-βAC.

[0042] Preferably, the peptide may have the formula CβA-IKVAV(spacer)PVGLIG(spacer)YIGSR-βAC, allowing the incorporation of a single peptide into the hydrogel prior to crosslinking and formation of the composite matrix.

[0043] In a preferred embodiment, the peptide that allows the introduction of a single peptide into the composite matrix prior to crosslinking and formation of said composite matrix has the formula: CβA-IKVAV-GGG-PVGLIG-GGG-YIGSR-βAC, CβA-YIGSR-GGG-PVGLIG-GGG-IKVAV-βAC, CβA-PVGLIG-GGG-YIGSR-GGG-IKVAV-βAC, CβA-PVGLIG-GGG-IKVAV-GGG-YIGSR-βAC, CβA-IKVAV-GGG-YIGSR-GGG-PVGLIG-βAC, or CβA-YIGSR-GGG-IKVAV-GGG-PVGLIG-βAC.

[0044] Particularly preferred is a peptide prior to crosslinking and formation of the composite matrix, allowing the incorporation of a single peptide into said composite matrix, the peptide having the formula CβA-IKVAV-GGG-PVGLIG-GGG-YIGSR-βAC.

[0045] In a particular embodiment, the amount of peptide used to introduce the single peptide into the complex 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, for a given complex matrix concentration, the skilled artisan can of course adjust the amount of the single peptide.

[0046] 2.2. Peptides capable of inducing calcium phosphate nucleation Peptides capable of inducing calcium phosphate nucleation constitute a second category of bioactive peptides that can be introduced into the composite matrix according to the invention. The experiments presented below show that in the presence of such peptides capable of inducing calcium phosphate nucleation in the composite matrix according to the invention, the distribution uniformity and retention of calcium phosphate is improved. Unexpectedly, in the presence of SNA15 peptide, approximately 10 times more hydroxyapatite particles are found in the matrix, which is particularly shown to have a beneficial effect on the osteoconductive and / or osteoinductive properties of the composite matrix.

[0047] For the purpose of composite matrix formation, it can be included in a single peptide that also contains at least one adhesion peptide, or it can constitute an independent peptide. In a preferred embodiment, the peptide capable of inducing calcium phosphate nucleation is a peptide that is not bound to an adhesion peptide prior to the formation of the composite matrix.

[0048] By way of example, among peptides capable of inducing calcium phosphate nucleation there may be mentioned: - peptides derived from statherin, in particular DDDEEKFLRRIGRFG (SNA15) and SNA15 analogues, more particularly the peptides DSSEEKFLRRIGRFG (SNS15) and EFLRRIGRFG (SN11) described in Raj et al. JBC, vol. 267(9), 25 March 1992, pp. 5968-5976; - the peptide DHTKE (Sun et al., Journal of Agricultural and Food Chemistry 2017 65 (44), pp. 9782-9789), - the peptide SSSEEIVPN (Meisel et al., Biol Chem Hoppe Seyler. 1988 December;369(12):1275-9), - the peptide DEGEQPRPFPFP (Lv et al., Food Chemistry, vol. 141(3), 2013, pp. 1645-1650), - the peptide WEWLHYW (Charoenphun et al., Eur Food Res Technol 236, 57-63 (2013)), - the peptide DGDDGEAGKIG (Chen et al., Journal of Functional Foods, Vol. 6, 2014, pp. 575-584), - the peptide GPAGPHGPPG (Guo et al., Food Chemistry, Vol. 173, 2015, pp. 536-542), - the peptide VLSGGTTMYASLYAG (Jung et al., Eur Food Res Technol 224, 763-767 (2007)), - the peptide TCH (Huang et al., Eur Food Res Technol 232, 281-287 (2011)), - the peptide VLGYIQIR (Hou et al., Food Chemistry, Vol. 243, 2018, pp. 389-395), and - Peptide DNLPNPEDNKNYQ (Choi et al., Food Sci Biotechnol 21, 1663-1667 (2012)).

[0049] According to a preferred embodiment, the peptide capable of inducing calcium phosphate nucleation is a peptide derived from statherin, more particularly an SNA15 analogue, even more particularly a peptide selected from SNA15, SNS15 and SN11. Preferably, the peptide capable of inducing calcium phosphate nucleation is an SNA15 peptide.

[0050] The peptide capable of inducing calcium phosphate nucleation may contain a cysteine ​​residue at each of its termini. The cysteine ​​residue may be covalently linked to the amino acid sequence of the peptide directly or via a spacer, in particular a peptide or pseudopeptide spacer. In particular, the spacer may 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, the peptide allowing the incorporation of the peptide capable of inducing calcium phosphate nucleation into the composite matrix of the invention prior to crosslinking and hydrogel formation may have the formula CβA-DDDEEKFLRRIGRFG-βAC.

[0051] According to a particular embodiment, the amount of peptide capable of inducing calcium phosphate nucleation in the composite matrix according to the invention is comprised between 99.9 and 0.1% (w / v). As mentioned above, according to a particular embodiment, for a composite matrix having a concentration between 3 and 5% (w / v) and for a thiol:alkene ratio of 1:1, said matrix may in particular comprise 0.1 to 3% (w / v) of at least one bioactive peptide.

[0052] 3. Calcium phosphate The composite matrix according to the invention also comprises an inorganic phase comprising calcium phosphate. Illustrative examples include calcium phosphate in the form of apatite, tricalcium phosphate, dicalcium phosphate and mixtures thereof. These calcium phosphate sources can be used in any available crystalline form. Furthermore, derivatives of these calcium phosphate sources can also be used, in particular carbonaceous derivatives. According to a particular embodiment, said calcium phosphate is in the form of apatite, more particularly hydroxyapatite. Advantageously, such calcium phosphate sources make it possible to enhance the osteogenic properties of the composite matrix.

[0053] According to a particular embodiment, the composite matrix according to the invention comprises between 0.1 and 50% (w / v) calcium phosphate. According to a particular embodiment, the composite matrix according to the invention comprises between 0.1 and 50% (w / v) hydroxyapatite. More particularly, the composite matrix may comprise between 0.2 and 20% (w / v) 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) hydroxyapatite.

[0054] 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.

[0055] 4. Exemplary embodiments of the composite matrix according to the present invention In particular, a composite matrix according to the present invention may comprise an elastin-like peptide, at least one bioactive peptide and calcium phosphate.

[0056] According to one embodiment, the concentration of the complex matrix is ​​comprised between 1 and 10% by mass (w / v), in particular between 3 and 5% (w / v).

[0057] According to another embodiment, the thiol:alkene molar ratio is comprised between 3:1 and 1:3, more particularly between 2:1 and 1:2, this ratio being more particularly equal to 1:1.

[0058] According to certain embodiments, the composite matrix comprises: (i) elastin-like peptides, (ii) at least one adhesion peptide; (iii) at least one peptide capable of inducing calcium phosphate nucleation, and (iv) Calcium phosphate.

