Porous 3D matrix based on polyelectrolyte complexes and uses thereof

Abstract

The present invention relates to a porous matrix of polyelectrolyte complexes (PECs) based on alginate and chitosan or derivatives thereof, particularly suitable for cell therapy and notably for soft tissue healing and tissue repair. The matrix according to the invention can be advantageously used in combination with macrophages to promote wound healing. The invention also relates to a hybrid biomaterial comprising such a porous matrix and pro-healing macrophages, and optionally mesenchymal stromal cells, and to a kit comprising this matrix. The present invention also relates to a method for preparing such a matrix and also to a method for preparing such a hybrid biomaterial and to the use of such a hybrid biomaterial in regenerative medicine.

Description

[0001] Porous 3D matrix based on polyelectrolyte complexes and its applications

[0002] TECHNICAL FIELD

[0003] The present invention relates to a porous 3D matrix obtained from polyelectrolyte complexes (PEC) formed between anionic and cationic polymers, particularly suitable for cell therapy and in particular for the healing of soft tissues and tissue repair. The matrix according to the invention can be advantageously used in combination with macrophages to promote wound healing. The invention also relates to a biomaterial comprising such a porous matrix and cells of interest, such as macrophages and / or mesenchymal stromal cells, and a kit comprising this matrix. The present invention also relates to a method for preparing such a matrix as well as a method for preparing such a biomaterial and the use of such a hybrid biomaterial in regenerative medicine.

[0004] TECHNOLOGICAL BACKGROUND

[0005] Tissue engineering, or regenerative medicine, generally uses therapeutic cells, substances necessary for tissue development (signal molecules, growth factors, etc.) and / or implantable biomaterials.

[0006] Generally speaking, an implantable biomaterial intended for deep cell seeding must have biocompatibility characteristics so that, once implanted, it does not generate an excessive inflammatory reaction. On the contrary, the material must ideally allow cell adhesion, normal function, migration, and the production of a new extracellular matrix. The biomaterial must also promote the viability of therapeutic cells. In addition, the biomaterial must have a highly macroporous and interconnected structure to allow cell colonization and nutrient diffusion. Finally, the material must have mechanical properties related to those of the target tissue, so as to reproduce a biomimetic environment for the cells it hosts.

[0007] In the case of wound treatment, particularly chronic wounds, therapeutic cells can be used. It may be particularly interesting to administer them via an implantable biomaterial, in order to provide a protective 3D environment ensuring the local delivery of viable cells to the wound or more generally to the injured tissue or organ. Chronic wounds, and particularly diabetic foot wounds, are "stuck" in a low-intensity inflammatory phase, preventing them from accessing the subsequent stages of healing and suggesting defective resolution of inflammation (Miao et al., 2012). The pathophysiological microenvironment of chronic wounds is itself associated with functional deregulation of macrophages. The deregulation and loss of functionality of macrophages contribute significantly to the failure of healing in chronic wounds, including diabetic foot wounds.

[0008] Thus, macrophages occupy a central place in the orchestration of the healing process. Relevantly and adapted to the context of healing where macrophages play a central role, the following distinction has recently been proposed (Krzyszczyk et al., 2018): pro-inflammatory macrophages, engaged in the elimination of pathogens by phagocytosis and which secrete pro-inflammatory cytokines, toxic intermediates and reactive oxygen species, on the one hand, and pro-healing macrophages and pro-resolving macrophages, involved in tissue repair and remodeling processes, on the other hand.

[0009] One of the most supported hypotheses to explain the maintenance of inflammation in chronic wounds is the absence of a phenotype change (switch) of proinflammatory macrophages towards a pro-resolving phenotype.

[0010] Macrophages therefore represent an increasingly attractive therapeutic target in regenerative medicine. Indeed, given the dysfunction of macrophages in chronic wounds and the deleterious impact of the absence of transition from a pro-inflammatory to a pro-resolving phenotype, it appears essential to restore the pro-resolving and pro-repair functionality of macrophages.

[0011] Work has focused on developing strategies to control inflammation in chronic wounds by more or less direct intervention on the phenotype and activity of macrophages. In particular, several complementary approaches have been considered, such as modulating the phenotype of endogenous macrophages (by promoting a pro-resolving phenotype or by attenuating the pro-inflammatory phenotype), or directly by local delivery of pro-resolving macrophages to the wound.

[0012] However, to date, none of the therapeutic healing strategies developed involving macrophages have allowed a return to satisfactory anatomical and functional integrity of wounds.

[0013] SUMMARY OF THE INVENTION

[0014] By working on wound healing, particularly chronic wounds in diabetics, the inventors have developed a PEC matrix based on anionic and cationic polymers that is particularly suitable for soft tissue healing and tissue repair. The matrix according to the invention has mechanical and structural properties such that it can simultaneously serve as a 3D support for cells, particularly macrophages, and as a dressing. The matrix according to the invention is particularly suitable for use in association with pro-resolving macrophages. In particular, the inventors have developed an alginate / chitosan PEC matrix that can be used as a cell therapy support suitable for soft tissues.

[0015] The subject of the invention is therefore a three-dimensional biomaterial comprising a matrix of polyelectrolyte complexes (PEC) based on alginate and chitosan, modified or not, and macrophages, said matrix having an interconnected open macroporosity in which the alginate / chitosan mass ratio is between 20 / 80 and 80 / 20.

[0016] In one embodiment, the PEC matrix is ​​based on alginate and chitosan, the alginate / chitosan mass ratio being 40 / 60.

[0017] The alginate may in particular have an M / G ratio of between 1.4 and 2.7, preferably equal to 2 +7-0.2, a molecular weight (Mw) of between 150,000 and 250,000, and a polydispersity index (PI) of less than 2, preferably approximately 1.5.

[0018] Chitosan advantageously has a degree of deacetylation (DD A) of between 75% and 90%, preferably between 75 and 85%, an Mw of between 130,000 and 400,000, preferably between 145,000 and 400,000, more preferably between 200,000 and 400,000, and a polydispersity index of less than 2, preferably of approximately 1.7, more preferably of approximately 1.8.

[0019] According to the invention, the biomaterial may be a hybrid biomaterial comprising pro-healing macrophages. The pro-healing macrophages are, for example, pro-resolving macrophages.

[0020] In one embodiment, the hybrid biomaterial comprises pro-healing macrophages and mesenchymal stromal cells (MSCs), the ratio of pro-healing macrophages to mesenchymal stromal cells preferably being between 1 / 99 and 99 / 1. For example, the ratio of pro-healing macrophages to mesenchymal stromal cells is 50 / 50.

[0021] The invention also relates to a method for preparing a hybrid biomaterial according to the invention, comprising the steps:

[0022] (a) culturing monocytes and / or macrophages under conditions allowing pro-healing macrophages to be obtained; (b) preparing a matrix of polyelectrolyte complexes (PEC) based on alginate and chitosan, in which the two polymers are in a relative proportion of between 40 / 60 and 60 / 40;

[0023] (c) optionally drying the PEC matrix;

[0024] (d) sterilize the PEC matrix;

[0025] (e) seeding the PEC matrix with pro-healing macrophages obtained in step (a) optionally combined with MSCs.

[0026] In one embodiment, sterilization step (d) is sterilization by low energy pulsed electron beam irradiation or sterilization by low energy continuous electron beam irradiation.

[0027] As indicated above, in one embodiment, the matrix resulting from step (b) undergoes a drying step before the sterilization step (d).

[0028] The invention also relates to a skin dressing intended for regenerative medicine comprising a three-dimensional biomaterial according to the invention.

[0029] The invention also relates to pro-healing macrophages optionally combined with MSCs for their use in regenerative medicine in a subject, characterized in that said pro-healing macrophages and the possible MSCs are in a form suitable for their administration to said subject by means of a three-dimensional biomaterial according to the invention.

[0030] Such pro-healing macrophages, possibly combined with MSCs, can be used in particular for the treatment of a wound, particularly a chronic wound.

[0031] For example, the subject has diabetes and / or is elderly.

[0032] In one embodiment, the three-dimensional biomaterial is in the form of a dressing, a patch, or an implantable matrix.

[0033] Said pro-healing macrophages and any CSM may have been previously obtained from cells of said subject among, bone marrow cells, iPS or blood monocytes, and adipose tissue (ASC: “Adipose tissue-derived mesenchymal Stroma Cell”).

[0034] The invention also relates to a kit intended for regenerative medicine, and in particular for the treatment of a wound, such as a chronic wound, said kit comprising: a PEC matrix of alginate and chitosan, said matrix having an interconnected open macroporosity in which the anionic polymer / cationic polymer mass ratio is between 20 / 80 and 80 / 20, preferably between 40 / 60 and 60 / 40; a culture medium suitable for the culture of pro-healing monocytes or macrophages and / or a culture medium suitable for the differentiation of bone marrow cells into macrophages with a pro-healing phenotype.

[0035] Advantageously, the PEC matrix is ​​sterilized.

[0036] DESCRIPTION OF DRAWINGS

[0037] [Fig. 1] shows the chemical structure of sodium alginate and its monomeric units M and G (A), and an example of an alginate chain sequence (B).

[0038] [Fig. 2] shows the chemical structure of chitin or chitosan. When R = -COCH3 and x > 50% it is chitin, and when R = H and y > 60-70% it is chitosan.

[0039] [Fig. 3A] represents the distribution of the different batches studied in an in vivo murine study model of the cell therapy strategy for chronic wounds.

