3D porous matrix based on polyelectrolyte complexes and its applications

Abstract

The present invention relates to a porous matrix of polyelectrolyte complexes (PECs) based on alginate and chitosan or their derivatives, particularly suited to cell therapy and especially to soft tissue healing and tissue repair. The matrix according to the invention can advantageously be 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 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 hybrid biomaterial and the use of such a hybrid biomaterial in regenerative medicine.

Description

Title of the invention: Porous 3D matrix based on polyelectrolyte complexes and its applications technical field

[0001] The present invention relates to a porous 3D matrix obtained from polyelectrolyte complexes (PECs) formed between anionic and cationic polymers, particularly suited to cell therapy and especially to soft tissue healing and tissue repair. The matrix according to the invention can advantageously be 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. TECHNOLOGICAL BACKGROUND

[0002] Tissue engineering, or regenerative medicine, generally uses therapeutic cells, substances necessary for tissue development (signal molecules, growth factors, etc.) and / or implantable biomaterials.

[0003] In general, an implantable biomaterial intended for deep cell seeding must exhibit biocompatibility characteristics so as not to generate an excessive inflammatory reaction once implanted. On the contrary, the material should 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. Furthermore, 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.

[0004] In the case of wound treatment, and particularly chronic wounds, therapeutic cells can be used. It can be particularly advantageous 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 ulcers, are "stuck" in a low-intensity inflammatory phase, preventing them from progressing to the subsequent stages of healing. This suggests impaired resolution of inflammation (Miao et al., 2012). The pathophysiological microenvironment of chronic wounds is itself associated with functional dysregulation of macrophages. This dysregulation and loss of macrophage functionality largely contribute to impaired wound healing in chronic wounds, including diabetic foot ulcers.

[0005] Thus, macrophages occupy a central place in the orchestration of the healing process. In a relevant and appropriate manner 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 the processes of tissue repair and remodeling, on the other hand.

[0006] One of the most supported hypotheses to explain the maintenance of inflammation at the level of chronic wounds is the absence of a change in phenotype (switch) of pro-inflammatory macrophages towards a pro-resolving phenotype.

[0007] Macrophages therefore constitute an increasingly attractive therapeutic target in regenerative medicine. Indeed, in view of macrophage dysfunctions 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-reparative functionality of macrophages.

[0008] Research has focused on developing strategies to control inflammation in chronic wounds through 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 site.

[0009] However, to date, none of the therapeutic wound healing strategies developed involving macrophages has allowed a return to satisfactory anatomical and functional integrity of wounds. Summary of the invention

[0010] While working on wound healing, particularly chronic wounds in diabetics, the inventors developed a PEC matrix based on anionic and cationic polymers particularly suited to 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 dressings. 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.

[0011] The invention therefore relates to 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.

[0012] In one embodiment, the PEC matrix is ​​based on alginate and chitosan, the alginate / chitosan mass ratio being 40 / 60.

[0013] Alginate may in particular have an M / G ratio between 1.4 and 2.7, preferably typically equal to 2 + / -0.2, a molecular weight (Mw) between 150,000 and 250,000, and a polydispersity index (PI) less than 2, preferably around 1.5.

[0014] Chitosan advantageously has a degree of deacetylation (DDA) between 75% and 90%, preferably between 75% and 85%, an Mw between 130,000 and 400,000, preferably between 200,000 and 400,000, and a polydispersity index of less than 2, preferably about 1.8.

[0015] According to the invention, the biomaterial may be a hybrid biomaterial comprising pro-healing macrophages. The pro-healing macrophages are, for example, pro-resolvent macrophages.

[0016] 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 being preferably between 1 / 99 and 99 / 1.

[0017] The invention also relates to a process for preparing a hybrid biomaterial according to the invention, comprising the steps:

[0018] (a) culture monocytes and / or macrophages under conditions allowing to obtain pro-healing macrophages;

[0019] (b) prepare a matrix of polyelectrolyte complexes (PEC) based on alginate and of chitosan, in which the two polymers are in relative proportions between 40 / 60 and 60 / 40;

[0020] (c) optionally dry the PEC matrix;

[0021] (d) sterilize the PEC matrix;

[0022] (e) seed the PEC matrix with pro-healing macrophages obtained at step (a) possibly combined with CSMs.

[0023] In one embodiment, the sterilization step (d) is sterilization by irradiation with pulsed low-energy electron beams or sterilization by irradiation with continuous low-energy electron beams.

[0024] As indicated above, in one embodiment, the matrix from step (b) undergoes a drying step before the sterilization step (d).

[0025] The invention also relates to a skin dressing intended for regenerative medicine comprising a three-dimensional biomaterial according to the invention.

[0026] The invention also relates to pro-healing macrophages possibly 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 adapted for their administration to said subject by means of a three-dimensional biomaterial according to the invention.

[0027] Such pro-healing macrophages possibly combined with MSCs can in particular be used for the treatment of a wound, in particular a chronic wound.

[0028] For example, the subject has diabetes and / or is an elderly subject.

[0029] In one embodiment, the three-dimensional biomaterial is in the form of a dressing, a patch, or an implantable matrix.

[0030] Said pro-healing macrophages and possible MSCs may have been previously obtained from cells of said subject including bone marrow cells, iPS or blood monocytes, and ASCs from adipose tissue.

[0031] The invention also relates to a kit for regenerative medicine, and in particular for the treatment of a wound, such as a chronic wound, said kit comprising:

[0032] 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 between 40 / 60 and 60 / 40;

[0033] 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 of pro-healing phenotype.

[0034] Advantageously, the PEC matrix is ​​sterilized. DESCRIPTION OF THE DRAWINGS

[0035] [Fig.1] shows the chemical structure of sodium alginate and its monomer units M and G (A), and an example of an alginate chain sequence (B).

[0036] [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. chitosan.

[0037] [Fig.3A] represents the distribution of the different batches studied in a murine in vivo study model of the cell therapy strategy for chronic wounds.

[0038] [Fig.3B] represents the timeline of the test of [Fig.3A] and the experimental conditions.