[0059] Components (i)-(iv) may be selected from the components described in parts 1-3 above.

[0060] More particularly, the composite matrix according to the invention may comprise: (i) a peptide derived from the elastin-like peptide ELPM80, (ii) peptide IKVAV and peptide YIGSR; (iii) peptide SNA15 or a peptide similar to SNA15, and (iv) Hydroxyapatite.

[0061] Preferably, the composite matrix according to the invention comprises: (i) peptide ELPM80, (ii) peptide IKVAV(spacer)PVGLIG(spacer)YIGSR; (iii) peptide SNA15, and (iv) Hydroxyapatite.

[0062] More preferably, the composite matrix according to the present invention comprises: (i) peptide ELPM80, (ii) peptide IKVAV-GGG-PVGLIG-GGG-YIGSR; (iii) peptide SNA15, and (iv) Hydroxyapatite.

[0063] 5. How to Prepare the Composite Matrix The composite matrix of the present invention can be prepared by mixing its various components and any other optional ingredients. The components of the composite matrix and the amounts of these components are selected to prepare a composite matrix having physical and support properties adapted to the requirements of the user.

[0064] The skilled artisan is familiar with the crosslinking techniques used in the state of the art for producing composite matrices by crosslinking. Thus, composite matrices can be produced by crosslinking under the action of a stimulus such as a change in temperature or pH, or by a crosslinking agent, in particular a photosensitive crosslinking agent (or photoinitiator). As an illustrative example, one can cite the induction of photopolymerization by a photoinitiator, for example the compound Irgacure 2959, in particular used at a density of 0.5% (w / v) in the mixture and activated by UV-visible light (λ=305-405 nm, in particular 305 nm) for between 5 and 12 minutes, more particularly between 6 and 10 minutes, in particular for about 8 minutes. In another alternative, the photoinitiator can be selected 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 wavelengths comprised between 365 and 475 nm for 5 to 12 minutes, more particularly between 6 and 10 minutes, and especially for approximately 8 minutes.

[0065] Advantageously, the skilled person may also refer to the preparation process described in the examples of the present application, where the hydrogel is produced by forming a cryomatrix. In one alternative embodiment, a pre-matrix solution containing the various matrix components is frozen to form water crystals. When a cross-linking reagent (i.e. a component having thiol and alkenyl moieties) surrounds said water crystals and when the solution is subjected to UV irradiation, the alkene and thiol moieties of the various components of the mixture react at the interface of said crystals. When the solution thaws, the spaces occupied by the water crystals become pores in the matrix. This process may be followed by a freeze-drying process. For example, after cross-linking, the matrix can be rehydrated, in particular for 24 hours, and freeze-dried under vacuum after low-temperature freezing, thus allowing the ice to sublimate without melting and generating new pores. The combination of these two techniques has the advantage of allowing the formation of a wide distribution of pore sizes and the colonization of different cell types useful for the induction of angiogenesis, innervation and osteogenesis.

[0066] Thus, according to a particular embodiment of the process according to the invention, the freezing step is carried out at a temperature between 0 and -80°C, more particularly at a temperature of about -20°C, for at least 2 hours, in particular for about 24 hours. The prematrix can then be subjected to UV radiation suitable for inducing photopolymerization, in particular for about 5 to 12 minutes, more particularly for about 6 to 10 minutes, in particular for about 8 minutes. After crosslinking, according to a particular embodiment, a rehydration step can be carried out, in particular for at least 2 hours, in particular for about 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, and then a freeze-drying step lasting for approximately 24 to 48 hours.

[0067] As mentioned above, these processes allow the formation of a composite matrix containing pores with a broad, controlled pore size distribution. The composite matrix according to the invention may in particular have macropores with an average size greater than 100 μm.

[0068] 6. Use of the Composite Matrix According to the Invention Advantageously, the properties of the composite matrix can be very finely defined. Moreover, said composite matrix according to the invention is biodegradable and has a porous structure suitable for colonization by different cell types, useful for inducing angiogenesis, innervation and osteogenesis. The inventors have also been able to show that the composite matrix according to the invention is not cytotoxic. It therefore collects advantageous properties useful for the development of tools suitable for tissue regeneration.

[0069] The composite matrix according to the invention can therefore efficiently support the in vitro culture of different cell types. In a particular embodiment, the invention therefore relates to a novel three-dimensional support capable of hosting in vitro various cells of interest for bone regeneration, in particular neural, osteocyte or endothelial cells. The invention therefore provides the skilled artisan with a particularly advantageous 3D cell culture system that not only allows cells to develop in a favorable environment, but also to study the interaction of different cell types with each other. This parameter is important for studying regeneration phenomena, which may require a complex dialogue between different cell types. The carrier of the invention can be used in particular to house osteogenic and endothelial cells, and to study angiogenic, osteogenic and innervation effects in in vitro cell culture methods that involve culturing cells on the carrier as defined above. The use of the support according to the invention may also include the addition of an agent to the culture, such as a growth factor or any other agent having or likely to have a biological effect (candidate agent), to determine its effect on one or more parameters and cellular responses, such as cell proliferation, induction of quiescence, cell death, secretion of proteins or other molecules or ions (in particular calcium or potassium ions) or expression of some genes.

[0070] 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 ​​used in a treatment method of regenerative medicine. It can be used to stimulate the innervation of tissues, in particular bone tissue, and is particularly suitable for use in bone tissue engineering. Advantageously, the composite matrix according to the invention promotes innervation, in particular in regenerative situations. More particularly, the composite matrix according to the invention can be advantageously 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 tissues.

[0071] According to another embodiment, the composite matrix according to the present invention is used to repair complex lesions exhibiting vascular and neural damage.

[0072] Furthermore, the composite matrix according to the present invention can also be used to regenerate the interface with the peripheral nervous system and to direct bone development and repair.

[0073] According to another embodiment, the composite matrix can be used as a cell therapy medium.Thus, the composite matrix as defined above can be pre-populated with cells of therapeutic interest, for example with stem cells, in particular artificial stem cells of the lineage of interest, hematopoietic stem cells, mesenchymal stromal stem cells from bone marrow or adipose tissue, neural stem cells or a mixture of cells of different lineages.Such composite matrices can be used in cell or tissue regeneration treatment methods.