[0040] [Fig. 3B] represents the timeline of the test in Figure 3A and the experimental conditions.

[0041] [Fig. 4] shows the gene expression profile of macrophage (mO) MO, Ml and M2 after 24 hours of culture or in contact with PEC 40 / 60 matrices. (A) Anti- and proinflammatory cytokines. (B) Membrane receptors. Analyses were performed with 3 replicates per condition (1-way ANOVA test; *P < 0.05; **P < 0.01; *** P < 0.001; **** P < 0.0001). For clarity on the graphs, only significant differences between m in contact with the matrices in relation to their reference group (m in culture) are indicated.

[0042] [Fig. 5 AB] shows the results of the flow cytometry study of the expression of the surface markers F4 / 80, CD206 (MR) and CD86 by the m <l» M2 après 24 heures de culture (A) ou au contact des matrices PEC 40 / 60 (B).

[0043] [Fig. 5C] is the histogram showing the corresponding percentages of the different F480+CD206+ and F480+CD86+ populations of Figures 5AB (n=3 replicates).

[0044] [Fig 6] is a diagram showing the urea concentrations per 100,000 mO MO, mO Ml or m M2 after 24 hours of culture or in contact with PEC 40 / 60 matrices (m <l>: macrophage). Analyses were performed with 3 replicates per condition (n = 3; 1-way ANOVA test; *P < 0.05; **P < 0.01; **** P < 0.0001)

[0045] [Fig. 7] is a diagram showing the luminescence monitoring of reactive oxygen species production by macrophages (m<5) MO, Ml and M2 after 24 hours of culture or in contact with 3D matrices after stimulation with 100 mM TPA.

[0046] [Fig 8] shows a PEC 40 / 60 matrix in confocal microscopy after 24 hours of culture and after differential labeling of live / dead cells. The upper left inset indicates the imaged faces of the matrix. The 3D matrix as a whole is presented in (A), it is a 3D reconstruction at 10X magnification with phase contrast and z-projection of the channels. The BCD images correspond to the z-projection of a cross-section of the matrix at 10X. The EFG then HIJ images are z-projections of the matrix imaged longitudinally at 10X and 40X respectively.

[0047] [Fig 9] shows the wound closure kinetics from the first day of treatment (D2). Statistical analysis between the 3D matrix + m group M2 and HFD control group is indicated by * (p<0.05).

[0048] [Fig 10AB] shows representative images of histological sections from the control group (A) and the group treated with 3D matrices + mO M2 (B).

[0049] [Fig. 10C] shows the histological scores associated with the images in Figures 10A and 10B.

[0050] [Fig 11] shows the viability of different cell types (human M2-Mcp and human ASC) in the matrix after 2 days and 6 days of culture after labeling with the “ReadyProbes™ Cell Viability Imaging Kit” and fluorescence analysis by confocal microscopy.

[0051] [Fig 12] shows the functionality of human M(p) and ASC seeded in the matrix in the basal state or after infectious stimuli (LPS). After 24h of culture, the concentrations of IL-10, IL-6 and IL-ip are determined by ELISA.

[0052] [Fig 13] shows the effects of a matrix according to the invention seeded with different murine therapeutic cells on the repair of chronic wounds. A 1 cm wound 2 is performed on the back of diabetic male C57BL / 6 mice over 10 months old. Matrices alone (A) or matrices containing pro-resolving macrophages (M2-Mcp) + associated with adipose tissue mesenchymal stromal cells (ASCs) (B) are applied to the wound. The matrices are placed on the wound for 24 hours and then replaced with a new matrix for 3 days. A picture of the wound is taken every day and the animals are sacrificed when a group reaches 90% wound closure. At different times, the wound surface is measured and the percentage of closure is determined relative to the wound surface at D0. (C) Area under the curve after the different treatments.

[0053] [Fig 14] shows a comparison of the number of days required to achieve 25, 50, 75, 95, and 100% wound closure between untreated mice and mice treated with therapeutic cells contained in the matrix. The number indicated on the histograms represents the difference in days required to achieve X% wound closure between untreated mice (black histogram) and mice treated with matrix alone (A), matrix containing M2-Mcp (B), or matrix containing M2-Mcp combined with ASCs (C).

[0054] [Fig 15] shows a histological analysis of the wounds after treatment with the matrix associated with different therapeutic cells. 10 days post-injury, the animals are sacrificed, the dorsal skin is removed and a histological analysis (hemalun Eosin staining) is carried out. In order to evaluate wound closure, the distance between the paniculus camosus present on each side of the wound is measured on the histological sections of mice treated with the matrix alone (A) or with the matrix containing M2-M <p et des ASCs (B). (C) Analyse macroscopiques des coupes histologiques (marquage hemalun Eosin) des plaies après traitement (10 jours postlésion).

[0055] [Fig 16] shows the effects of matrices containing different human pro-resolving cells on the proliferation of human skin fibroblasts. M2-Mcp (A) or M2-Mcp associated with ASCs (M2Mq > + ASCs) (B) were cultured in culture plates (2D) (black dotted line) or on the matrix (3D) (black solid line) for 24 h in a-MEM + 0.1% fetal calf serum (FCS). After 24 h of culture, the supernatants were collected and then added to human primary fibroblasts cultured at low density. The proliferation of fibroblasts cultured in these culture supernatants is evaluated for 7 days using a real-time imaging system (Incucyte S3, Sartorius). A picture of each well is taken every 6 hours and the proliferation rate is analyzed by comparing the percentage of the area occupied by the cells on each image at different times. Negative control: 0.1% FCS (light gray line-round) positive control: 10% FCS (dark gray line-square).

[0056] [Fig 17] shows that the matrix promotes the anti-inflammatory profile of macrophages. Human macrophages are differentiated into anti-inflammatory macrophages (M2-Mcp) (IL-4 for 24h). Then the M2-Mcp are cultured in plates (2D) or on the matrix (3D). After 24h, the culture supernatants are collected and the IL-10 concentration is evaluated by ELISA. DETAILED DESCRIPTION OF THE INVENTION

[0057] The present invention relates to a matrix of polyelectrolyte complexes particularly suitable for use in a wound dressing in the context of tissue regeneration. In particular, the matrix can be used as a biomaterial in combination with therapeutic cells, such as macrophages and / or mesenchymal stromal cells (MSCs), to aid in wound healing.

[0058] Definitions

[0059] For the purposes of this application, the following definitions apply.

[0060] By "matrix" is meant a three-dimensional polymeric structure, capable of being worked to take the desired shape, depending on the purpose of its use. In the context of the invention, a matrix is ​​advantageously biocompatible and / or biodegradable.

[0061] “Biocompatible” means a material, matrix or substance that does not interfere with or degrade the biological environment in which it is used.

[0062] Biodegradable means a material or matrix capable of being naturally broken down by living organisms. In the case of an implantable material or matrix, this means a material or matrix capable of being naturally broken down by the organism in which it is to be implanted.

[0063] By "biomaterial" is meant a non-living material, intended to interact with a biological system, with an appropriate host response in a specific application. In the context of the invention, a "hybrid biomaterial" means the combination of a matrix and therapeutic cells, which can be used in particular as a dressing or temporary skin substitute, capable of delivering therapeutic substances (bioactive molecules secreted by the cells, active ingredients) and / or stimulating a cellular target.

[0064] "Chronic wounds" refer to wounds that are difficult to heal or do not follow the classic physiological process. Generally, a wound is considered chronic if it has not managed to restore anatomical and functional integrity after three months. Chronic wounds generally include vascular ulcers (venous and arterial), pressure ulcers, and diabetic feet.

[0065] The term “approximately” in conjunction with a numerical value means the indicated value + / - 10%, preferably + / - 5%. Unless otherwise indicated, where ranges are indicated, the values ​​at the limits are included within said ranges.

[0066] Matrix

[0067] The present invention is mainly based on a biocompatible and advantageously biodegradable polymer matrix. More precisely, the matrix is ​​a matrix of polyelectrolyte complexes (PEC) based on anionic polymer(s) and cationic polymer(s). PECs have many advantages such as maintaining the very good biocompatibility of the polymers (here biosourced) from which they are derived. This is why PECs are already conventionally used in tissue engineering with promising results in this field (Ishihara 2019).

[0068] The matrix is ​​a three-dimensional (3D) matrix. More specifically, the matrix according to the invention is a PEC matrix having interconnected open macroporosity.

[0069] By "interconnected open macroporosity" is meant that the matrix more particularly comprises pores with an average diameter of between 50 and 300 pm, that said pores communicate with each other and that it is possible to access said pores from the external surface of the matrix.

[0070] According to the invention, the anionic polymer / cationic polymer mass ratio in the matrix is ​​between 20 / 80 and 80 / 20. In particular, said ratio is approximately 20 / 80, 30 / 70, 40 / 60, 50 / 50, 60 / 40, 70 / 30, 80 / 20. Preferably, said ratio is between 35 / 65 and 65 / 35, more preferably between 35 / 65 and 45 / 55, and in particular approximately 40 / 60.

[0071] Biocompatible anionic polymers are advantageously chosen from alginates and modified alginates. Alginates form a family of linear copolymers consisting of a chain of PD-mannuronic acid units (M unit) and aL-guluronic acid (G unit), linked together by P (1-4) glycodisic bonds. It is possible to vary the rigidity of alginate gels by varying the M / G ratio. For example, it is possible to use sodium alginate. Modified alginates are understood to be alginates whose basic structure is functionalized by one or more groups, in particular by one or more peptides. For example, it is possible to use alginate functionalized by RGD tripeptides (L-arginine, glycine, L-aspartic acid) participating in cell adhesion.