[0039] [Fig.4] shows the macrophage gene expression profile (m <e>MO, M1, and M2 after 24 hours of culture or upon contact with PEC 40 / 60 matrices. (A) Anti- and pro-inflammatory cytokines. (B) Membrane receptors. Analyses were performed with 3 replicates per condition (1-way ANOVA; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). For clarity in the graphs, only significant differences between the m <e>in contact with the matrices with respect to their reference group (m <e>(in cultivation) are indicated.

[0040] [Fig. 5AB] shows the results of the flow cytometry study of the expression of the surface markers F4 / 80, CD206 (MR) and CD86 by the m <e>M2 after 24 hours of culture (A) or in contact with PEC 40 / 60 matrices (B).

[0041] [Fig.5C] is the histogram showing the corresponding percentages of the different populations F480+CD206+ and F480+CD86+ of figures 5AB (n=3 replicates).

[0042] [Fig.6] is a diagram showing urea concentrations per 100,000 m <e>MO, m <e>You are mine. <e>M2 after 24 hours of culture or in contact with PEC 40 / 60 matrices (m <e>: macrophage). Analyses were performed with 3 replicates per condition (n= 3; one-way ANOVA test; *P < 0.05; **P < 0.01; ****P < 0.0001)

[0043] [Fig.7] is a diagram showing the luminescence monitoring of the production of reactive oxygen species by macrophages (m <e>) MO, Ml and M2 after 24 hours of culture or in contact with 3D matrices after stimulation with 100 mM of TPA.

[0044] [Fig. 8] shows a confocal microscopy image of a PEC 40 / 60 matrix after 24 hours of culture and differential labeling of live / dead cells. The inset in the upper left corner indicates the imaged faces of the matrix. The entire 3D matrix is ​​shown in (A); this is a 3D reconstruction at xlO (10X) magnification with phase contrast and the z-projection of the channels. The BCD images correspond to the z-projection of a transverse section of the matrix at 10X. The EFG and HIJ images are z-projections of the matrix imaged longitudinally at 10X and 40X, respectively.

[0045] [Fig. 9] shows the wound closure kinetics from the first day of treatment (D2). Statistical analysis between the 3D matrix group + m <e>M2 and the HFD control group is indicated by * (p<0.05).

[0046] [Fig. 1OAB] shows representative images of histological sections of the control group (A) and the group treated with 3D matrices + m <e>M2 (B).

[0047] [Fig. 1OC] shows the histological scores associated with the images in Figures 10A and 10B.

[0048] [Fig. 11] shows the viability of different cell types (human M2-Mq> and human ASC) in the matrix after 2 days and 6 days of culture after labeling with the "Ready Probes™ Cell Viability Imaging Kit" and fluorescence analysis by confocal microscopy.

[0049] [Fig. 12] shows the functionality of human Mq and ASCs seeded in the matrix at baseline or after infectious stimuli (LPS). After 24 hours of culture, the concentrations of IL-10, IL-6, and IL-1 are determined by ELISA. DETAILED DESCRIPTION OF THE INVENTION

[0050] The present invention relates to a polyelectrolyte complex matrix particularly suited for use in a dressing for 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 wound healing.

[0051] Definitions

[0052] In the context of this application, the following definitions apply.

[0053] By "matrix" is meant a three-dimensional polymeric structure, capable of being processed to take the desired shape, depending on its intended use. In the context of the invention, a matrix is ​​advantageously biocompatible and / or biodegradable.

[0054] The term “biocompatible” means a material, matrix or substance that does not interfere with or degrade the biological environment in which it is used.

[0055] By "biodegradable" is meant a material or matrix capable of being naturally decomposed by living organisms. In the case of an implantable material or matrix, this means a material or matrix capable of being naturally decomposed by the organism into which it is to be implanted.

[0056] The term "biomaterial" refers to a non-living material intended to interact with a biological system, eliciting an appropriate host response in a specific application. In the context of the invention, a "hybrid biomaterial" refers to a 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 cells, active ingredients) and / or stimulating a cellular target.

[0057] "Chronic wounds" refer to wounds that are difficult to heal or that do not follow the classic physiological process. Generally, a wound is considered chronic if it has not regained its anatomical and functional integrity after three months. Vascular ulcers are generally grouped together under the category of chronic wounds. cular (venous and arterial), bedsores, diabetic feet.

[0058] The term "approximately" associated with a numerical value means the indicated value + / -10%, preferably + / -5%.

[0059] Unless otherwise indicated, when ranges are indicated, the values ​​at the terminals are within said ranges.

[0060] Matrix

[0061] The present invention is based primarily on a biocompatible and advantageously biodegradable polymeric matrix. More specifically, the matrix is ​​a polyelectrolyte complex (PEC) matrix based on anionic polymer(s) and cationic polymer(s). PECs offer numerous advantages, such as maintaining the very good biocompatibility of the polymers (here, bio-based) from which they are derived. Therefore, PECs are already commonly used in tissue engineering with promising results in this field (Ishihara 2019).

[0062] The matrix is ​​a three-dimensional (3D) matrix. More precisely, the matrix according to the invention is a PEC matrix having an interconnected open macroporosity.

[0063] By "interconnected open macroporosity", it is understood that the matrix more particularly comprises pores with an average diameter 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.

[0064] 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.

[0065] Biocompatible anionic polymers are advantageously chosen from alginates and modified alginates. Alginates form a family of linear copolymers consisting of a chain of [3-D-mannuronic acid (M unit) and [3-G-guluronic acid (G unit)] units linked together by [3(1-4) glycodissociated bonds. The rigidity of alginate gels can be varied by changing the M / G ratio. For example, sodium alginate can be used. 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, alginate functionalized with RGD tripeptides (L-arginine, glycine, L-aspartic acid) that participate in cell adhesion can be used.

[0066] Biocompatible cationic polymers are advantageously chosen from chitosan, and any chitosan derivative that is possibly functionalized but retains an overall positive charge. Chitosan is a polysaccharide consisting of a... Chitosan consists of a chain of N-acetyl-D-glucosamine groups linked together by [3(1-4)] bonds. Its properties vary depending on its molecular weight and degree of deacetylation (DDA).