[0074] The composite matrix according to the invention can also be used to modify the implant system as well as to improve its biocompatibility and integration. [Brief description of the drawings]

[0075] [Figure 1] Fluorescence scattering and pore size quantification of hydrated matrices. A) Pore size quantification of hydrated hydrogels YIGSR+ELPM80. N=2-5 hydrogels (502<pores<2100) Kruskal-Wallis; Dunn's post-hoc; p<0.001. C) Pore size classification of hydrogels ELPM80+YIGSR into micropores (required for angiogenesis and innervation) and macropores (required for bone formation). [Diagram 2] Figure 1 shows the quantification of vascular and neural structures formed within the hydrogel constructs of the composition IKVAV+YIGSR and its scrambled version VKAIV+GYSRI. Each placement is represented by a point and the average is represented by a bar (n=5-6). It is noted that within the same group, high variability was observed, confirming heterogeneous cell colonization within the hydrogel constructs. No statistical differences were detected. [Diagram 3]Matrix manufacturing process: A diagram showing the preparation of a pre-matrix solution frozen at -20°C for 24 hours, followed by (2) cross-linking under UV light. (3) The cryogel is hydrated for 24 hours and then (4) lyophilized. [Figure 4] Figure showing the quantification of the structure and pore size of a matrix dehydrated to 4% (w / v) using 0% - 5% (w / v) HA. The pore size was quantified using ImageJ. Data are shown as ±SD, 10 < n < 32, and statistical differences are shown as ***p < 0.001 (ANOVA with post hoc Bonferroni test). [Diagram 5] Figure showing the structure and pore size of dehydrated matrices generated with different final mass concentrations (% w / v) and different HA concentrations (% w / v) with a cross-linking time of 8 minutes. Pore size quantification. Data are presented as mean ± SD, 7 < n < 26, and no statistical differences were observed (ANOVA with Bonferroni test or Mann-Whitney test as post hoc test). [Figure 6] Figure showing the internal porosity of matrices hydrated to 3% (w / v) and 4% (w / v) using different HA concentrations. (B) The pore size was quantified using ImageJ. Data are shown as ±SD, 21 < n < 65, and statistical differences are shown by *p < 0.05 and ***p < 0.001 (ANOVA with Bonferroni post hoc test). Bar: 100 μm. [Figure 7]Figure 2 shows the mineralization potential of 3% w / v matrix in mice containing no HA or 1%, 2% and 2.5% w / v HA placed subcutaneously for 2 and 4 weeks. Quantification of microCT acquisition using MicroView software at day 0 (day of placement) and at 2 and 4 weeks. Note that the volume of mineralized tissue increased over time, with matrices containing 2% and 2.5% w / v HA resulting in a greater volume of mineralized tissue than matrices containing no HA and 1% w / v HA. Data are presented as mean ± SD, n=4-5 mice per group, and statistical differences are indicated by *p<0.05 and **p<0.01 (Bonferroni test, ANOVA with post-hoc). [Figure 8] Figure 1 shows the analysis of scanning electron microscopy combined with EDX on ELP matrices + IKVAV / YIGSR peptide with or without peptide SNA15. ELP matrices + IKVAV / YIGSR peptide with 0, 1, 2 and 2.5% w / v HA with or without peptide SNA15 were lyophilized and cut in half. The inside of the cut matrices was analyzed. The signals in the form of white dots correspond to calcium particles that make up the hydroxyapatite. The bar shown in the figure corresponds to 2 mm. [Figure 9] Figure 1: Longitudinal micro-CT monitoring of matrix mineralization. Representative micro-CT images of subcutaneous placement of matrices without HA or containing 1%, 2% and 2.5% (w / v) HA at 0, 15 and 30 days after placement. Mineral volume / total volume (MV / TV) ratios were measured from three-dimensional micro-CT images reconstructed using Microview® software. ANOVA statistical test followed by Bonferroni's post-hoc test with *p<0.05 and **p<0.01 was performed. [Figure 10] FIG. 1 shows micro-CT monitoring of mineralization of subcutaneously placed ELP matrices containing 2% (w / v) HA using MicroView® software. [Figure 11] It is a figure showing histological analysis of a heterotopically implanted matrix stained with Masson's trichrome. Matrices containing 0%, 1%, 2% and 2.5% HA were implanted subcutaneously and stained with Masson's trichrome. Quantitative analysis of the formed osteoid tissue (15 < n < 23 per sample) using the Kruskal-Wallis multiple comparison test with p **<0.01 and p ***<0.001. [Figure 12] It is a figure showing immunostaining of blood vessel networks and nerve structures in a composite matrix containing 2% HA at 0, 3, 7, 15 and 30 days after implantation at a heterotopic site. (A) Quantification of the surface area occupied by blood vessels × 1000 relative to the matrix surface area. (B) Quantification of the surface area occupied by nerve structures × 1000 relative to the matrix surface area. Quantitative analysis using the Kruskal-Wallis multiple comparison test. [Figure 13] It is a figure showing micro-CT analysis of mineralization induced by a composite matrix in a femoral condyle lesion model in rats. (A) Representative micro-CT images of bone lesions at 7, 15, 30 and 60 days after implantation, by group: empty lesions, those filled with an ELP matrix containing 2% HA and the positive control Collapat®. (B) For three groups (empty, ELP, Collapat®), the mineral volume / total volume (MV / TV) ratio was measured from three-dimensional micro-CT images reconstructed using Microview® software for all implantation times (D7, D15, D30 and D60). Quantitative analysis of mineralization using the Kruskal-Wallis multiple comparison test with p*<0.1 and ns = not significant. [Figure 14] It is a figure showing histological sections of lesions in the femoral condyle filled with an ELP matrix / peptide containing 2% HA. It is a figure showing staining of a bone lesion filled with a composite matrix using Masson's trichrome 7 days after implantation. The area corresponding to the lesion is demarcated by a black dotted circle. [Figure 15]Figure 10: Matrix mineralization in a mandibular lesion model.3D micro-CT images showing the reconstruction of a mandibular defect in the presence of a composite matrix. EXAMPLES

[0076] Example 1 Preparation of hydrogels with improved properties Our team recently evaluated biomaterials considering the importance of vascularization and innervation during bone regeneration (Paiva dos Santos et al., Acta Biomater. 2019, 99, 154). The aim of this study was to develop an acellular, growth factor-free hydrogel that promotes angiogenesis and innervation. Hydrogels containing 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 studies could show that the 50% IKVAV composition has a greater potential to support osteogenesis, angiogenesis and innervation. In vivo, this composition made it possible to induce a higher vascular density and the formation of nerve endings in the surrounding tissue after subcutaneous placement in mice without inducing any inflammatory reaction. However, this previous study had some limitations. First, the porosity of the hydrogel was not sufficient to allow uniform cell colonization in the graft. Furthermore, the hydrogel does not appear to degrade after placement, possibly because PEG is not sufficiently biodegradable over the long term.

[0077] Therefore, we sought to modify the composition and structure of the hydrogel to improve the uniformity of cell colonization and the biodegradability of the material.

[0078] Development of methods to induce improved porosity First, we sought to determine the best way to induce porosity in the hydrogel matrix to allow its colonization by cells. These structures were analyzed by scanning electron microscopy (SEM).

[0079] State-of-the-art hydrogels consisting of ELPM40 and PEG were prepared and their pore sizes were characterized.

[0080] The first method used to induce pores, hereafter referred to as "CryoUV", involves the formation of a cryomatrix. A prematrix solution was frozen to form water crystals. When a cross-linking reagent surrounds the water crystals and the solution is subjected to UV irradiation, the alkene and thiol moieties of the various components of the mixture react at the interface of the crystals. When the solution thaws, the spaces occupied by the water crystals become pores in the matrix.