[0072] Biocompatible cationic polymers are advantageously chosen from chitosan, and any chitosan derivative possibly functionalized but retaining an overall positive charge. Chitosan is a polysaccharide consisting of a chain of N-acetyl-D-glucosamine groups linked together by P(l-4) bonds. The properties of chitosan vary in particular according to its molecular mass and its degree of deacetylation (DD A).

[0073] The matrix according to the invention may in particular be a PEC matrix based on alginate and chitosan. The alginate / chitosan mass ratio is between 20 / 80 and 80 / 20. Preferably, said ratio is between 30 / 70 and 70 / 30, more preferably between 35 / 65 and 45 / 55, and in particular approximately 40 / 60.

[0074] The alginate used may in particular have an M / G ratio of between 1.4 and 2.7, in particular between 1.5 and 2.5, for example, approximately equal to 1.5, to 1.6, to 1.7, to 1.8, to 1.9, to 2, to 2.1, to 2.2, to 2.3, to 2.4, to 2.5.

[0075] Advantageously, the alginate has a molecular weight (Mw) of between 150,000 and 250,000, + / - 10%, for example, approximately 150,000, 155,00, 160,000, 165,000, 170,000, 175,000, 180,000, 185,000, 190,000, 195,000, 200,000, 210,000, 220,000, 230,000, 240,000, 250,000.

[0076] The alginate may also have a polydispersity index (PI), preferably less than 2, in particular between 1.9 and 1, preferably approximately 1.9, approximately 1.8, approximately 1.7, approximately 1.6, approximately 1.5, approximately 1.4, approximately 1.3, approximately 1.2, approximately 1.1, approximately 1.

[0077] The chitosan used can have a degree of deacetylation (DD A) between 50% and 90%, in particular between 75% and 90%, preferably between 75 and 85%.

[0078] The molecular weight (Mw) of chitosan is advantageously between 100,000 and 500,000 + / - 10%, preferably between 130,000 and 400,000, between 140,000 and 350,000, between 150,000 and 300,000, between 175,000 and 270,000, between 200,000 and 250,000, in particular approximately 135,000, approximately 140,000, approximately 145,000, approximately 150,000, approximately 155,000, approximately 160,000, approximately 165,000, approximately 170,000, approximately 175,000, approximately 180,000, approximately 185,000, approximately

[0079] 190,000, about 195,000, about 200,000, about 210,000, about 220,000, about

[0080] 230,000, about 240,000, about 250,000, about 260,000, about 270,000, about

[0081] 2800,000, about 290,000, about 300,000, about 350,000, about 400,000.

[0082] The polydispersity index (PI) of the chitosan is preferably less than 2, in particular between 1.9 and 1, preferably approximately 1.9, approximately 1.8, approximately 1.7, approximately 1.6, approximately 1.5, approximately 1.4, approximately 1.3, approximately 1.2, approximately 1.1, approximately 1. Preferably, the PEC matrix comprises alginate and chitosan according to the above characteristics, in an alginate / chitosan mass ratio of between 35 / 65 and 45 / 55, preferably approximately 40 / 60.

[0083] In the context of the invention, the alginates advantageously have an M / G ratio of between 1.4 and 2.7, preferably equal to 2 +7-0.2, a molecular weight (Mw) of between 150,000 and 250,000, in particular approximately 195,000, and a polydispersity index (PI) of less than 2, preferably approximately 1.5.

[0084] Likewise, in the context of the invention, chitosan advantageously has a degree of deacetylation (DDA) of between 75% and 90%, preferably between 75 and 85%, an Mw of between 130,000 and 400,000, preferably between 140,000 and 250,000 or between 200,000 and 400,000, and a polydispersity index of less than 2, preferably of approximately 1.8 or approximately 1.7.

[0085] Thus, in one example, the alginate used may have a M / G ratio of 2.1 + / - 0.1, a molecular weight of about 193,100 and a polydispersity index of about 1.5. Similarly, the chitosan used may have a DDA of 80% + / - 5%, a molecular weight of about 216,300 and a PI of about 1.8.

[0086] In another example, the alginate used may have a M / G ratio of 1.4 + / - 0.1, a molecular weight of about 162,000 and a polydispersity index of about 1.7. Similarly, the chitosan used may have a DDA of 88% + / - 5%, a molecular weight of about 146,700 and a PI of about 1.7.

[0087] In a particular embodiment, the PEC matrix comprises an alginate / chitosan mass ratio of between 35 / 65 and 45 / 55, preferably of approximately 40 / 60. The alginate of the matrix has a M / G ratio of 2.1 -+7-0.1, a molecular weight of approximately 193,100 and a PI of approximately 1.5, and the chitosan has a DDA of 80% + / -5%, a molecular weight of approximately 216,300 and a PI of approximately 1.8.

[0088] The matrix according to the invention is suitable for deep seeding and immobilization of therapeutic cells and has absorbent properties, particularly suitable for use as a dressing for the absorption of exudates.

[0089] The matrix according to the invention can be prepared by any suitable method known to those skilled in the art. In particular, it is possible to implement the preparation method described in application WO2017 / 017379. After obtaining, the matrix can be sterilized and optionally dried or lyophilized, this drying or lyophilization being preferably carried out before sterilization for its subsequent use as a biomaterial. Advantageously, the polyelectrolyte complex (PEC) matrix is ​​produced by mixing the anionic polysaccharide, such as alginate, and the cationic polymer, such as chitosan, in proportions of between 40 / 60 and 60 / 40. In particular, the cationic polymers and the anionic polymer are in mass proportions of between 40 / 60 and 60 / 40. In particular, the cationic polymers and the anionic polymer are in proportions allowing a maximum level of ionic interaction (+ / -10%).

[0090] For example, the step of preparing a PEC matrix based on chitosan and alginate includes

[0091] - mix an alginate solution and a chitosan solution;

[0092] - mold and freeze the resulting PEC mixture;

[0093] - freeze-dry the PEC mixture;

[0094] - gel the lyophilized PEC matrix by adding a 0.1M CaCl2 solution;

[0095] - rinse, optionally refreeze then optionally re-dry said matrix.

[0096] The matrix according to the invention can be dried, for example by supercritical CO2 drying, and / or freeze-dried, in order to promote its preservation and storage before use.

[0097] The matrix according to the invention can advantageously be sterilized, preferably dry. Sterilization can be carried out by any means, physical or chemical, known to those skilled in the art and suitable for PEC matrices. In particular, it is possible to sterilize the matrix by irradiation with low-energy pulsed electron beams or by irradiation with low-energy continuous electron beams. Low-energy irradiation means an energy of less than 500 keV. The sterilization method described in Farno et al 2021 (“Low-energy electron beam sterilization of solid alginate and chitosan, and their polyelectrolyte complexes”, Carbohydrate Polymers 261 (2021) 117578) is particularly suitable.

[0098] In one embodiment, the sterilization is carried out by irradiation at a dose of less than 5 kGy, in particular between 2 and 3 kGy, preferably 2.5 kGy, by pulsed technology with a beam energy of between 200 and 350 keV, preferably between 250 and 300 keV, in particular at 280 keV.

[0099] In another embodiment, the sterilization is carried out by irradiation at a dose of between 10 and 25 kGy, preferably between 12 and 20 kGy, more preferably 15 kGy, by pulsed technology with a beam energy of between 350 and 550 keV, preferably between 400 and 450 keV. For example, the sterilization is carried out by irradiation at a dose of 15 kGy, with a beam energy of 430 keV.

[0100] Such sterilizations advantageously make it possible to preserve the porosity and architecture of the matrix according to the invention.

[0101] Hybrid biomaterial

[0102] The invention also relates to a biomaterial combining a matrix as described above, and therapeutic cells and / or therapeutic substances. The matrix according to the invention has mechanical and biological properties particularly suited to cell therapy. In particular, the matrix according to the invention has properties favorable to the seeding and survival of particular cells, such as macrophages and mesenchymal stromal cells. Indeed, the porosity generated allows deep seeding of the matrix, and the architecture recreates a biomimetic environment capable of maintaining cell viability, a particular phenotype and / or metabolic activity.

[0103] The matrix according to the invention is thus particularly suitable for the seeding and survival of macrophages and / or mesenchymal stromal cells (MSCs). It is thus possible to use the matrix according to the invention with macrophages, or with a combination of macrophages and MSCs.

[0104] Chronic wound infection is known to cause significant delays in healing. Indeed, in 90% of chronic wounds, a biofilm develops, which represents a major obstacle to healing. Thus, the transition to the inflammation resolution phase can only occur if this biofilm is eliminated. Pro-inflammatory macrophages, engaged in the elimination of pathogens by phagocytosis and by secreting proinflammatory cytokines, toxic intermediates and reactive oxygen species, are therefore essential in the control of wound infection.