[0067] 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.

[0068] In the context of the invention, the alginates advantageously have an M / G ratio between 1.4 and 2.7, preferably equal to 2 + / -0.2, a molecular weight (Mw) between 150,000 and 250,000, in particular about 195,000, and a polydispersity index (PI) less than 2, preferably about 1.5.

[0069] Similarly, in the context of the invention, the chitosan advantageously has a degree of deacetylation (DDA) of between 75% and 90%, preferably between 75% and 85%, a Mw of between 130,000 and 400,000, preferably between 200,000 and 400,000, and a polydispersity index of less than 2, preferably of about 1.8. Thus, the alginate used can have an M / G ratio of 2.1 + / -0.1, a molecular weight of about 193,100 and a polydispersity index of about 1.5. Likewise, the chitosan used can have a DDA of 80% + / -5%, a molecular weight of about 216,300 and a polydispersity index of about 1.8.

[0070] In a particular embodiment, the PEC matrix comprises an alginate / chitosan mass ratio of between 35 / 65 and 45 / 55, preferably of about 40 / 60. The alginate of the matrix has a M / G ratio of 2.1 + / -0.1, a molecular weight of about 193.100 and an IP of about 1.5, and the chitosan has a DDA of 80% + / -5%, a molecular weight of about 216.300 and an IP of about 1.8.

[0071] 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.

[0072] The matrix according to the invention can be prepared by any suitable process known to those skilled in the art. In particular, it is possible to implement the preparation process described in application WO2017 / 017379. After obtaining the matrix, it can be sterilized and optionally dried or freeze-dried, this drying or freeze-drying being preferably carried out before sterilization for its subsequent use as a biomaterial.

[0073] Advantageously, the polyelectrolyte complex (PEC) matrix is ​​made by mixing the anionic polysaccharide, such as alginate, and the cationic polymer, such as chitosan, in proportions between 40 / 60 and 60 / 40. In particular, the cationic polymers and the anionic polymer are in proportions mass ratios 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%).

[0074] For example, the step of preparing a chitosan and alginate-based PEC matrix includes

[0075] - mix an alginate solution and a chitosan solution;

[0076] - mold and freeze the resulting PEC mixture;

[0077] - freeze-dry the PEC mixture;

[0078] - gel the lyophilized PEC matrix by adding a 0.1M CaC12 solution;

[0079] - rinse, optionally refreeze and then optionally hydrate said matrix.

[0080] 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.

[0081] The matrix according to the invention can advantageously be sterilized, preferably dry. Sterilization can be carried out by any physical or chemical means 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 is understood to mean an energy of less than 500 keV. The sterilization method described in Famo 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.

[0082] In one embodiment, 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 between 200 and 350 keV, preferably between 250 and 300 keV, in particular 280 keV.

[0083] In another embodiment, sterilization is achieved by irradiation at a dose of between 10 and 25 kGy, preferably between 12 and 20 kGy, more preferably 15 kGy, using pulsed technology with a beam energy of between 350 and 550 keV, preferably between 400 and 450 keV. For example, sterilization is achieved by irradiation at a dose of 15 kGy, with a beam energy of 430 keV.

[0084] Such sterilizations advantageously allow the preservation of the porosity and architecture of the matrix according to the invention.

[0085] Hybrid biomaterial

[0086] 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 exhibits 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 specific cells, such as macrophages and mesenchymal stromal cells. Indeed, the generated porosity allows for deep seeding of the matrix, and the architecture recreates a biomimetic environment suitable for maintaining cell viability, a particular phenotype, and / or metabolic activity.

[0087] The matrix according to the invention is thus particularly suited to the seeding and survival of macrophages and / or mesenchymal stromal cells (MSCs). It is therefore possible to use the matrix according to the invention with macrophages, or with a combination of macrophages and MSCs.

[0088] It is known that chronic wound infection causes 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 resolution phase of inflammation can only occur if this biofilm is eliminated. Pro-inflammatory macrophages, involved in eliminating pathogens by phagocytosis and by secreting pro-inflammatory cytokines, toxic intermediates, and reactive oxygen species, are therefore essential in controlling wound infection.

[0089] Furthermore, in the wound healing process, pro-resolving macrophages are essential for terminating inflammation and directing healing towards the repair phase. In the later stages of healing, these macrophages aim to restore homeostasis and mature the skin back to its initial state. The resolution of inflammation is partly activated by the phagocytosis of apoptotic PMNs by macrophages, which consequently secrete TGF-3 and VEGF. Pro-resolving macrophages also produce high quantities of IL-10, 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 are involved in the apoptosis of myofibroblasts, thus minimizing the risk of excessive fibrosis.Pro-resolving macrophages are characterized by the presence of PRR (Pattern Recognition Receptor) receptors capable of recognizing molecular patterns associated with pathogens (PAMPs) and / or molecular patterns associated with hazards (DAMPs).

[0090] Thus, the matrix according to the invention advantageously comprises pro-healing macrophages. According to the invention, pro-healing macrophages refer to pro-inflammatory macrophages, pro-resolving macrophages, or a mixture thereof.

[0091] The addition of pro-inflammatory / antibacterial and / or pro-resolving macrophages to the matrix is ​​advantageously determined by the infectious and inflammatory status of the wound to be treated. Those skilled in the art can decide which type of pro-healing macrophages to use with the matrix, depending on the wound being treated.

[0092] In a particularly surprising way, the inventors have shown that the matrix according to the invention makes it possible to maintain macrophages of the matrix in a pro-resolvent state.

[0093] Advantageously, the matrix according to the invention, comprising pro-resolving macrophages, can 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 site towards a pro-resolving phenotype.

[0094] 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 allow the induction of a pro-resolving phenotype in 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 site of the wound to 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, thereby accelerating 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 and stimulating VEGF production by cells in the wound microenvironment (Shingyochi, Y, 2015, Exp Opinion mol Ther). MSCs thus have significant potential to accelerate wound closure through their anti-inflammatory properties, the stimulation of angiogenesis, and the production of soluble factors that improve the wound microenvironment and promote tissue regeneration. Thus, matrix CMS can play a role in the different phases of wound healing, whether the wound is healthy or infected.