[0081] The other method used is based on the generation of matrices using the CryoUV method followed by a freeze-drying step (hereafter called "CryoUV+Lyoph"). After crosslinking, the matrices are rehydrated for 24 hours and freeze-dried under vacuum after 24 hours of cryo-freezing, thus sublimating the ice without melting, thus generating new pores. The combination of these two techniques has the advantage of allowing the generation of a wide distribution of pore sizes and allowing the colonization of different cell types useful for the induction of angiogenesis, innervation and osteogenesis.

[0082] We used environmental SEM to evaluate the external structure of the matrix. Prior to SEM analysis, CryoUV hydrogels were generated and their water content evaporated to allow SEM visualization. For the CryoUV+Lyoph method, freeze-drying allowed the three-dimensional structure of the hydrogel to be revealed. The results show that both methods allow the generation of hydrogels with a porous structure.

[0083] The inventors then investigated the possibility of modifying the hydrogel composition to improve the retention and biodegradability properties described above.

[0084] Generation of peptide ELPM(alkene)-80 ELPM40 MW [(VPGVG)(VPGMG)(VPGVG)2], previously described (Petitdemange et al., Biomacromolecules. 2017 Feb 13;18(2):544-550). 15 The plasmid ELPM40-pUC19 encoding ELPM40 was digested with the restriction enzyme BsmF1 to linearize it, and then 5' dephosphorylated with Antarctic phosphatase Antarctic. The nucleotide sequence encoding the amino acid sequence [(VPGVG)(VPGMG)(VPGVG)2]5 was extracted from the plasmid pUC19 encoding ELPM20, also as described above, by digestion with the restriction enzymes BsmF1 and BtgZI. This sequence was used as an insert introduced by ligation into the linearized plasmid ELPM40-pUC19, thus obtaining the plasmids pUC19-ELPM60 and pUC19-ELPM80 encoding ELPM60 and ELPM80, respectively. The sequence encoding ELPM80 was then introduced into the expression vector pET44a after digestion of the plasmid pUC19-ELPM80 with the restriction enzymes NdeI and BamHI, allowing IPTG-inducible expression of ELPM80. Petitdemange et al. (Biomacromolecules. 2017 February 13; 18(2):544-550) report that the peptide MX [VPGVGVPGMG (VPGVG)2] 10 The construction of an expression vector for (ELPM40), its expression in E. coli, its isolation from bacterial lysates, its purification and characterization have been described. These conditions were adapted for the production and purification of ELPM80. The correct structure and molecular weight were confirmed by 1H NMR and MALDI mass spectrometry.

[0085] An alkene moiety was then added to peptide ELPM80 by chemoselective thioalkylation of the methionine side chain of peptide ELPM80 using allyl glycidyl ether to generate peptide ELPM(alkene)-80, hereafter referred to as ELPMa-80, also following the conditions described in Petitdemange 2017. The structure of the resulting alkenylated peptide was confirmed by 1H NMR.

[0086] Preparation and characterization of composite matrices containing ELPM80. To improve the hydrogel composition, we replaced the ELPM40 contained in the prior art composition with the elastin-like peptide ELPM80. Incidentally, the adhesion peptide YIGSR was also introduced into the hydrogel composition. Its scrambled version GYSRI was used as a control. The resulting hydrogel composition is shown in Table 1.

[0087] [Table 1]

[0088] Diffusion of FITC-dextran 500 kDa into the matrix structure and evaluation of fluorescence in the core material by confocal microscopy were used to quantify pore size and porosity using ImageJ. The lowest measured pore size for both CryoUV and CryoUV+Lyoph methods was 2.16 μm, while the highest was 382.5 μm using the CryoUV method and 523.5 μm using the CryoUV=Lyoph method, indicating that the latter produced larger pores (Figure 1).

[0089] To satisfy angiogenesis, innervation and osteogenesis, the range of pores needs to be extremely wide. Data from literature suggests that the optimal size is 0.005-0.3 μm for innervation, 5-15 μm for fibroblast proliferation, 50-400 μm for rapid angiogenesis, and 100-400 μm for bone regeneration. Considering the pore size for inducing osteogenesis, we evaluated the pore frequency by size and named them as micropores (smaller than 100 μm) and macropores (larger than 100 μm). In general, macropores are more frequent when the sample is freeze-dried compared to the CryoUV method (Figure 1), which satisfies angiogenesis, innervation and bone regeneration.

[0090] Based on the previous sections, the CryoUV+Lyoph method was standardized to consider the following criteria: (i) large pore size to meet the prerequisites for cell colonization by different cells allowing angiogenesis, innervation and osteogenesis; (ii) ensuring high porosity; (iii) compositional modifications were made to generate all-natural polymeric materials to facilitate matrix degradation, meaning that it was possible to generate PEG-free matrices while maintaining alkene / thiol equimolarity; and (iv) ELPM40 was replaced with ELPM80. We subcutaneously implanted the IKVAV+YIGSR composition and also its scrambled version in mice to evaluate angiogenesis and innervation potential in vivo. After 4 weeks of implantation, samples were collected and histologically analyzed by hematoxylin-eosin (HE) staining to evaluate tissue architecture, cell colonization and persistent inflammation. For angiogenesis potential, CD31 immunohistochemistry (IHC) was performed, and for innervation potential, β3 tubulin IHC was used. For both compositions, no persistent signals of inflammation were observed, and nerves and blood vessels were found inside the hydrogel. However, heterogeneous cell colonization occurred, indicating the need to optimize and standardize material production. Quantification of blood vessel and nerve density also confirmed these observations, showing greater variability within the same group (Figure 2).

[0091] Based on the data outlined above, modifications seemed necessary to increase the functionality of the hydrogel in vivo. Given the limited reproducibility of the hydrogel and the resulting cell colonization, the inventors decided to optimize the matrix generation protocol and modify the composition to combine osteogenic potential with the de novo generation of the material. As described above, using hydrogels based on ELP and adhesion peptides, the inventors have already shown that the hydrogels can induce angiogenesis and innervation around the implantation site.