[0105] Furthermore, in the wound healing process, pro-resolving macrophages are essential to stop inflammation and direct healing towards the repair phase. In the final phases of healing, these macrophages aim to return homeostasis and mature the skin towards its initial state. The resolution of inflammation is partly activated by the phagocytosis of apoptotic PNNs by macrophages, which consequently secrete TGF-0 and VEGF. Pro-resolving macrophages also produce IL-10 in high quantities, which is known to be immunosuppressive. In addition, they actively contribute to the regulation of the extracellular matrix (ECM) and its remodeling by secreting various proteases such as MMPs and their inhibitors TIMPs. Similarly, these macrophages intervene in the apoptosis of myofibroblasts to minimize the risk of excessive fibrosis.Proresolving macrophages are characterized by the presence of Pattern Recognition Receptors (PRRs) capable of recognizing pathogen-associated molecular patterns (PAMPs) and / or danger-associated molecular patterns (DAMPs).

[0106] Thus, the matrix according to the invention advantageously comprises pro-healing macrophages. According to the invention, pro-healing macrophages designate pro-inflammatory macrophages, pro-resolving macrophages, or a mixture thereof.

[0107] The addition of pro-inflammatory / anti-bacterial macrophages and / or pro-resolving macrophages to the matrix is ​​advantageously conditioned by the infectious and inflammatory status of the wound to be treated. A person skilled in the art is able to decide on the type of pro-healing macrophages to be used with the matrix, depending on the wound to be treated.

[0108] Particularly surprisingly, the inventors have demonstrated that the matrix according to the invention makes it possible to maintain macrophages of the matrix in a pro-resolving state.

[0109] Advantageously, the matrix according to the invention comprising pro-resolving macrophages can make it possible to force endogenous macrophages, in contact with said matrix, towards a pro-resolving phenotype. Thus, in the case of use of the matrix as a dressing applied to a wound, said matrix can promote healing by driving the macrophages present at the wound towards a pro-resolving phenotype.

[0110] The matrix according to the invention can also be used with other types of therapeutic cells, such as MSCs and / or with therapeutic molecules, and can optionally be functionalized with antibiotics, natural substances (propolis, proanthocyanidin A). MSCs are particularly interesting in the context of wound treatment, in combination with macrophages, because they make it possible to induce a pro-resolving phenotype of macrophages. The polarization of macrophages towards a pro-resolving phenotype by MSCs can concern both exogenous macrophages, provided by the biomaterial, and endogenous macrophages, present at the level of the wound on which the hybrid biomaterial is applied.Furthermore, MSCs, through the secretion of paracrine factors, can modulate and recruit endogenous wound cells such as fibroblasts and keratinocytes and thus accelerate wound closure (Shingyochi, Y, 2015, Exp Opinion mol Ther; Krzyszczyk, P, 2018, Front Physiol). MSCs also play a central role in wound neovascularization by secreting anti-inflammatory factors but also by stimulating the production of VEGF by cells in the microenvironment (Shingyochi, Y, 2015, Exp Opinion mol Ther). MSCs therefore have a strong potential to accelerate the wound closure process through their anti-inflammatory properties, by stimulating angiogenesis as well as by producing soluble factors that improve the wound microenvironment and thus promote tissue reconstruction. Thus, matrix CMS can play a role in the different phases of healing of healthy or infected wounds.

[0111] The CMS used can be derived for example from adipose tissue (ASC), bone marrow, umbilical cord, or derived from IPS

[0112] Interestingly, the inventors demonstrated that the association between pro-resolving macrophages and ASCs significantly increases the number of viable cells in the matrix.

[0113] Advantageously, the association between pro-resolving macrophages and ASCs potentiates the pro-resolving and therapeutic activity of cells seeded in the matrix.

[0114] In a particular embodiment, the biomaterial according to the invention comprises a matrix based on alginate and chitosan, advantageously with an alginate / chitosan mass ratio of approximately 40 / 60, and macrophages, preferably pro-healing macrophages, in particular pro-resolving macrophages.

[0115] In a particular embodiment, the hybrid biomaterial according to the invention comprises a matrix based on alginate and chitosan, advantageously with an alginate / chitosan mass ratio of approximately 40 / 60, pro-healing macrophages and mesenchymal stromal cells.

[0116] In a particular embodiment, the hybrid biomaterial according to the invention comprises a matrix based on alginate and chitosan, advantageously with an alginate / chitosan mass ratio of approximately 40 / 60, pro-healing macrophages, preferably pro-resolving macrophages, and mesenchymal stromal cells.

[0117] In one embodiment, the hybrid biomaterial according to the invention consists of a matrix as described above and pro-healing macrophages, preferably pro-resolving macrophages. Alternatively, the hybrid biomaterial according to the invention consists of a matrix as described above, pro-resolving macrophages and MSCs.

[0118] When the hybrid biomaterial according to the invention contains macrophages, the volume concentration of cells in the PEC matrix is ​​between 800 and 8000 cells / mm3 of PEC matrix, preferably between 500 cells and 4000 cells / mm3 of PEC matrix. The macrophages having a pro-healing phenotype, and in particular a pro-resolving phenotype, preferably represent at least 60% in number of the macrophages contained in the PEC matrix, more preferably at least 70%, 80%, 85%, 90%, 95%.

[0119] When the hybrid biomaterial according to the invention contains mesenchymal stromal cells, the volume concentration of MSCs in the PEC matrix is ​​preferably between 800 and 8000 MSCs / mm3 of PEC matrix.

[0120] When the hybrid biomaterial according to the invention contains macrophages and MSCs, the macrophage / mesenchymal cell ratio is advantageously between 1 / 99 and 99 / 1. For example, the macrophage / mesenchymal cell ratio may be 50 / 50.

[0121] The hybrid biomaterial according to the invention can be prepared by any method known to those skilled in the art.

[0122] Generally speaking, the method for preparing a hybrid biomaterial according to the invention may comprise the steps of preparing a matrix of polyelectrolyte complexes (PEC) according to the invention, optionally drying, sterilization of said PEC matrix and seeding of the PEC matrix with therapeutic cells, in particular pro-healing macrophages.

[0123] In particular, once the matrix as described above is manufactured, dried and sterilized, said matrix is ​​seeded with therapeutic cells, by bringing the matrix into contact with a culture medium comprising said therapeutic cells of interest. Of course, it is possible to directly seed the matrix according to the invention, without going through a drying step.

[0124] Thus, the method for preparing a hybrid biomaterial according to the invention may comprise the steps:

[0125] (a) culturing monocytes and / or macrophages under conditions enabling pro-healing macrophages to be obtained;

[0126] (b) preparing a matrix of alginate-chitosan polyelectrolyte complexes (PECs), in which the two polymers are in relative proportions between 40 / 60 and 60 / 40;

[0127] (c) optionally drying the PEC matrix;

[0128] (d) advantageously sterilizing the PEC matrix; Y1

[0129] (d) seeding the PEC matrix with pro-healing macrophages obtained in step (a) optionally combined with MSCs.

[0130] Depending on the intended use, and in particular depending on the stage of healing of the wound to be treated, step (a) may comprise the culture of the macrophages under conditions making it possible to obtain macrophages with a pro-resolving phenotype. For example, step (a) of culturing the macrophages comprises the culture of bone marrow cells previously taken from a human or non-human mammal, in a culture medium making it possible to obtain naive macrophages, the polarization towards a pro-resolving phenotype by culturing them in a culture medium supplemented with IL-4, IL-13 and dexamethasone for several days, in particular between 2 and 10 days, for example at least 4 days. The polarization of the macrophages towards a pro-inflammatory phenotype is obtained by culturing them in a culture medium supplemented with Interferon-γ and / or LPS for several days, in particular between 2 and 10 days, for example at least 4 days.

[0131] Alternatively, it is possible to culture iPS, blood monocytes, etc., in a culture medium adapted to differentiate said cells into macrophages and polarize them towards a pro-resolving phenotype (Douthwaite H. et al. 2022 Bio Protoc.). The person skilled in the art is able to adapt this cell preparation step, depending on the origin of the cells and the intended final use.

[0132] If the matrix is ​​to be seeded with pro-inflammatory macrophages, step (a) may comprise culturing macrophages under conditions that produce macrophages with a pro-inflammatory phenotype. For example, macrophage culture step (a) comprises a differentiation step in the presence of MSCF and a 24-hour culture step in the presence of inflammatory cytokines, such as gamma interferon, and / or bacterial cell walls in a defined medium.

[0133] In the case where the cells to be seeded are MSCs, step (a) may comprise the isolation of cells from the adipose tissue, bone marrow or umbilical cord of a human or non-human mammal and their culture in a suitable culture medium. The MSCs are obtained after a tissue digestion step. The cells obtained are cultured in alpha MEM medium supplemented with human platelet lysate or fetal calf serum. The cells will be used after at least one culture passage.

[0134] Of course, it is possible to proceed in several steps (a), so as to seed several cell types in the matrix.

[0135] The PEC matrix preparation step (b) and the sterilization step (d) can be carried out as described above. Of course, the cell culture step (a) and steps (b) and (d) can be carried out independently and in different time periods. For example, it is possible to prepare the matrix and sterilize it several hours, days, months, etc. before the cell culture step. Before seeding, the matrices can be stored in a lyophilized and sterilized state in a sterilization package.

[0136] Advantageously, seeding step (e) comprises bringing the lyophilized and sterilized PEC matrix into contact with a cell pellet (for example a cell pellet of macrophages and / or mesenchymal cells) and then centrifuging said matrix before adding complete medium.

[0137] The seeded matrices can be preserved in culture. For example, the seeded matrices are placed in multi-well cell culture dishes, to which a volume of medium, adapted according to the size of the wells and matrices, is added so as to immerse the seeded matrix. The dishes are kept in an incubator at approximately 37°C and 5% CO2. The storage time may depend on the cell type. The culture medium can be renewed at a frequency depending on the cell type. A person skilled in the art knows how to adapt the culture medium, the volumes and the renewal of the medium to the cell types and the intended end use of the biomaterial.