[0095] The CMS used can be derived, for example, from adipose tissue, bone marrow or umbilical cord.

[0096] Interestingly, the inventors have highlighted that the association between Pro-resolving macrophages and ASCs significantly increase the number of viable cells in the matrix.

[0097] Advantageously, the association between pro-resolving macrophages and ASCs potentiates the pro-resolving and therapeutic activity of cells seeded in the matrix.

[0098] In a particular embodiment, the biomaterial according to the invention comprises an alginate and chitosan-based matrix, advantageously with an alginate / chitosan mass ratio of about 40 / 60, and macrophages, preferably pro-healing macrophages, in particular pro-resolving macrophages.

[0099] In a particular embodiment, the hybrid biomaterial according to the invention comprises an alginate and chitosan-based matrix, advantageously with an alginate / chitosan mass ratio of about 40 / 60, pro-healing macrophages and mesenchymal stromal cells.

[0100] In a particular embodiment, the hybrid biomaterial according to the invention comprises an alginate and chitosan-based matrix, advantageously with an alginate / chitosan mass ratio of about 40 / 60, pro-healing macrophages, preferably pro-resolving macrophages, and mesenchymal stromal cells.

[0101] 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.

[0102] When the hybrid biomaterial according to the invention contains macrophages, the volume concentration of cells in the PEC matrix is ​​preferably between 500 cells and 4000 cells / mm3 of PEC matrix.

[0103] Macrophages exhibiting a pro-healing phenotype, and in particular a pro-resolving phenotype, preferentially represent at least 60% by number of macrophages contained in the PEC matrix, more preferably at least 70%, 80%, 85%, 90%, 95%.

[0104] 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

[0105] 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.

[0106] The hybrid biomaterial according to the invention can be prepared by any known process of The man in the trade.

[0107] In general, the process for preparing a hybrid biomaterial according to the invention may include the steps of preparing a polyelectrolyte complex (PEC) matrix according to the invention, optionally drying, sterilizing said PEC matrix and seeding the PEC matrix with therapeutic cells, in particular pro-healing macrophages.

[0108] In particular, once the matrix as described above is manufactured, dried, and sterilized, said matrix is ​​seeded with therapeutic cells by contacting the matrix with a culture medium containing said therapeutic cells of interest. Of course, it is possible to seed the matrix directly according to the invention, without going through a drying step.

[0109] Thus, the process for preparing a hybrid biomaterial according to the invention may comprise the following steps:

[0110] (a) culture monocytes and / or macrophages under conditions allowing to obtain pro-healing macrophages;

[0111] (b) prepare a matrix of alginate-chitosan polyelectrolyte complexes (PECs), in which the two polymers are in relative proportions between 40 / 60 and 60 / 40;

[0112] (c) possibly the drying of the PEC matrix;

[0113] (d) sterilize the PEC matrix;

[0114] (d) seed the PEC matrix with pro-healing macrophages obtained at step (a) possibly combined with CSMs.

[0115] Depending on the intended use, and in particular depending on the stage of wound healing of the wound to be treated, step (a) may include culturing macrophages under conditions that produce macrophages with a pro-resolving phenotype. For example, step (a) of macrophage culture includes culturing bone marrow cells previously collected from a human or non-human mammal in a culture medium that produces naïve macrophages, polarizing them 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. Polarizing the macrophages towards a pro-inflammatory phenotype is achieved 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.

[0116] Alternatively, it is possible to culture iPS cells, blood monocytes, etc., in a suitable culture medium to differentiate these cells into macrophages and polarize them towards a pro-resolving phenotype (Douthwaite H. et al. 2022 Bio Protoc.). Those skilled in the art can adapt this preparation step of the cells, depending on the origin of the cells and the intended end use.

[0117] If the matrix is ​​to be seeded with pro-inflammatory macrophages, step (a) may include culturing macrophages under conditions that produce macrophages with a pro-inflammatory phenotype. For example, macrophage culture step (a) includes a differentiation step in the presence of MSCF and a 24-hour culture step in the presence of inflammatory cytokines, such as interferon gamma, and / or bacterial cell walls in a defined medium.

[0118] In the case where the cells to be seeded are MSCs, step (a) may include isolating cells from adipose tissue, bone marrow, or umbilical cord of a human or non-human mammal and culturing them 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. The cells will be used after at least one passage in culture.

[0119] Of course, it is possible to proceed in several steps (a), so as to seed several cell types in the matrix.

[0120] Step (b) of preparing the PEC matrix and step (d) of sterilization can be carried out as described above.

[0121] Of course, the cell culture step (a) and steps (b) and (d) can be carried out independently and at different times. For example, it is possible to prepare the matrix and sterilize it several hours, days, months, etc., before the cell culture step. Before inoculation, the matrices can be stored in a lyophilized and sterilized state in a sterilization package.

[0122] Advantageously, the seeding step (e) includes contacting the lyophilized and sterilized PEC matrix with a cell pellet (for example, a cell pellet of macrophages and / or mesenchymal cells) and then centrifuging said matrix before adding complete medium.

[0123] Seeded matrices can be stored 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 stored 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. Persons skilled in the art know 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.

[0124] In the case of a matrix seeded with pro-resolving macrophages, preservation in culture can extend over a period of several days, and in particular 10 days, 15 days, 20 days, 21 days or more, with a viability rate of the ma- very high crophages.

[0125] It is also possible to freeze the matrix after seeding.

[0126] Kit

[0127] The invention also relates to a kit for regenerative medicine, and in particular for the treatment of a wound, such as a chronic wound, said kit comprising:

[0128] - a PEC matrix based on alginate and chitosan or their derivatives, such as described above;

[0129] - a culture medium suitable for cells intended to seed the matrix.

[0130] For example, the culture medium is suitable for the culture of pro-resolving macrophages and / or MSCs.

[0131] For example, macrophages are cultured in supplemented RPMI 1640, and MSCs are cultured in supplemented alpha-MEM.