[0092] To increase biological functions, we explored other biomimetic peptides to enhance neural and vascular cell recruitment, matrix degradation, cell anchorage and mineral content retention: (i) the basic adhesive peptides CβA-IKVAV-βAC and CβA-YIGSR-βAC were replaced with the sequence CβA-IKVAV-GGG-PVGLIG-GGG-YIGSR-βAC, which retained the same cross-linking chemistry strategy, with a thiol group present on the adjacent cysteine ​​to allow cross-linking with the alkene grafted onto ELPM80. We also used a control peptide in which the scrambled versions VKAIV and GYSRI replaced their original sequences IKVAV and YIGSR, respectively. In this control peptide, the sequence PVGLIG was retained. Three glycines were used as spacers between the functional units, and (ii) PVGLIG, a target degradation site for matrix metalloproteinases 2 and 9, was included between the two adhesive sequences. Thus, once cleaved, each adhesive sequence is still attached to the matrix and available for cell attachment. Moreover, this sequence induces matrix degradation and thus aids cell colonization. (ii) To maintain and ensure a uniform distribution of hydroxyapatite particles, the peptide SNA15, CβA-DDDEEKFLRRIGRFG-βAC, a calcium phosphate nucleating unit derived from statherin, was used. As a result, (iii) hydroxyapatite (HA) corresponding to the synthetic bone mineral content is included in the material composition to provide a biochemical signal of osteoconduction properties. Our group has already synthesized and characterized them, with 50-100 nm rod-shaped HA crystals that form aggregates with an average diameter of 3.26 ± 0.62 μm. We used the CryoUV+Lyoph method to standardize the incubation time in the matrix generation protocol (Figure 3).

[0093] Considering the importance of porosity and pore size, the next step in the characterization process was to optimize the matrix according to 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 since it has a direct effect on the creation of the matrix. 4% (w / v) matrices (Table 4) consisting of ELPM80, a biomimetic peptide containing the sequences IKVAV, PVGLIG and YIGSR, SNA15, with or without HA were generated and crosslinked for 5, 8 and 15 minutes. Using scanning electron microscopy, images were obtained from the matrix and the pore size could be quantified (Figure 4).

[0094] [Table 2]

[0095] The results obtained suggest that shorter crosslinking times (5 min) result in larger pores. Indeed, when a crosslinking time of 15 min was used, almost no pores were observed. Without wishing to be bound by any theory, we hypothesize that with longer crosslinking times, the cryomatrix is ​​more likely to start thawing. This would result in a stiffer matrix due to a higher crosslinking rate and smaller pores due to thawing. Indeed, this could explain why very few pores could be observed when a crosslinking time of 15 min was used. In general, the pore size differs as a function of crosslinking time in matrices without HA, whereas there is no difference in pore size when a 5% (w / v) HA matrix was used. This suggests that HA interferes with pore formation regardless of the crosslinking time. Considering the pore size in matrices with and without HA as well as the fact that the matrices were dehydrated, we decided to standardize the crosslinking time to 8 min for subsequent experiments.

[0096] We then evaluated the effect of final matrix concentration on 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).

[0097] The 3% (w / v) matrix tended to retain its structure depending on the different HA concentrations, while the 2% (w / v) and 4% (w / v) matrices had a powder-like structure with potentially reduced reproducibility. SEM analysis confirmed the external porosity of the dehydrated matrices produced with a crosslinking time of 8 min, demonstrating that the volumetric density of the mesh increased in proportion to the matrix concentration. The 4% (w / v) matrix appears to contain more pores but with smaller diameters, while the 2% (w / v) and 3% (w / v) matrices appear to contain fewer pores but with larger diameters. The pore size of dehydrated matrices with the same HA concentration did not change as a function of the final concentration. Again, the HA content appears to interfere with the pore size in matrices with the same final concentration, but this trend does not appear to be statistically significant.

[0098] To confirm these results and evaluate the internal porosity, we quantified the pore size in the hydrated matrices by cutting them in the middle and generating histological sections by cryostat. Figure 6 shows that in 3% (w / v) matrices, 5% HA matrices have smaller pore sizes than matrices with 0% and 2.5% HA. Furthermore, when comparing 3% (w / v) and 4% (w / v) matrices, 3% (w / v) matrices with 0% HA, 2.5% HA and 5% HA have significantly larger pores than 4% (w / v) matrices with the same HA concentration. Importantly, in the 3% (w / v) matrices, there is no difference in pore size for matrices without HA and with 2.5% (w / v) HA, and the pore size is larger than 100 μm, which is the minimum pore size described in the literature that satisfies bone formation. From then on, the study was focused on 3% (w / v) matrices containing up to 2.5% (w / v) HA.

[0099] We then evaluated the overall porosity of matrices without HA and with 2.5% (w / v) HA after rehydration. They used the methodology described by Ma and Zang, which is based on the overall density of the fibrous matrix and the skeletal density (Ma and Zhang, J. Biomed. Mater. Res. 1999, 46, 60). The skeletal density is taken as the density of the polymer and HA. Matrices without HA and with 2.5% w / v HA did not differ in terms of porosity. The matrix without HA was 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 matrix.

[0100] The retention of HA in the matrix structure was then studied by thermogravimetric analysis (TGA). The HA content retained in the matrix structure reached a plateau at 2.5% (w / v). Therefore, we decided to use a maximum final HA concentration of 2.5% (w / v) for the remainder of the study, but did not exclude the possibility of using matrices containing 1 or 2% HA, taking into account the results obtained at 2.5%.

[0101] We then analyzed the HA distribution by EDX on the surface and inside the matrix. SEM images confirmed the surface and internal porosity of the matrix with and without HA. EDX analysis is an X-ray technique used to identify the elemental composition of a material, in this case calcium content appears as white dots. Experiments could show that hydroxyapatite is uniformly distributed on the surface and in the matrix.

[0102] In parallel, biological evaluations were performed to analyze cell survival and distribution in the matrix. Rat primary endothelial cells (EC)-RFP and mesenchymal stem cells derived from bone marrow (BMSC) were co-cultured with the matrix and observed by confocal microscopy. The acquisitions performed by the latter had a z-volume of 130-190 μm and were analyzed using Imaris for 3D display. After 7 days of co-culture, EC-RFP were observed at the periphery and in the matrix, indicating that the matrix provides a support for cell culture. To evaluate whether cells settle in the matrix nuclei, they were cut and then histological sections were prepared using a cryostat. The cores of the samples were counterstained with DAPI (40,60-diamidino-2-phenylindole) and images were captured to detect RFP and DAPI to evaluate cell settlement in the matrix cores. For matrices without HA, no RFP or DAPI staining was observed in the core of the matrix but only at its periphery, whereas within matrices containing 2.5% HA, RFP+ cells were evenly distributed.

[0103] After detailed matrix characterization, we subcutaneously implanted 3% w / v matrices without HA or containing 1%, 2% and 2.5% w / v HA in mice. We then monitored implantation on the day of implantation (day 0), 2 weeks (2W) and 4 weeks (4W) and assessed the mineralization potential of the matrices using micro-CT. Micro-CT confirmed the formation of ectopic mineralized tissue within 2 and 4 weeks of implantation (Figure 7). Once quantified, the volume of mineralized neo-tissue 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 in inducing mineralized neo-tissue formed after 2 or 4 weeks of implantation. Matrices with 2% and 2.5% led to a greater volume of mineralized tissue after 2 weeks than matrices with 1% w / v HA. A p=0.059 was achieved compared to the volume of mineralized tissue produced by matrices containing 1% and 2% w / v HA. Matrices without HA did not result in visible or significant mineralized tissue over time. A small amount of mineralized tissue was observed in some animals at 2 weeks, but was likely resorbed and not detectable by 4 weeks. 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.