[0138] In the case of a matrix seeded with pro-resolving macrophages, preservation in culture can extend over a period of several days, including 10 days, 15 days, 20 days, 21 days or more, with a very high rate of macrophage viability.

[0139] It is also possible to freeze the matrix after seeding.

[0140] Kit

[0141] The invention also relates to a kit intended for regenerative medicine, and in particular for the treatment of a wound, such as a chronic wound, said kit comprising:

[0142] - a PEC matrix based on alginate and chitosan or their derivatives, as described above;

[0143] - a culture medium suitable for the cells intended to seed the matrix.

[0144] For example, the culture medium is suitable for the culture of pro-resolving macrophages and / or MSCs.

[0145] For example, macrophages are cultured in supplemented RPMI 1640, and MSCs are cultured in supplemented alpha-MEM. The kit may also include a culture medium suitable for the differentiation of cells, such as bone marrow cells, iPS cells, etc., into macrophages with a pro-resolving and / or pro-inflammatory phenotype.

[0146] Advantageously, the PEC matrix is ​​an alginate and chitosan based matrix, as described above.

[0147] The PEC matrix is ​​advantageously sterilized, and optionally packaged in a sterilization package, in which it can be stored until use for cell seeding.

[0148] The kit according to the invention may also comprise a matrix as described above, already seeded with pro-healing macrophages and possibly MSCs. Such a seeded matrix may be supplied frozen. The kit may then comprise a medium suitable for thawing the matrix and the survival of the therapeutic cells.

[0149] According to the invention, the kit may comprise a matrix as described above, shaped for use as a dressing, or provided with means enabling use as a dressing. Thus, the kit may contain a strip or any adhesive, to enable the matrix to be held in position over a wound. In another embodiment, the matrix may be provided in a format enabling it to be cut to fit the size / shape of the wound.

[0150] Uses

[0151] The matrix according to the invention can be used alone, or in the form of a biomaterial, in combination with pro-healing macrophages and / or therapeutic molecules.

[0152] The matrix according to the invention can in particular be used as a dressing to be applied to a wound. Indeed, as indicated above, such a matrix is ​​capable of promoting the polarization of endogenous macrophages, present at the level of a wound to be treated for example, towards a pro-resolving phenotype. Thus, the application of a dressing comprising such a matrix can help the healing of a wound.

[0153] Likewise, it is possible to use the matrix according to the invention in combination with one or more therapeutic molecules and / or biological substances. For example, it is possible to load the matrix with antibiotics or natural substances, intended to reduce the risks of infection of a wound on which said matrix is ​​intended to be applied. It is also possible to load the matrix with molecules capable of promoting a cellular phenotype, and in particular of promoting the pro-resolving phenotype for macrophages (for example, IL-13 / IL-4, dexamethasone, eicosanoids). Thus, when the matrix is ​​applied to a wound, said molecules will help healing by promoting the polarization of endogenous macrophages towards a pro-resolving phenotype.

[0154] The matrix according to the invention can advantageously be used in combination with macrophages and more particularly pro-resolving and / or pro-inflammatory macrophages, and / or MSCs, in regenerative medicine, and more particularly in the context of the treatment of chronic wounds.

[0155] Regenerative medicine refers to the repair, replacement, or regeneration of damaged cells or tissues. The main targets of the invention are tissue repair and the healing of skin wounds.

[0156] Thus, the invention relates to pro-healing macrophages for their use in regenerative medicine in a subject, said healing macrophages being in a form suitable for their administration to said subject by means of a hybrid biomaterial according to the invention. The invention relates in particular to pro-resolving macrophages, optionally in combination with MSCs, for their use in regenerative medicine in a subject, said pro-resolving macrophages being in a form suitable for their administration to said subject by means of a hybrid biomaterial according to the invention.

[0157] A subject is understood to be a mammal, human or non-human, in need of medical treatment, such as the treatment of a wound, in particular a chronic wound. Preferably, the subject is a human mammal, child, adolescent or adult. Preferably, the subject has a disease limiting the healing mechanisms and / or is an elderly subject. In particular, the subject is a diabetic, preferably a type II diabetic. An elderly subject is understood to be a human mammal aged 70 years or older.

[0158] The invention particularly relates to pro-resolving macrophages, optionally in combination with MSCs, in a hybrid biomaterial according to the invention, for their use in the treatment of a wound, in particular a chronic wound.

[0159] The invention also relates to pro-inflammatory macrophages, optionally in combination with MSCs, in a hybrid biomaterial according to the invention, for their use in the treatment of a wound, in particular a chronic wound.

[0160] Such use is particularly suitable for subjects with a disease limiting healing mechanisms, such as diabetes. Said pro-healing macrophages and / or MSCs can be obtained from cells of said subject such as blood monocytes, MSCs from adipose tissue, bone marrow or umbilical cord, macrophages, bone marrow cells and / or iPS.

[0161] Advantageously, the three-dimensional biomaterial is in the form of a dressing, a patch, or an implantable matrix.

[0162] The invention also relates to the use of a biomaterial according to the invention, in particular in the form of a dressing or implantable matrix, in regenerative medicine. In particular, the invention relates to the use of such a biomaterial according to which said biomaterial is brought into contact with injured tissue, such as a skin wound.

[0163] The invention also relates to a method for treating injured tissue according to which a biomaterial as described above is applied against the injured tissue to promote the repair of said tissue. In particular, the invention relates to a method for treating a wound, and particularly a chronic wound, according to which said biomaterial is applied against the wound to promote healing. In such a case, the hybrid biomaterial advantageously contains pro-healing macrophages and in particular pro-resolving macrophages, optionally in combination with MSCs. The biomaterial can be applied in the form of a dressing capable of being held on the wound.

[0164] The invention also relates to the use of macrophages for the preparation of a biomaterial according to the invention, for the treatment of injured tissue. In particular, the invention relates to the use of macrophages for the preparation of a biomaterial according to the invention, for the treatment of a wound, more particularly a chronic wound, such as a chronic diabetic wound.

[0165] EXAMPLES

[0166] MATERIALS AND METHODS

[0167] A) Matrix

[0168] The polymers used are a “medium viscosity” alginate marketed by Sigma (reference A-2033; lot 051M0054V) or by Algaia (Satialgine XPS 170NS) and a “medium molecular weight” chitosan also supplied by Sigma (reference 448877, lot STBF8484V or lot BCCJ1419).

[0169] For Sigma alginate, a M / G ratio of 2.1 was determined by the method described by Vilén et al. (Vilén et al., 2011). The mass-average (Mw) and number-average (Mn) molecular weights of alginate were determined by size exclusion chromatography and were 193,100 and 126,900 g / mol, respectively, with a polydispersity index of 1.5.

[0170] For chitosan, the degree of deacetylation (DD A) was calculated by applying the method of Heux et al. (Heux et al., 2000) and was estimated at 80% + / -5%. The Mw and Mn values ​​obtained by size exclusion chromatography are respectively 216,300 and 120,300 g / mol with a polydispersity index of 1.8, for the alginate of batch STBF8484V. For the second alginate, the Mw and Mn values ​​obtained by size exclusion chromatography are respectively 146,700 and 86,500 g / mol with a polydispersity index of 1.7.

[0171] PEC matrices are obtained by mixing an alginate solution with a fixed concentration of 3% (weight / volume) and a chitosan solution with 1.5% acetic acid and a variable chitosan concentration depending on the final polymer ratio desired in the matrices.

[0172] Briefly, the alginate and chitosan solutions are homogenized by mechanical stirring and then mixed in equivalent mass proportions. After the addition of the two polyelectrolytes, the mixture is also mechanically stirred. The final PEC mixture is then placed in a culture plate, frozen for at least 24 hours at -20°C, and the matrices are then lyophilized for 24 hours. The PEC matrices are gelled for 1 hour with a 0.1M calcium chloride solution. The matrices are then rinsed, frozen and lyophilized again.

[0173] This protocol allows the production of a family of 3D matrices with an alginate / chitosan ratio ranging from 80 / 20 to 20 / 80.

[0174] B) Cells

[0175] The murine macrophages used were derived from a primary culture. They were extracted from the bone marrow of C57BL6 / JRj mice (Janvier Labs). Murine ASCs were obtained by enzymatic and mechanical digestion of inguinal adipose tissue from C57BL6 / JRj mice (Janvier Labs).

[0176] After euthanasia of the mice by carbon dioxide overdose, the femurs and tibias are removed and cleaned to remove the surrounding muscles. After disinfection of the bones in 70% ethanol, the two epiphyses of each bone are removed and then the medullary canal is washed by injection of PBS in order to recover the bone marrow cells. Mechanical dissociation of the cell suspension is carried out with a pipette by successive pushing and pulling, then the suspension is filtered at 40 pm and centrifuged for 10 minutes at 1500 rpm. The cell pellet is then taken up in 1 mL of ACK buffer in order to lyse the red blood cells. The pellet is taken up in 9 mL of complete medium (DMEM glutamax, 10% FCS, 1% penicillin / streptomycin and 1% L-glutamine) and centrifuged a second time. The final pellet is taken up in 5 mL; 10 pL are taken for trypan blue labeling and counting on Malassez cells.On average, between 20 and 40 million bone marrow cells are recovered per mouse. The cells are taken up in their entirety in complete medium supplemented with M-CSF (30 ng / mL); they are seeded in petri dishes so as to have 300,000 cells / cm. 2 After 4 days of adhesion, MO macrophages (mO MO) are obtained (Martinez et al., 2006; Xia Zhang et al., 2008).