[0132] The kit may also include a culture medium adapted to the differentiation of cells, such as bone marrow cells, iPS cells, etc., into macrophages with a pro-resolving and / or pro-inflammatory phenotype.

[0133] Advantageously, the PEC matrix is ​​an alginate and chitosan-based matrix, as described above.

[0134] The PEC matrix is ​​advantageously sterilized, and optionally packaged in a sterilization package, in which it can be stored until use for cell seeding.

[0135] The kit according to the invention may also include a matrix as described above, already seeded with pro-healing macrophages and optionally MSCs. Such a seeded matrix may be supplied frozen. The kit may then include a medium suitable for thawing the matrix and ensuring the survival of the therapeutic cells.

[0136] According to the invention, the kit may include a matrix as described above, shaped for use as a dressing, or supplied with means for use as a dressing. Thus, the kit may contain a strip or any adhesive to hold the matrix in position on a wound. In another embodiment, the matrix may be supplied in a format that allows it to be cut to fit the size / shape of the wound.

[0137] Uses

[0138] The matrix according to the invention can be used alone, or as a biomaterial, in combination with pro-healing macrophages and / or therapeutic molecules.

[0139] 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 ​​suitable for This promotes the polarization of endogenous macrophages, present at the site of a wound being treated, for example, towards a pro-resolving phenotype. Thus, the application of a dressing containing such a matrix can aid in wound healing.

[0140] Similarly, the matrix according to the invention can be used in combination with one or more therapeutic molecules and / or biological substances. For example, the matrix can be loaded with antibiotics or natural substances intended to reduce the risk of infection of a wound to which the 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, these molecules will aid healing by promoting the polarization of endogenous macrophages towards a pro-resolving phenotype.

[0141] 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.

[0142] Regenerative medicine refers to the repair, replacement, or regeneration of damaged cells or tissues. According to the invention, tissue repair and wound healing are primarily targeted.

[0143] Thus, the invention relates to pro-healing macrophages for use in regenerative medicine in a subject, said healing macrophages being in a form adapted for administration to said subject by means of a hybrid biomaterial according to the invention. The invention further relates to pro-resolving macrophages, optionally in combination with MSCs, for use in regenerative medicine in a subject, said pro-resolving macrophages being in a form adapted for administration to said subject by means of a hybrid biomaterial according to the invention.

[0144] A subject is understood to mean a mammal, human or non-human, in need of medical treatment, such as the treatment of a wound, particularly 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 mean a human mammal aged 70 years or older.

[0145] The invention relates particularly to pro-resolving macrophages, possibly 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.

[0146] The invention also relates to pro-inflammatory macrophages, possibly 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.

[0147] Such use is particularly suitable for subjects with a disease limiting healing mechanisms, such as diabetes.

[0148] Said pro-healing macrophages and / or MSCs may 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 cells.

[0149] Advantageously, the three-dimensional biomaterial is in the form of a dressing, a patch, or an implantable matrix.

[0150] The invention also relates to the use of a biomaterial according to the invention, particularly in the form of a dressing or implantable matrix, in regenerative medicine. In particular, the invention relates to the use of such a biomaterial wherein said biomaterial is brought into contact with damaged tissue, such as a wound.

[0151] The invention also relates to a method for treating damaged tissue, in which a biomaterial as described above is applied against the damaged tissue to promote its repair. In particular, the invention relates to a method for treating a wound, and especially a chronic wound, in 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, possibly in combination with mesenchymal stem cells (MSCs). The biomaterial can be applied in the form of a dressing suitable for maintaining its position on the wound. EXAMPLES MATERIALS AND METHODS

[0152] A) Matrix

[0153] The polymers used are a "medium viscosity" alginate marketed by Sigma (reference A-2033; lot 051M0054V) and a "medium molecular weight" chitosan also supplied by Sigma (reference 448877; lot STBF8484V).

[0154] For 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 are 193,100 and 126,900 g / mol, respectively, with a polydispersity index of 1.5.

[0155] For chitosan, the degree of deacetylation (DDA) was calculated using 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 dispersity index of 1.8.

[0156] The PEC matrices are obtained by mixing an alginate solution of fixed concentration of 3% (weight / volume) and a chitosan solution with 1.5% acetic acid and chitosan concentration variable according to the desired final polymer ratio in the matrices.

[0157] Briefly, the alginate and chitosan solutions are homogenized by mechanical stirring and then mixed in equal 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.

[0158] This protocol allows the obtaining of a family of 3D matrices with alginate / chitosan ratios ranging from 80 / 20 to 20 / 80. Figure 3 shows the case of mixing to obtain a matrix with a ratio of 40 / 60.

[0159] B) Cells

[0160] The murine macrophages used are derived from a primary culture. They are extracted from the bone marrow of C57BL6 / JRj mice (Janvier Labs). The murine ASCs are obtained by enzymatic and mechanical digestion of the inguinal adipose tissue of C57BL6 / JRj mice (Janvier Labs).

[0161] After euthanasia of the mice by carbon dioxide overdose, the femurs and tibias are removed and cleaned to remove the surrounding muscles. After disinfecting the bones in 70% ethanol, both epiphyses of each bone are removed, and then the medullary canal is washed by injection of PBS to recover the bone marrow cells. Mechanical dissociation of the cell suspension is performed by pipetting using successive ejections, and then the suspension is filtered to 40 µm and centrifuged for 10 minutes at 1500 rpm. The cell pellet is then resuspended in 1 mL of ACK buffer to lyse the red blood cells. The pellet is resuspended in 9 mL of complete medium (DMEM glutamax, 10% FBS, 1% penicillin / streptomycin, and 1% L-glutamine) and centrifuged a second time. The final pellet is resuspended in 5 mL; 10 qL are taken for trypan blue labeling and counting on a Malassez cell.On average, between 20 and 40 million bone marrow cells are collected per mouse. The cells are taken in whole into complete medium supplemented with M-CSF (30 ng / mL); they are seeded in petri dishes to obtain 300,000 cells / cm2. After 4 days of adhesion, MO macrophages (mO MO) are obtained (Martinez et al., 2006; Xia Zhang et al., 2008).