[0104] In conclusion, we have described a novel cell-free and growth factor-free matrix composite capable of inducing the formation of ectopic mineralized tissue in mice. The matrix is ​​composed of ELPM80, a biomimetic peptide involved in the recruitment of neural and vascular cells, stimulates cell colonization, and retains HA. Combined with 1-2.5% (w / v) HA, the functionalized matrix has osteoinductive properties.

[0105] Example 2 Evidence for involvement of peptide SNA15 in the retention and distribution of hydroxyapatite particles. We have generated a composite matrix as described above, whose organic part consists of a biomimetic peptide containing ELPM80-alkene, the adhesion sequences IKVAV, YIGSR, the proteolytic cleavage motif PVGLIG and also the calcium nucleating peptide SNA15, and whose inorganic phase consists of hydroxyapatite microparticles.

[0106] The calcium phosphate nucleating peptide SNA15 with the sequence DDDEEKFLRRIGRFG is derived from the salivary protein statherin. A modified SNA15 peptide with the sequence CβA-DDDEEKFLRRIGRFG-βAC was used.

[0107] Composite matrices were generated with or without this peptide SNA15 by using the same ELP matrix concentration and varying the HA concentration from 0% to 2.5%.

[0108] Energy dispersive X-ray (EDX) analysis was performed to determine the surface elemental composition of the material, more specifically the element calcium (identified as white dots in FIG. 8). It can be seen that the matrix without HA contains little or no calcium. As expected, the more HA particles are added to the matrix, the more the signal increases. This phenomenon is even more visible when the gel contains peptide SNA15.

[0109] Quantitative analysis shows that the atomic percentage of calcium in matrices containing SNA15 is greater than in matrices not containing the peptide SNA15, more than 8-fold greater in the 1% HA+SNA15 matrix, more than 8.9-fold greater in the 2% HA+SNA15 matrix, and more than 12.6-fold greater in the 2.5% HA+SNA15 matrix.

[0110] It can be concluded that peptide SNA15 is able to substantially improve the retention of calcium phosphate particles since there are 8 to 12.6 times more calcium phosphate particles in its presence.

[0111] Example 3 In vivo evaluation of ELP-based composite matrices in ectopic sites and bone lesions I. Ectopic placement of ELP matrices supplemented with different concentrations of hydroxyapatite ELP and peptide matrices containing different concentrations of HA particles were placed subcutaneously to select the one with the best mineralization, vascularization and innervation potential. The four matrices differed only in their HA content (0%, 1%, 2% and 2.5% particles).

[0112] I.1. Study of mineralization ability of composite matrices Mineralization of matrices placed at subcutaneous sites was monitored by X-ray microtomography (micro-CT) immediately after matrix placement (D0) and then 15 and 30 days later (D15 and D30) (Figure 9). At day 0, no mineralization was observed in 0%, 1% and 2% HA implants, while very little mineralization was observed in the 2.5% HA matrix (Figure 8). These results indicate that HA particles at the concentrations studied are only poorly visible using the micro-CT technique.

[0113] After 15 and 30 days of placement, ectopic mineralized tissue was formed in 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 placement of HA-free matrices, demonstrating the importance of these particles in the mineralization process. After 1 month of placement (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 any significant difference in the amount of mineralized tissue formed between matrices containing 2% and 2.5% HA after 30 days of placement. Mineralization of 2% HA matrix is ​​shown in Figure 10.

[0114] I.2. Study of the osteoinductive properties of the composite matrix After 4 weeks of placement, the matrices were excised, paraffin embedded and analyzed histologically using staining with Masson's Trichrome to assess tissue architecture, cell colonization, inflammation and osteoid tissue formation.

[0115] Histological sections of the material in association with the surrounding epithelium, fat and muscle tissue show that after one month of placement, very little fibrosis occurred around the ELP matrices, regardless of formulation. This indicates that the matrices induce little inflammation and are not rejected. These results are consistent with the biocompatibility demonstrated in vitro. Again, the presence of sheet-like structures and pores can be seen in these sections.

[0116] The presence of cell nuclei within the deposited matrices was also observed, indicating that cells were also able to invade these matrices.

[0117] Since the tissue and matrix were not demineralized, some areas of hypermineralization were damaged during microtome sectioning. However, the presence of mineralization in the form of dark purple crystals is noteworthy. Osteoid tissue is also visible at the periphery and within the matrix.

[0118] The amount of newly formed osteoid tissue (Figure 11) increases significantly with the amount of HA in the matrix from 0 to 2%. In contrast, the values ​​measured in matrices containing a 2.5% mineral fraction were not significantly different from those containing a 2% mineral fraction. Thus, matrices containing 2% HA particles appear to be a formulation favorable for mineralization and osteoinduction processes.

[0119] I.3. Study of angiogenesis and innervation potential of ELP matrices The formation of vascular and neural structures was observed by immunostaining for vascular (endomucin) and neural (β III tubulin) markers. During this study, histological sections of the matrices were taken at 0, 3, 7, 15 days and 1 month after placement to determine the kinetics of the formation of these structures.

[0120] Regarding the formation of vascular structures on day 0, no blood vessels were observed in the matrix approximately 3-4 hours after placement. After 3 days of placement, blood vessels appeared mainly on the periphery of the matrix, and then their infiltration was observed on day 7. After 15 days of placement, blood vessels were present both on the periphery and inside of the matrix. By day 30, the vascular network within the matrix became denser.

[0121] These observations are confirmed by quantification of blood vessels in the studied matrices (Fig. 1eA). One month after placement, vasculature increases by approximately 10% in all matrices, whatever the HA concentration. The only significant difference is observed between matrices without HA (0%) and those containing 1% HA.

[0122] The ability of the matrix to stimulate innervation was also demonstrated by the presence of nerves at the periphery of the material, especially at early stages after placement. During bone regeneration, nerve structures first appear even at early stages after bone damage. This explains the presence of some nerve fibers visible at the periphery of the composite matrix from the third day of placement. After one month, nerve structures are still detectable at the periphery of the matrix, but their diameter appears to have increased.

[0123] Quantitative results obtained by measuring the surface area occupied by neural structures located at the periphery of the matrix in relation to the total surface area of ​​the matrix confirmed previous observations (Figure 12B). In terms of formulation, the composite matrix containing 1% HA appears to significantly stimulate the formation of neural structures after one month of placement. This is particularly evident when comparing these results with those obtained with matrices without HA or supplemented with 2.5% HA.

[0124] Since the measured density of nerves or blood vessels may vary depending on the position and / or angle of the tissue section, we also used a technique that does not require cutting the sample and allows the whole tissue to be visualized in three dimensions after immunofluorescent labeling of the neural network. This technique allows observing in three dimensions the autoluminescence of the matrix and the presence of a network of neural structures expressing β III tubulin. In particular, the strong presence of neural structures in close proximity to the matrix is ​​visible. Incidentally, the vascular network appears to be uniformly distributed.