[0177] Four days after their isolation and differentiation, MO macrophages are not activated and can be polarized towards a pro-resolving M2 phenotype (m$ M2) with complete medium supplemented with M-CSF (30 ng / mL), IL-4 (10 ng / mL) and dexamethasone (10-7M) for 3 days, or towards a pro-inflammatory Ml phenotype (m Ml) with complete medium supplemented with M-CSF (30 ng / mL), IFN-y (2 ng / mL) and LPS (100 ng / mL) for 24 hours.

[0178] Human ASCs are isolated from human adipose tissue obtained after abdominal dermolipectomies. The adipose tissue is digested mechanically and then enzymatically. The cell pellet represents the stromal vascular fraction of the adipose tissue that contains the ASCs. The SVF is then cultured and the ASCs are obtained after 8 days of culture.

[0179] Human monocytes are isolated from peripheral blood mononuclear cells (PBMCs) obtained from white blood cell concentrates obtained from different healthy donors. PBMCs are isolated by Ficoll gradient centrifugation. The PBMC ring is collected and monocytes are isolated by adherence or after magnetic sorting. After one week of culture, the obtained macrophages are stimulated for 24 hours either with 100 ng / mL of M-CSF to obtain non-polarized macrophages (MO-Mcp) or 20 ng / mL of IL-4 to induce a pro-resolving phenotype (M2-Mcp).

[0180] C) Seeding

[0181] To recover the macrophages, the culture medium is removed and replaced with cold PB S containing 2 mM EDTA, a calcium chelator. After 5 minutes on ice, the macrophages are gently detached using a rake. The suspension is centrifuged for 5 minutes at 1500 rpm, before being labeled with trypan blue for counting on Malassez cells.

[0182] In the 3D matrix contact experiments, non-activated MO or polarized M1 (pro-inflammatory phenotype) or M2 (pro-resolving phenotype) macrophages are cultured for two hours. After macrophage adhesion, the matrices are placed in the well so that they are in direct contact with the cells. The cells or culture supernatant are collected 24 hours later for the various analyses. For the in vitro test, the matrixes are seeded in a lyophilized state by depositing a 15 pL cell pellet containing 400,000 macrophages. The matrix rapidly absorbs the cell pellet and is then centrifuged for 1 minute at 400 g to facilitate cell penetration over the entire depth of the 3D matrices. Finally, complete medium is gently added to the edge of the well and the matrices are incubated (37°C, 5% CO2).

[0183] D) Recovery of macrophages after seeding in 3D matrices

[0184] The first step is to cut the matrices into 4 in order to maximize the contact between the cells attached to the matrix and the detachment solution.

[0185] The samples are then incubated with a detachment solution, before filtration. The debris is successively filtered through a 100 µm and then a 40 µm sieve, followed by centrifugation of the cell suspensions.

[0186] E) Study of gene expression by RT-PCR

[0187] The Reverse Transcription-Polymerase Chain Reaction (RT-PCR) is carried out in three successive steps: extraction of messenger RNA from cells, reverse transcription of mRNA into cDNA and then the cDNA amplification reaction by PCR. First, the macrophages seeded in the 3D matrices were detached. The detached macrophage pellet as well as the macrophages cultured in wells (so-called "2D" controls) are frozen at -80°C in 100µl of lysis buffer (Promega).

[0188] mRNA extraction

[0189] The ReliaPrep™ RNA Cell Miniprep System (Promega) is used to extract mRNA following the protocol provided by the supplier. The mRNA is then assayed using the Nanodrop® (ND-1000).

[0190] Reverse transcription

[0191] Reverse transcription of mRNA into cDNA is performed using the Superscript Vilo cDNA synthesis kit (Invitrogen). For each experiment, a pool of pure cDNA is created by mixing 2 μL of each sample. The pool is then diluted to V5e, E10e, E20e, and E40e to create a standard curve. The remaining cDNAs are then diluted to ESe in RNAse / DNAse-free water.

[0192] PCR cDNA amplification is performed using the “SYBR Green I-based real-time PCR” kit (Roche) in a LightCycler® 480. The sequences of the primers used are detailed in the Table below. The relative expression of each gene in the samples is determined relative to the standard curve and relative to the housekeeping gene (Gapdh).

[0193] [Table 1]

[0194] F) Study of surface markers by flow cytometry

[0195] To study the expression of certain surface markers, macrophages cultured in wells (2D control) and those detached from 3D matrices are analyzed by flow cytometry. The cell suspensions are centrifuged, then the cell pellets are incubated in the dark for 20 minutes at 4°C in Fc block buffer (saturation of non-specific sites). Labeling is then carried out in the dark for 15 minutes at room temperature with the following antibodies diluted in FACS buffer: F4 / 80+ FITC Biolegend), CD206 APC (Biorad) and CD86 PE (Biolegend). Then the cell suspensions are rinsed and centrifuged, before being incubated with Live / dead violet (miltenyibiotec). Finally, the samples are taken up in 186 FACS buffer before acquisition on a BD Fortessa cytometer (BD Biosciences). Analyses are performed using BD FACS DVIA™ software (BD Biosciences).

[0196] Fc block buffer: FACS buffer supplemented with 3% mouse serum, 3% rat serum and the anti-CD16 / CD32 antibody pair at ' / O 6

[0197] FACS buffer: PBS supplemented with 5% FCS and 5 mM EDTA G) Quantification of reactive oxygen species (ROS) production

[0198] Reactive oxygen species (ROS) production by macrophages seeded or not in PEC 40 / 60 matrices was measured by chemiluminescence in the presence of 5-amino-2,3-dihydro-1,4-phthalazinedione (luminol) using a luminometer (Envision, PerkinElmer). Chemiluminescence generation was continuously monitored for 30 minutes after incubation of cells and / or biopolymeric matrices with luminol (66 pM) and then for 1 hour after stimulation with tetradecanoylphorbol acetate (TPA; 100 nM). Statistical analysis was performed using the area under the curve expressed as number x seconds.

[0199] H) Study of arginase activity

[0200] The measurement of arginase activity is determined from cell lysates by colorimetric assay of urea production.

[0201] I) In vivo experimental protocol

[0202] All experiments on mice were carried out in compliance with current regulations concerning the use of animals for scientific purposes.

[0203] The mice used in this in vivo assay are 8-week-old male C57B1 / 6NRJ mice (Janvier Labs). All animals are maintained on a daily cycle divided into 12 hours of darkness, followed by 12 hours of light at 22°C, with unlimited access to water and food. To induce type II diabetes, a high-fat diet (HFD) for 4 months (260 HF diet, Safe) is fed to the mice (Gâlvez et al., 2019). Mice are weighed at regular intervals and their fasting blood glucose levels are measured to check for hyperglycemia.

[0204] For the first protocol, the mice are randomly divided into 4 groups of six mice (Figure 3A). The protocol carried out extends over about ten days (Figure 3B) and begins with the creation of a wound on day D0. To do this, the mice are anesthetized using isoflurane. The dorsal part and the flanks are shaved and disinfected then a circular excision of approximately 1 cm2 is carried out. Immediately after, a non-invasive experimental device is implanted, developed by PharmaDev (FR2984719 / FR2984722). It allows, among other things, the maintenance of the 3D matrices at the level of the wounds.

[0205] After surgery, the mice are placed in a heated cage until they wake up and food is placed directly in their cage to facilitate feeding.

[0206] After 48 hours (day D2), the first topical treatments are applied: Control group: 30 pL of culture medium (DMEM glutamax);

[0207] “3D Matrix Only” group: 3D matrix sterilized by pulsed electron irradiation and hydrated in culture medium;

[0208] “M2 macrophages alone” group: 200,000 M2 polarized macrophages in vitro;

[0209] “3D Matrix with M2 Macrophages” group: 3D matrix sterilized by pulsed electron irradiation, seeded with polarized M2 macrophages in vitro and hydrated.

[0210] These treatments are left for 24 hours and are repeated twice, on days D3 and D4.

[0211] On the day of the mice sacrifice, skin explants are removed and then embedded in paraffin. After cutting with a microtome (HM 3 40 E Rotary Microtome, Thermo Scientific ™), the sections are stained with hematoxylin and eosin (HE stain). Finally, the slides are scanned for analysis (NanoZoomer Digital Pathology, Hamamatsu).

[0212] For the second in vivo protocol, the mice are randomly divided into 4 groups of five mice. The protocol carried out extends over about ten days and begins with the creation of a wound on day D0 (see in vivo protocol Figure 3). After 48 hours (day D2), the first topical treatments are applied:

[0213] Control group: 30 pL of culture medium (DMEM glutamax);

[0214] “3D Matrix Only” group: 3D matrix irradiated with UV and hydrated in culture medium;

[0215] “3D Matrix with M2 Macrophages” group: UV-irradiated 3D matrix, seeded with in vitro polarized M2 macrophages and hydrated.

[0216] “3D Matrix with M2 Macrophages and ASC” Group: UV-irradiated 3D matrix seeded with in vitro polarized M2 macrophages and ASC and hydrated.

[0217] These treatments are left for 24 hours and are repeated twice, on days D3 and D4.