[0162] Four days after their isolation and differentiation, MO macrophages are not activated and can be polarized towards a pro-resolving M2 (mO M2) phenotype with a 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 phenotype Ml (m <b ml) avec un milieu complet supplémenté en m-csf (30 ng ml), ifn-y (2 ml) et lps (100 pendant 24 heures.

[0163] les ascs humaines sont isolées à partir de tissu adipeux humain obtenu après der-molipectomies abdominales. le est digéré par action mécanique puis enzymatique. culot cellulaire représente la fraction stromale vasculaire du qui contient les ascs. la fsv mise ensuite culture asc obtenues 8 jours culture.

[0164] monocytes humains isolés cellules mononuclées sang périphérique (pbmc) obtenus concentrés leucocytaires différents donneurs sains. pbmc centrifugation sur gradient ficoll. l’anneau collecté adhérence ou tri magnétique. après une semaine culture, macrophages stimulés heures soit 100 ml pour obtenir des non polarisés (m0-mq>) or 20 ng / mL of IL-4 to induce a pro-resolving phenotype (M2-M <p).

[0165] C) Seeding

[0166] To recover the macrophages, the culture medium is removed and replaced with cold PBS 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 a Malas sez cell.

[0167] In the 3D matrix contact experiments, unactivated MO or polarized M1 (pro-inflammatory phenotype) or M2 (pro-resolvent phenotype) macrophages are cultured for two hours. After macrophage adhesion, the matrices are deposited in the well so that they are in direct contact with the cells. The cells or the culture supernatant are collected 24 hours later for the various analyses.

[0168] The lyophilized matrices are seeded 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 to the full depth of the 3D matrices. Finally, complete medium is carefully added to the edge of the well, and the matrices are incubated (37°C, 5% CO2).

[0169] D) Recovery of macrophages after seeding in 3D matrices

[0170] The first step consists of cutting the matrices into 4 in order to maximize the contact between the cells attached to the matrix and the detachment solution.

[0171] The samples are then incubated with a detachment solution, before filtration. Filtration of debris successively on a 100 qm sieve then a 40 qm sieve, then centrifugation of the cell suspensions.

[0172] E) Study of gene expression by RT-PCR

[0173] Reverse Transcription-Polymerase Chain Reaction (RT-PCR) takes place in three successive steps: extraction of messenger RNA from cells, reverse transcription of TRNA into cDNA, and then amplification of the cDNA by PCR. Prior to this, macrophages seeded in 3D matrices were detached. The pellet of detached macrophages, as well as macrophages cultured in wells (so-called "2D" controls), were frozen at -80°C in 1000 L of lysis buffer (Promega).

[0174] Extraction of mRNA

[0175] The “ReliaPrep™ RNA Cell Miniprep System” kit (Promega) is used to extract mRNAs following the protocol provided by the supplier. The mRNAs are then quantified using Nanodrop® (ND-1000).

[0176] Reverse transcription

[0177] Reverse transcription of mRNAs to cDNA is performed using the "Superscript Vilo cDNA synthesis kit" (Invitrogen). For each experiment, a pure cDNA pool is prepared by mixing 2 qL of each sample. The pool is then diluted 1 / 5, 1 / 10, 1 / 20, and 1 / 40 to establish a standard curve. The remaining cDNAs are then diluted 1 / 5 in RNase / DNAse-free water.

[0178] PCR

[0179] cDNA amplification was performed using the "SYBR Green Lbased real-time PCR" kit (Roche) in a LightCycler® 480. The primer sequences used are detailed in the table below. The relative expression of each gene in the samples was determined with respect to the standard curve and with respect to the housekeeping gene (Gapdh).

[0180] [Tables 1] Gene Primer Sequence Gapdh Sens 5' AAC-TTT-GGC-ATT-GTG-GAA-GG 3' Antisense 5' ACA-CAT-TGG-GGG-TAG-GAA-CA 3' 11-10 Sens 5' TTT-TCA-CAG-GGG-AGA-AAT-CG 3' Antisense 5' CCA-AGC-CTT-ATC-GGA-AAT-GA 3' Tgf-^1 Sens 5' AGG-TTG-GCA-TTC-CAC-TTC-AC 3' Antisense 5' AGG-GGC-CTC-TAA-GAG-CAG-TC 3' Tnf-a Sens 5' AGG-CTG-TGC-ATT-GCA-CCT-CA 3' Antisense 5' GGG-ACA-GTG-ACC-TGC-ACT-GT 3' Mrcl Sens 5' ATG-CCA-AGT-GGG-AAA-ATC-TG 3' Antisense 5' TGT-AGC-AGT-GGC-CTG-CAT-AG 3' Cd36 Sens 5' GCA-GAA-TCA-AGG-GAG-AGC-AC 3' Antisense 5' GAG-CAA-CTG-GTG-GAT-GGT-TT 3'

[0181] F) Study of surface markers by flow cytometry

[0182] In order to study the expression of certain surface markers, macrophages Cells cultured in wells (2D control) and those detached from the 3D matrices are analyzed by flow cytometry. Cell suspensions are centrifuged, and 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 performed 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). The cell suspensions are then rinsed and centrifuged before being incubated with Live / dead violet (miltenyibiotec). Finally, the samples are resuspended in FACS 186 buffer before acquisition using the BD Fortessa flow cytometer (BD Biosciences). Analyses are performed using BD FACS DVIA™ software (BD Biosciences).

[0183] Fc block buffer: FACS buffer supplemented with 3% mouse serum, 3% rat serum and the anti-CD16 / CD32 antibody pair at 11006

[0184] FACS buffer: PB S supplemented with 5% S VF and 5 mM EDTA

[0185] G) Quantification of the production of reactive oxygen species (ROS)

[0186] The production of reactive oxygen species (ROS) 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 using a luminometer (Envision, PerkinElmer). Chemiluminescence generation was continuously monitored for 30 minutes after incubation of cells and / or biopolymer matrices with luminol (66 µM) 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 a number x seconds.

[0187] H) Study of arginase activity

[0188] Arginase activity is determined from cell lysates by colorimetric assay of urea production.