[0125] I.4. Study of inflammatory response after subcutaneous placement of composite matrix When the inflammatory reaction is moderate, it is an essential process for tissue regeneration and angiogenesis, whereas when it is too strong, the response from the immune system can lead to rejection of the graft.

[0126] In the case of composite matrices, histological analysis using staining with Masson's Trichrome (MT) did not reveal any inflammatory fibrous capsule around the implants, regardless of their composition.

[0127] To confirm these data, we performed immunolabeling of immune cell populations. As inflammatory markers, we used CD11b, which is expressed by the entire myeloid cell line (monocytes, macrophages, neutrophils), and CD45, a more specific marker for lymphocytes. We found that immune cells were recruited to the periphery of the matrix a few hours after placement. These cells are detectable up to 7 days after placement. Then, after 3 and 6 months, immune cells are only present inside the matrix. They may be macrophages, which may play a role in matrix degradation.

[0128] I.5. Future in vivo studies of composite matrices To determine the longevity of the matrices, subcutaneous placement was performed and their future was monitored for up to 6 months after placement.

[0129] Immediately after placement (D0), the composite matrix containing 2% HA particles is clearly visible (reddish color) and blue HA crystals can be identified. After 1 month of placement, the free spaces left by the pores have been filled as a result of cellular colonization. Several nuclei can be identified within the matrix. After 3 months of placement, the structure of the matrix is ​​no longer visible, probably due to its degradation by the immune system. One can note the presence of a strong cellular component and of an extracellular matrix filling the space previously occupied by the composite material. After 6 months, similar results are observed, but with increased mineralization of the entire area previously occupied by the composite matrix.

[0130] In conclusion, with regard to these studies carried out at ectopic sites, matrices developed from ELP, peptides IKVAV / YIGSR and SNA15 demonstrated their ability to promote angiogenesis and innervation. Supplementing the matrices with HA endows them with osteoinductive properties so that they can then form mineralized and osteoid tissue.

[0131] In conclusion, the composite matrix containing 2% HA was selected for implantation in bone sites since it exerted the best potential in terms of bone formation, neovascularization and creation of neural structures.

[0132] II. Placement of the composite matrix into the bone site The regenerative potential of a composite matrix containing 2% HA was evaluated in rat models of femoral condyle and mandibular defects.

[0133] II.1. Placement in the rat femoral condyle Circular defects of 3 mm diameter were created in the femoral condyles of rats. Three conditions were tested: an unfilled defect corresponding to a negative control, a defect filled with our composite matrix, and a defect filled with the commercially available Collapat® matrix used as a positive control.

[0134] Micro-CT analysis was performed at different times after placement (7, 15, 30 and 60 days) to monitor the mineralization of the condylar defect. 2D images of the femoral condyle reconstructed by 3D micro-CT are shown in Figure 13A, and quantification of the volume of mineralized tissue relative to the total volume (MV / TV) is shown in Figure 13B.

[0135] Micro-CT images show the formation of mineralized volumes in lesions filled with composite matrix and Collapat®, whereas less mineralization is detected in unfilled defects (FIG. 13A).

[0136] The results of the quantitative analysis (FIG. 13B) show that there is partial mineralization of the unfilled defects and a non-significant increase in MV / TV as a function of time. At day 7, this ratio is 8.6%±4.8%, then increases to reach 10.3%±5.6% at day 15, 14.7%±5.9% at day 30, and 37.5%±3.0% at day 60. However, we observed fracture of the site in some animals, which complicated the quantification of MV / TV. Our previous study showed that bone lesions with a diameter of 2.8 mm can repair naturally (Schlaubitz et al., 2014). In this case, we created a lesion with a diameter of 3 mm, which probably weakened the lesion site and caused a fracture that delayed or altered the kinetics of repair. Nevertheless, the micro-CT images show mineralization at the periphery of the lesions, explaining the high MV / TV ratio in the region of interest (ROI) (Figure 13B). In all cases, these images observed for the unfilled lesions differ from those obtained after placement of the composite matrix.

[0137] Indeed, in this case, a mineralization is observed that begins at the inner edge of the lesion and increases with time after placement. Initially, the mineralization is essentially peripheral and then progresses towards the interior of the lesion over time. After 60 days of placement, it is visible that the cortical portion is remodeled in contrast to the unfilled defect (Figure 13A). Quantitative analysis shows that the MV / TV values ​​increase significantly over time (Figure 13B). They are 8.0±3.9% after 7 days of placement, 15.6±7.0% after 15 days, 28.8±14.2% after 30 days and 56.0±6.9% after 60 days.

[0138] With the control material Collapat®, the MV / TV is 40.2±9.4% from day 7 of placement, which is significantly higher than that measured with the ELP / peptide / HA composite matrix. This result was expected since this commercial material contains a high content of calcium phosphate particles, which induces an X-ray signal (FIG. 13A). No significant increase in mineralization was observed from D7 to D30. There is little or no change in the MV / TV ratio, so the MV / TV ratio is 67.3±6.3% after 60 days.

[0139] Thus, comparing the three conditions studied, we observe that the amount of mineralized tissue measured between days 7 and 60 increases by 4.4-fold in unfilled defects, 7.1-fold with our composite matrix, and 1.7-fold with Collapat®.

[0140] Histological analysis of the bone formation induced by the composite matrix was then performed.

[0141] At 7 days, 15 days, 1 month, and 2 months after placement, femurs were removed, decalcified, and histological sections were taken, which were then stained with Masson's trichrome (Figure 14).

[0142] Histological sections of the lesion areas were observed. For unfilled lesions, the defect borders were still clearly visible 7 days after surgery. After 15 days, the lesions were filled with primarily collagenous tissue. By 30 days, the lesion borders were difficult to distinguish, and organized osteoid tissue appeared at the periphery.

[0143] Histological sections show that the ELP / peptide / HA composite matrix was well integrated into the bone defect. There is no visible demarcation between the lesion and the material. The presence of numerous cell nuclei within the matrix confirms cell colonization. Furthermore, the absence of fibrosis is a sign of good biocompatibility of the matrix used. Quantification of the newly formed osteoid tissue shows an increase from day 15, with the maximum amount measured at day 30. However, by 2 months, the amount of osteoid tissue had decreased. Analysis of the histological sections shows that bone formation occurs from the periphery towards the center of the defect, confirming the images observed by micro-CT (Figure 13A).

[0144] Good bone integration was also observed for the Collapat® material, as well as bone tissue formation from the periphery towards the center of the defect. Quantification of newly formed osteoid tissue also showed maximum neogenesis after 30 days, which is comparable to the kinetics observed with our composite matrix.

[0145] To specifically observe the presence of bone cells, immunolabeling of osteocalcin, a specific marker for bone tissue, was performed. The images show that the unfilled lesions have almost no osteoblastic cells 7 and 15 days after surgery. After 30 days, few osteoblastic cells are detected at the edge of the area delimiting the bone lesion.