[0218] Viability of human macrophages and human ASCs in matrix

[0219] Human pro-resolving macrophages (M2-M(p: 2xl0 5 cells) and human ASCs (2 x 10 5 cells) are seeded alone or in combination in the 3D matrix and are cultured. After 2 and 6 days of seeding, the cells present in the matrix are labeled with the kit "ReadyProbes™ Cell Viability Imaging Kit" (Invitrogen) according to the manufacturer's instructions. The NucBlue™ Live reagent labels the nuclei of all cells while the NucGreen™ Dead labels only the nuclei of dead cells. After 20 minutes of labeling, the matrices are washed 3 times with PBS and the fluorescence is detected by confocal microscopy (LSM880, Zeiss). The total cell number as well as the number of dead cells are evaluated by an automated analysis of the photos on Fidji (Macro developed in the Restore laboratory) (Analysis of 5 photos per condition).

[0220] Measurement of cytokine production by human macrophages and human ASCs seeded in matrix

[0221] Human ASCs (1x10 5 cells) and different populations of human Mcp (IxlO 5 cells) are seeded alone or combined in the 3D matrix. After 12 hours of seeding, the medium is changed and the matrices containing the cells are re-cultured and stimulated or not with LPS (10 ng / mL). After 24 hours of culture, the supernatants are collected and the concentrations of IL-6, IL-ip and IL-10 are determined by ELISA following the supplier's instructions (Invitrogen).

[0222] RESULTS

[0223] A) Analysis of retention, viability and distribution of macrophages in 3D matrices

[0224] To validate 3D matrices as pro-resolving M2 macrophage carriers for cell therapy, the retention, viability, and distribution of these macrophages within the 3D matrices were studied. The retention rate of M2 mOs after seeding was assessed by quantification of the total extracted proteins.

[0225] The results show that the retention rate is high (around 85%).

[0226] The study of M2 macrophage viability within the 3D PEC40 / 60 matrix was qualitatively monitored by confocal microscopy in live imaging. The hydrated 3D matrices were imaged longitudinally and transversely (Figure 8). Analysis of the reconstructions showed a viability of M2 macrophages of 89±3% after 24 hours of seeding in the 3D matrix.

[0227] This marking also provides information on the spatial distribution of the M2 mOs in the matrices. No regionalization is observed. On the contrary, the m M2 are distributed homogeneously both on the surface and in depth. Interestingly, after 21 days of culture, the M2 mO still show good viability in the 3D matrices.

[0228] In conclusion, 3D matrices combining alginate and PEC 40 / 60 chitosan constitute an optimal 3D support of M2 macrophages for cell therapy.

[0229] B) Effect of the matrix according to the invention on the phenotype of macrophages

[0230] The effect of 3D alginate-chitosan matrices (on the polarization of activated macrophages towards an MO, Ml and M2 phenotype was studied. To do this, alginate-chitosan matrices (40 / 60) were placed in contact with macrophages (mO MO, Ml or M2) for 24 hours and the level of expression of mRNAs coding for pro- and anti-inflammatory cytokines (Tnf-o., 11-10, Tgf-01) was studied (Figure 4). The gene and protein expression of membrane receptors characteristic of the Ml (Cd86) and M2 (Cd206, Cd36) phenotypes was also studied (Figure 4 and Figure 5).

[0231] This first phenotypic characterization was then completed by functional analyses such as the study of arginine metabolism or the production of reactive oxygen species (ROS).

[0232] First, these analyses validated the MO, Ml or M2 polarization of macrophages in culture after their isolation from the bone marrow and treatment with different inducers. In addition, they show that upon contact with 3D alginate-chitosan matrices, macrophages orient themselves towards an M2 phenotype. Indeed, 3D alginate-chitosan matrices seem on the one hand to potentiate the phenotype of M2 macrophages by increasing the expression of M2 markers (11-10, Tgf-pi, Cd206, Cd36) and by decreasing the expression of Ml markers (Tnf-a, Cd86); and on the other hand to orient non-activated MO macrophages towards an M2 phenotype. Equally interestingly, Ml macrophages in contact with matrices exhibit lower expression of pro-inflammatory markers characteristic of Ml macrophages (Tnf-a and Cd86).

[0233] All these results demonstrate that contact of M2 macrophages with 3D alginate-chitosan matrices does not modify their phenotype. Moreover, the matrices seem to orient MO and Ml macrophages towards an M2 phenotype and potentiate the M2 phenotype of M2 macrophages.

[0234] To explore the impact of alginate-chitosan matrices on macrophage effector functions, arginine metabolism (Figure 6) and oxidative stress (Figure 7) were assessed. Arginine metabolism is central to the different functions associated with the M1 and M2 phenotypes of macrophages (m). Indeed, there is competition between nitric oxide synthase (NOS) and arginase for the substrate L-arginine (Mills, 2012; Rath et al., 2014; Shearer et al., 1997). In particular, urea is frequently measured to assess the activity of arginase, a characteristic enzyme of M2 macrophages (Csonka et al., 2015). MO and M2 macrophages exhibit significantly higher urea production upon contact with 3D al inate-chitosan matrices, demonstrating greater arginase activity in these macrophages (Figure 6).

[0235] Regarding ROS production, as expected, the capacity of Ml macrophages to produce ROS is greater than that of MO and M2 macrophages after TPA stimulation. This capacity to produce ROS is strongly reduced in Ml and M2 macrophages placed in contact with the matrices (Figure 7). These results show that the 3D alginate-chitosan matrices are able to orient the functions of MO and Ml macrophages towards functions characteristic of M2 macrophages and to potentiate the specific functions of M2 macrophages.

[0236] To conclude, all of these initial results have made it possible to highlight the maintenance of the phenotype and functions of M2 macrophages by the 3D matrices according to the invention. In addition, the 3D matrices appear to orient MO macrophages towards an M2 phenotype and attenuate M1 polarization.

[0237] C) In vivo study of the association of pro-resolving macrophages / 3D matrices on a murine wound model in the context of type 2 diabetes according to the first protocol

[0238] Before wounding, fasting blood glucose and weight of mice on HFD diet were checked. These values ​​were compared to that of a "normal chow" control group consisting of C57B1 / 6 mice fed normally. The average mass of mice on high-fat diet (HFD) was higher than that of control mice (51.5 ± 0.5 g vs. 25.2 ± 0.9 g). As for their fasting blood glucose, it reached a significantly higher value than that of the control group (183.2 ± 6.3 mg / dl vs. 128.3 ± 4.5 mg / dl). Once this model was validated, skin wounds were made on the dorsal part by circular excision of approximately 1 cm2. Immediately after, a non-invasive experimental device (FR2984719 / FR2984722) was implanted. Among other things, it allows the maintenance of 3D matrices at the level of wounds.Treatments began 48 hours after the wounds were made, so as not to interfere with the inflammatory phase, the proper course of which allows the activation of the subsequent phases of healing (Jetten et al., 2014).

[0239] The treatments consist of topically applying to the wound either culture medium (control group), 200,000 M2 macrophages (mî> M2) or a hydrated 3D matrix alone or seeded with 200,000 M2 macrophages. Thanks to the non-invasive experimental device, wound closure was monitored daily by taking standardized photographs, from the creation of the wound on D0 until the sacrifice of the mice on D15.

[0240] Quantitatively, wound closure was determined by daily measurement of wound area for each mouse. Results are presented as percentage wound closure relative to wound area on the day treatment began (D2) (Figure 9).

[0241] It can thus be seen that the local addition of M2 macrophages at the wound level does not improve wound closure. Interestingly, the application of hydrated 3D matrices alone accelerates wound closure with a significant effect rather at the early stages (D4 to D7). This result correlates with the orientation of MO macrophages towards an M2 phenotype and the attenuation of the M1 profile observed when macrophages come into contact with the 3D matrices. From D4 to D15, the application of 3D matrices seeded with M2 macrophages significantly accelerates wound closure compared to the control group.

[0242] Thus, the beneficial pro-healing effect of the matrices alone is potentiated by the addition of M2 macrophages. These results show that the association of PEC 40 / 60 matrices with pro-resolving macrophages allows for faster wound closure. The association of the matrix according to the invention with pro-resolving macrophages shows a synergistic effect on wound closure.

[0243] The biomaterial according to the invention therefore makes it possible (i) to act as a barrier to infectious agents in the environment, (ii) upon contact with the wound to cause an orientation of the endogenous macrophages of the wound towards an anti-inflammatory phenotype but also (iii) to maintain the viability and pro-resolving functionality of the macrophages.

[0244] D) Assessment of scar quality

[0245] To investigate the quality of the newly formed skin, hematoxylin and eosin staining was performed on histological sections taken at day 15. Several scores have been developed to standardize the qualitative analysis of skin sections after healing (Gantwerker & Horn, 2011; Gupta & Kumar, 2015) (Figure 10).

[0246] The score was determined based on the following parameters: the presence of inflammatory cells; the quality of the epidermis (continuity and thickness compared to healthy skin); the quality of the dermis (continuity, presence of hair follicles); the presence of a crust (if yes, detachment and / or presence of inflammatory cells). Qualitative analysis of the slides was performed by two different operators, and scores ranging from 0 to 15 were assigned (the score is lower as the quality of the newly formed skin is high). The histological score is lower in mice treated with the 3D matrix seeded by pro-resolving macrophages.