[0189] I) In vivo experimental protocol

[0190] All experiments on mice were carried out in compliance with current regulations concerning the use of animals for scientific purposes.

[0191] The mice used in this in vivo assay were 8-week-old male C57B1 / 6NRJ mice (Janvier Labs). All animals were maintained on a daily cycle of 12 hours of darkness followed by 12 hours of light at 22°C, with unlimited access to food and water. To induce type 2 diabetes, the mice were fed a high-fat diet (HFD) for 4 months (260 HF diet, Safe) (Gâlvez et al., 2019). The mice were weighed at regular intervals, and their fasting blood glucose levels were measured to check for hyperglycemia.

[0192] Mice are randomly divided into 4 groups of six mice ([Fig. 3A]). The protocol is carried out over approximately ten days ([Fig. 3B]) and begins with the creation of a wound on day 0. To do this, the mice are anesthetized with isoflurane. The dorsal area and flanks are shaved and disinfected, and then a circular excision of approximately 1 cm² is performed. Immediately afterward, a non-invasive experimental device, developed by PharmaDev (FR2984719 / FR2984722), is implanted. This device allows, among other things, the retention of the 3D matrices at the wound site.

[0193] After the operation, the mice are placed in a heated cage until they wake up and food is placed directly in their cage to facilitate feeding.

[0194] After 48 hours (day D2), the first topical treatments are applied:

[0195] Control group: 30 qL of culture medium (DMEM glutamax);

[0196] Group “3D Matrix Only”: sterilized and hydrated 3D matrix in medium of culture ;

[0197] “M2 Macrophages alone” group: 200,000 M2 polarized macrophages in vitro;

[0198] Group “3D Matrix with M2 Macrophages”: sterilized 3D matrix, seeded with polarized M2 macrophages in vitro and hydrated.

[0199] These treatments are left for 24 hours and are renewed twice, on days J3 and J4.

[0200] On the day of the mouse sacrifice, skin expiations are collected and then included in Paraffin. After sectioning with a microtome (HM 340E Rotary Microtome, Thermo Scientific™), the sections are stained with hematoxylin and eosin (H&E staining). Finally, the slides are scanned for analysis (NanoZoomer Digital Pathology, Hamamatsu).

[0201] For the second in vivo protocol, the mice were randomly divided into 4 groups of five mice. The protocol lasted approximately ten days and began with the creation of a wound on day 0 (see in vivo protocol Figure 3). After 48 hours (day 02), the first topical treatments were applied:

[0202] Control group: 30 qL of culture medium (DMEM glutamax);

[0203] Group “3D Matrix Only”: sterilized and hydrated 3D matrix in medium of culture ;

[0204] Group “3D Matrix with M2 macrophages”: sterilized 3D matrix, seeded with in vitro polarized M2 macrophages and hydrated.

[0205] Group “3D Matrix with M2 macrophages and ASC”: sterilized 3D matrix, seeded with in vitro polarized M2 macrophages and ASC and hydrated.

[0206] These treatments are left for 24 hours and are renewed twice, on days J3 and J4.

[0207] Viability of human macrophages and human ASCs in the matrix

[0208] Human pro-resolving macrophages (M2-Mq>: 2x05 cells) and ASCs Human cells (2 x 10⁵ cells) are seeded individually or in combination in the 3D matrix and cultured. After 2 and 6 days of seeding, the cells in the matrix are labeled with the 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 reagent labels only the nuclei of dead cells. After 20 minutes of labeling, the matrices are washed three times with PBS, and fluorescence is detected by confocal microscopy (LSM88O, Zeiss). The total cell count and the number of dead cells are evaluated by automated image analysis on Fiji (Macro developed at the Restore laboratory) (analysis of 5 images per condition).

[0209] Measurement of cytokine production by human macrophages and human ASCs seeded in the matrix

[0210] Human ASCs (1xlO5 cells) and different human Mq> populations (1xlO5 cells) are seeded alone or in combination in the 3D matrix. After 12 h of seeding, the medium is changed and the matrices containing the cells are recultured and stimulated or not with LPS (10 ng / mL). After 24 h of culture, the supernatants are collected and the concentrations of IL-6, IL-1[3, and IL-10 are determined by ELISA according to the manufacturer's (Invitrogen) instructions. RESULTS

[0211] A) Analysis of the retention, viability and distribution of macrophages in 3D matrices

[0212] To validate 3D matrices as supports for pro-resolving M2 macrophages for cell therapy, the retention, viability, and distribution of these macrophages within the 3D matrices were studied. The retention rate of the m <e>M2 after seeding was evaluated by quantification of total extracted proteins.

[0213] The results show that the retention rate is high (approximately 85%).

[0214] The viability of M2 macrophages within the 3D PEC40 / 60 matrix was qualitatively monitored by confocal microscopy in live imaging. The hydrated 3D matrices were imaged longitudinally and transversely ([Fig. 8]). Analysis of the reconstructions showed an M2 macrophage viability of 89 ± 3% after 24 hours of seeding in the 3D matrix.

[0215] This labeling also provides information on the spatial distribution of M2 mO in the matrices. No regionalization is observed. On the contrary, the M2 mO are distributed homogeneously both on the surface and in depth.

[0216] Interestingly, after 21 days of culture, the mO M2 still exhibit good viability in 3D matrices.

[0217] In conclusion, 3D matrices combining alginate and chitosan PEC 40 / 60 constitute an optimal 3D support for M2 macrophages for cell therapy.

[0218] B) Effect of the matrix according to the invention on the phenotype of macrophages

[0219] The effect of 3D alginate-chitosan matrices on the polarization of activated macrophages towards MO, M1, and M2 phenotypes was investigated. To this end, alginate-chitosan matrices (40 / 60) were exposed to macrophages (mO, MO, M1, or M2) for 24 hours, and the expression levels of mRNAs encoding pro- and anti-inflammatory cytokines (Tnf-α, 11-10, Tgf-

[31] ) were studied ([Fig. 4]). The gene and protein expression of membrane receptors characteristic of the M1 (Cd86) and M2 (Cd206, Cd36) phenotypes was also investigated ([Fig. 4] and Figure 5).