[0146] In the case of composite matrix and Collapat®, osteocalcin was detected as early as day 15 and its presence was confirmed at days 30 and 60, confirming the presence of osteoblastic cells in the new tissue.

[0147] To evaluate vascularization and innervation in the condylar lesion, an analysis of vascular markers (endomucin, podocalyxin and Meca32) and neural markers (β III tubulin) was performed using the whole bone clearing technique. This analysis made it possible to visualize the organization of the vascular and neural networks in the initially damaged area. Multiple labeling was used to facilitate the visualization of thick samples as in the case of whole bones (Kirst et al., 2020, Cell 180, 780-795.e25).

[0148] 2D sections of lesions filled with composite matrix containing 2% HA were analyzed after 30 days of placement. The bone tissue appears in green due to its autofluorescence, the vascular network is in cyan blue and the nerve extension is in red. 2D images obtained for each condition after 15 and 30 days of placement were also analyzed: lesions not filled, lesions filled with composite matrix or with Collapat®.

[0149] In unfilled lesions, a vascular network developed, likely originating mainly from the peripheral muscle tissue. After 30 days, vascular and neural structures within the damaged area were detectable. However, histological analysis using TM staining showed that the damaged area was filled with soft, disorganized, collagen-rich tissue.

[0150] For lesions filled with the composite matrix, formation of a vascular network throughout the matrix was observed as early as day 15.

[0151] In contrast, for defects filled with Collapat®, vascularization was mainly present in the outer periphery of the defect. Moreover, this vascularization was only visible from day 30 after placement. Thus, these qualitative results indicate that the Collapat® matrix is ​​less effective than our composite matrix in promoting vascularization of the graft.

[0152] As far as innervation is concerned, the majority of the fibers are located in the periosteum covering the cortical bone surface, but for defects that are unfilled or filled with a composite matrix, few fibers are visible within the lesion. In contrast, when the condylar defect was filled with Collapat®, no nerve fibers were detected.

[0153] II. Placement of composite matrix in mandibular defects To evaluate the use of the developed materials for craniofacial reconstruction, we chose to create a mandibular defect model. Only two bones of the skull are mobile: the hyoid bone and the mandible. The mandible has the advantage of being the larger and more easily accessible bone.

[0154] Mandibular defects also offer the advantage of being subjected to high mechanical stresses compared to the parietal defects classically used to test bone repair materials. However, these mechanical stresses are necessary for effective bone repair. In the mandible, the stresses are intrinsically linked to chewing. Therefore, this model of mandibular pathology was chosen to test bone repair materials, as it better adapts to physiological reality than parietal defects.

[0155] To validate this model, a preliminary experiment was performed. A circular defect of 3 mm diameter was created and its repair was monitored using micro-CT. After 3 months, no bone repair was observed, indicating that the defect was indeed of the critical size.

[0156] Preliminary micro-CT observations were performed at different time points after placement (7, 15, and 30 days) to monitor the mineralization of the mandibular defect. The 3D images of the micro-CT reconstructed mandible filled with the composite matrix are shown in Figure 15.

[0157] Partial remodeling is observed at the periphery of the defect at day 30. Quantification of MV / TV is 0.037±0.010 at day 7 and 0.040±0.018 at day 15, i.e. comparable. At day 30, an increase in the MV / TV ratio (0.094±0.049) was measured.

[0158] Histological sections obtained after 15 days of placement of the composite matrix were analyzed by TM staining.

[0159] Interestingly, at the bone-matrix junction, areas were observed that highlighted the presence of new bone tissue with osteoblastic border cells, small squares containing osteocytes within a bone structure that appeared to be lamellar. Blood vessels were also seen in the new tissue.

[0160] Furthermore, when considering tissue remodeling from the periphery to the center of the lesion, it is important to note the tissue infiltration accompanied by angiogenesis and the formation of bone tissue in contact with the matrix, confirming its osteoconductive properties.

[0161] Example 4 Conclusions of the in vivo study In conclusion, different composite materials were tested in vivo in small animals at ectopic and orthotopic sites.

[0162] All matrices tested integrated well with the surrounding tissue and did not induce any significant inflammatory response. HA-containing matrices were also shown to induce mineralization. Vascular structures infiltrated the implants, demonstrating their angiogenic potential. The presence of nerves at the periphery of the materials, especially at early stages after placement, also demonstrated the ability of the matrices to stimulate innervation.

[0163] A matrix containing 2% HA was selected for its ability to promote mineralization and for its angiogenic potential, and was placed into the condylar and mandibular defects.

[0164] Bone remodeling was improved after placement of the composite matrix in condylar defects compared to unfilled defects. Organized bone structures expressing osteocalcin were identified in these hydrogels. Organized vascular networks developed throughout the bone lesions, but not in the case of the Collapat® control material.

[0165] The results obtained in the mandibular defect model show very satisfactory results regarding the osteoconductive and angiogenic properties of the composite matrix. Histological analysis showed that the material was well integrated. Bone and vascular structures were found as early as 15 days after placement.

[0166] These results therefore demonstrate the therapeutic value of the composite matrix according to the invention.

Claims

1. At least one elastin-like peptide, At least one bioactive peptide, A peptide that can induce calcium phosphate nucleation, and Calcium phosphate A composite material containing [a certain material].

2. The composite material according to claim 1, wherein the elastin-like peptide comprises at least one alkenylated residue prior to the formation of the composite material.

3. Elastin-like peptides are given by formula MW[VPGVGVPGMG(VPGVG)] 2 ] x The composite material according to claim 1, wherein x is a peptide of (wherein x is an integer between 15 and 25, particularly between 19 and 21, and more specifically, an integer equal to 20, the methionine residue of which is alkenylated before the formation of the composite material).

4. Elastin-like peptides are given by formula MW[(VPGVG)(VPGMaG)(VPGVG)] 2 ] 20 (In the formula, Ma is given by formula (III): 【Chemistry 1】 The composite material according to claim 1, wherein the peptide ELPM80 represents an alkenylated methionine residue prior to the formation of the composite material.

5. The composite material according to claim 1, wherein the at least one physiologically active peptide is an adhesion peptide capable of recruiting target cells.

6. The composite material according to claim 1, wherein the at least one physiologically active peptide is an adhesion peptide of the formula IKVAV-GGG-PVGLIG-GGG-YIGSR.

7. The composite material according to claim 1, wherein the peptide capable of inducing calcium phosphate nucleation is the peptide with sequence DSSEEKLFRRIGRFG.

8. The composite material according to claim 1, wherein the peptide capable of inducing calcium phosphate nucleation is the peptide with the sequence DDDEEKFLRRIGRFG.

9. The composite material according to claim 1, wherein the calcium phosphate corresponds to hydroxyapatite.

10. The composite material according to claim 9, comprising hydroxyapatite in an amount between 1% and 3% (w / v).

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

12. A composite material according to any one of claims 1 to 11, for use in promoting nerve innervation, bone formation and angiogenesis.

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

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

Images