[0247] Histological analysis of skin sections by hematoxylin and eosin staining reveals better quality of scar tissue in terms of continuity of the dermis and epidermis, as well as a lower presence of inflammatory cells in wounds treated with 3D matrices associated with pro-resolving macrophages.

[0248] E) Viability and functionality of the different associated cell types (human M2-Mcp and human ASC) in the matrix

[0249] The results show that, although macrophages and ASCs were seeded in identical quantities, from D2 the ASCs are present in greater numbers, thus revealing a slightly greater viability than the M2-M . Interestingly, the association between the two cell types significantly increases the number of both cell types as well as their viability in the matrix (Figure 11).

[0250] Interestingly, the results show that the association between ASCs and different types of macrophages induces a higher production of IL-10 and an explosive production of IL-6, two cytokines playing a central role in the resolution of inflammation and tissue repair. In the presence of LPS, the induction of IL-1beta production by matrix macrophages is strongly inhibited by co-seeding with ASCs (Figure 12).

[0251] All these results demonstrate the interest of associating macrophages and ASCs in the matrix to increase their viability and promote a pro-resolving and restorative environment.

[0252] F) In vivo study of the association of pro-resolving macrophages / ASC in the 3D matrix on a murine wound model in the context of type 2 diabetes

[0253] Following the results obtained previously (CE), a second murine in vivo protocol was carried out to evaluate the effect of the association of M2 macrophages with ASCs in the matrix on healing in a context of type 2 diabetes. Using the non-invasive experimental device used previously, wound closure was monitored daily by taking standardized photographs, from the creation of the wound on D0 until the sacrifice of the mice on D14. From a quantitative point of view, wound closure was determined by daily measurement of the wound surface for each mouse. The results are presented as percentage of wound closure on D14 (day of sacrifice) compared to the surface area of ​​the wounds on the day the wound was created. As expected (Figure 9), the application of 3D matrices seeded with M2 macrophages improves wound closure compared to the control group.Interestingly, the association of M2 macrophages with ASCs significantly increases the percentage of wound closure at D14 compared to mice that received no treatment.

[0254] Furthermore, quantification of the filling tissue clearly shows a greater filling tissue in the group receiving the matrix containing M2 macrophages and ASCs compared to the other groups.

[0255] Thus, the beneficial pro-healing effect of M2 macrophage-seeded matrices appears to be potentiated by the addition of ASCs. These results show that the association of PEC 40 / 60 matrices with pro-resolving macrophages and ASCs allows for faster wound filling and closure.

[0256] G) In vivo study of the effect of the 3D matrix according to the invention seeded with different therapeutic cells on the repair (closure) of chronic wounds according to the second protocol

[0257] Treatment of diabetic mouse wounds with matrix alone did not alter wound closure speed (Fig. 13A) or wound size compared to untreated mice (Fig. 15A). On the other hand, the association of M2-M(p) with ASCs in the matrix induced a significant improvement in wound closure speed (Fig. 13B), a decrease in wound size (Fig. 15B) and an improvement in the quality of the reconstructed tissue (Fig. 15C). Indeed, this treatment induced the presence of stable, highly vascularized granulation tissue with reduced inflammation. In addition, bridging of the superficial but also deep tissue was observed (Fig. 15C).

[0258] The time required for complete wound closure across treatments was also compared. Complete wound closure of wounds treated with matrices containing M2-M(p) occurred 4 days earlier than untreated wounds. Treatment of wounds with matrices containing M2-Mcp and ASCs induced their complete closure 6 days earlier than untreated wounds (Fig. 14).

[0259] M2-Mcp cells in the matrix retain a pro-resolving phenotype. Indeed, M2-Mcp cells cultured in the matrix (3D) produce more IL-10 than M2-Mcp cells cultured in a culture plate (2D) (Fig. 17). Moreover, culturing M2-Mcp cells on the matrix modifies their secretory profile. Indeed, culture supernatants of M2-Mcp cells cultured in 2D do not induce fibroblast proliferation, whereas culture supernatants of M2-Mcp cells cultured on the matrix (3D) significantly induce human fibroblast proliferation (Fig. 16A). Similar results are obtained when M2-Mcp cells are co-cultured with ASCs (Fig. 16B). These results show that cells cultured in the matrix change their secretory profile and produce molecules that promote fibroblast proliferation. (Fig.16), resolution of inflammation (Fig.17) and therefore tissue repair (Fig.13). < / l>

Claims

CLAIMS 1. Three-dimensional biomaterial comprising a matrix of polyelectrolyte complexes (PEC) based on modified or unmodified alginate and chitosan, and macrophages, said matrix having an interconnected open macroporosity in which the alginate / chitosan mass ratio is between 20 / 80 and 80 / 20.

2. Biomaterial according to claim 1, in which the alginate / chitosan mass ratio is 40 / 60.

3. Biomaterial according to claim 1 or 2, in which the alginate has an M / G ratio of between 1.4 and 2.7, and / or a molecular weight (Mw) of between 150,000 and 250,000, and / or a polydispersity index (PI) of less than 2.

4. Biomaterial according to claim 3, in which the alginate has an M / G ratio of between 1.4 and 2.7, preferably equal to 2 + / -0.2, a molecular weight (Mw) of between 150,000 and 250,000, and a polydispersity index (PI) of less than 2, preferably approximately 1.

5.

5. Biomaterial according to one of the preceding claims, in which the chitosan has a degree of deacetylation (DDA) of between 75% and 90% and / or an Mw of between 130,000 and 400,000, and / or a polydispersity index of less than 2.

6. Biomaterial according to claim 5, in which the chitosan has a degree of deacetylation (DDA) of between 75% and 90%, preferably between 75 and 85%, an Mw of between 130,000 and 400,000, preferably between 145,000 and 400,000, more preferably between 200,000 and 400,000, and a polydispersity index of less than 2, preferably of approximately 1.7, more preferably of approximately 1.

8.

7. Hybrid biomaterial according to one of the preceding claims, comprising pro-healing macrophages.

8. Hybrid biomaterial according to claim 7, wherein the pro-healing macrophages are pro-resolving macrophages.

9. Hybrid biomaterial according to one of the preceding claims, comprising pro-healing macrophages and mesenchymal stromal cells (MSCs), the ratio of pro-healing macrophages / mesenchymal stromal cells preferably being between 1 / 99 and 99 / 1, in particular approximately 50 / 50.

10. Method for preparing a hybrid biomaterial according to one of claims 1 to 9, comprising the steps: (a) culturing monocytes and / or macrophages under conditions that provide pro-healing macrophages; (b) preparing a matrix of polyelectrolyte complexes (PEC) based on alginate and chitosan in relative proportions between 40 / 60 and 60 / 40; (c) optionally drying the PEC matrix; (d) sterilize the PEC matrix; (e) seeding the PEC matrix with pro-healing macrophages obtained in step (a) optionally combined with MSCs.

11. A method of preparing a hybrid biomaterial according to claim 10, wherein the sterilization step (d) is sterilization by irradiation with low-energy pulsed electron beams or sterilization by irradiation with low-energy continuous electron beams.

12. Skin dressing intended for regenerative medicine comprising a three-dimensional biomaterial according to one of claims 1 to 9.

13. Pro-healing macrophages, optionally combined with MSCs, for their use in regenerative medicine in a subject, characterized in that said pro-healing macrophages and the possible MSCs are in a form suitable for their administration to said subject by means of a three-dimensional biomaterial according to one of claims 1 to 9.

14. Pro-healing macrophages, optionally combined with MSCs, for their use according to claim 13, for the treatment of a wound, in particular a chronic wound.

15. Pro-healing macrophages, optionally combined with MSCs, for their use according to claim 14, characterized in that the subject has diabetes and / or is an elderly subject.

16. Pro-healing macrophages, optionally combined with MSCs, for their use according to one of claims 13 to 15, characterized in that the three-dimensional biomaterial is in the form of a dressing, a patch, or an implantable matrix.

17. Pro-healing macrophages, optionally combined with MSCs, for their use according to one of claims 13 to 16, characterized in that said pro-healing macrophages and any MSCs have been previously obtained from cells of said subject among, bone marrow cells, iPS or blood monocytes, and ASCs from adipose tissue.

18. Kit intended for regenerative medicine, and in particular for the treatment of a wound, such as a chronic wound, said kit comprising: - a matrix of PEC alginate and chitosan, said matrix having an interconnected open macroporosity in which the anionic polymer / cationic polymer mass ratio is between 20 / 80 and 80 / 20, preferably 40 / 60; - a culture medium suitable for the culture of pro-healing monocytes or macrophages and / or a culture medium suitable for the differentiation of bone marrow cells into macrophages with a pro-healing phenotype.

19. The kit of claim 18, wherein the PEC matrix is ​​sterilized.

20. Method for treating damaged tissue according to which a hybrid biomaterial according to one of claims 1 to 9 is applied to the damaged tissue to promote the repair of said tissue.

21. A method of treatment according to claim 20, wherein the injured tissue is a chronic wound, in particular a chronic diabetic wound.

22. A method of treatment according to claim 20 or 21, wherein said hybrid biomaterial contains pro-healing macrophages, in particular pro-resolving macrophages.

23. The method of treatment of claim 22, wherein the biomaterial further comprises MSCs.

24. Treatment method according to one of claims 20 to 23, according to which the biomaterial is applied in the form of a dressing on the injured tissue.

25. Use of macrophages for the preparation of a biomaterial according to one of claims 1 to 9, for the treatment of injured tissue, in particular a wound, more particularly a chronic wound.

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