[0220] This initial phenotypic characterization was subsequently complemented by functional analyses such as the study of arginine metabolism or the production of reactive oxygen species (ROS).

[0221] First, these analyses validated the MO, M1, or M2 polarization of cultured macrophages after their isolation from bone marrow and treatment with various inducers. Furthermore, they show that upon contact with 3D alginate-chitosan matrices, macrophages shift towards an M2 phenotype. Indeed, the 3D alginate-chitosan matrices appear, on the one hand, to potentiate the M2 macrophage phenotype by increasing the expression of M2 markers (11-10, Tgf-[31, Cd206, Cd36) and decreasing the expression of M1 markers (Tnf-α, Cd86); and on the other hand, to direct non-activated MO macrophages towards an M2 phenotype. Equally interesting, M1 macrophages in contact with the matrices exhibit lower expression of pro-inflammatory markers characteristic of M1 macrophages (Tnf-α). and the CD86).

[0222] Taken together, these results demonstrate that contact between M2 macrophages and 3D alginate-chitosan matrices does not alter their phenotype. Furthermore, the matrices appear to direct MO and M1 macrophages towards an M2 phenotype and potentiate the M2 phenotype of M2 macrophages.

[0223] In order to explore the impact of alginate-chitosan matrices on macrophage effector functions, arginine metabolism ([Fig. 6]) and oxidative stress ([Fig. 7]) were evaluated. Arginine metabolism is central to the different functions associated with the M1 and M2 phenotypes of macrophages (m <e>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 alginate-chitosan matrices, demonstrating greater arginase activity in these macrophages ([Fig. 6]).

[0224] Regarding ROS production, as expected, the capacity of M1 macrophages to produce ROS is greater than that of MO and M2 macrophages after TPA stimulation. This capacity to produce ROS is significantly reduced in M1 and M2 macrophages exposed to the matrices ([Fig. 7]). These results show that the 3D alginate-chitosan matrices are capable of shifting the functions of MO and M1 macrophages towards functions characteristic of M2 macrophages and of potentiating the specific functions of M2 macrophages.

[0225] In conclusion, these initial results have demonstrated the maintenance of the phenotype and functions of M2 macrophages by the 3D matrices according to the invention. Furthermore, the 3D matrices appear to direct MO macrophages towards an M2 phenotype and attenuate ML polarization

[0226] C) In vivo study of the association between pro-resolving macrophages and 3D matrices on a murine wound model in the context of type 2 diabetes

[0227] Before the wounds were created, the fasting blood glucose and weight of the mice placed on a high-fat diet (HFD) were checked. These values ​​were compared to those of a "normal chow" control group consisting of normally fed C57B1 / 6 mice. The average weight of the mice on the high-fat diet (HFD) was greater than that of the control mice (51.5 ± 0.5 g vs. 25.2 ± 0.9 g). Their fasting blood glucose was significantly higher 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 created on the dorsal side by circular excision of approximately 1 cm². Immediately afterward, a non-invasive experimental device (FR2984719 / FR2984722) was implanted. Among other things, it allows the 3D matrices to be maintained at the wound site. Treatments began 48 hours after the wounds were created, so as not to interfere with the inflammatory phase, the proper development of which allows the subsequent phases of healing to be activated (Jetten et al., 2014).

[0228] The treatments consist of applying topically to the wound either culture medium (control group), 200,000 M2 macrophages (mO M2) or a hydrated 3D matrix alone or seeded with 200,000 M2 macrophages.

[0229] Thanks to the non-invasive experimental device, wound closure was monitored daily by taking standardized photographs, from the creation of the wound on J0 until the sacrifice of the mice on J15.

[0230] From a quantitative point of view, wound closure was determined by daily measurement of the wound surface area for each mouse. The results are presented as a percentage of wound closure relative to the surface area on the day treatment was started (D2) ([Fig.9]).

[0231] It can thus be observed that the local addition of M2 macrophages to wounds does not improve wound closure. Interestingly, the application of hydrated 3D matrices alone accelerates wound closure, with a significant effect occurring primarily in the early stages (days 4 to 7). This result correlates with the orientation of MO macrophages towards an M2 phenotype and the attenuation of the M1 profile observed upon contact of the macrophages with the 3D matrices. From day 4 to day 15, the application of 3D matrices seeded with M2 macrophages significantly accelerates wound closure compared to the control group.

[0232] Thus, the beneficial pro-healing effect of the matrices alone is potentiated by the addition of M2 macrophages. These results show that the combination of PEC 40 / 60 matrices with pro-resolving macrophages allows for faster wound closure. The combination of the matrix according to the invention with pro-resolving macrophages demonstrates a synergistic effect on wound closure.

[0233] The biomaterial according to the invention therefore allows (i) to act as a barrier to infectious agents in the environment, (ii) upon contact with the wound to induce an orientation of the endogenous macrophages of the wound towards an anti-inflammatory phenotype but also (iii) to maintain the viability and pro-resolutive functionality of the macrophages.

[0234] D) Evaluation of scar quality

[0235] To investigate the quality of the newly formed skin, hematoxylin and eosin stains were performed on histological sections taken at day 15. Several scoring systems have been developed to standardize the qualitative analysis of skin sections after healing (Gantwerker & Hom, 2011; Gupta & Kumar, 2015) (Figure 10).

[0236] The score was determined on the basis of 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 scab (if so, detachment and / or presence of inflammatory cells).

[0237] Qualitative analysis of the slides was performed by two different operators, and scores ranging from 0 to 15 were assigned (the lower the score, the higher the quality of the newly formed skin). The histological score was lower in mice treated with the 3D matrix seeded by pro-resolving macrophages.

[0238] 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 at the level of wounds treated by 3D matrices associated with pro-resolving macrophages.

[0239] E) Viability and functionality of different cell types (human M2-Mq and human ASCs) in the matrix

[0240] The results show that, although macrophages and ASCs were seeded in identical quantities, from day 2 onwards the ASCs are present in greater numbers, thus revealing a slightly higher viability than the M2-M <1> . Very interestingly, the association between the two cell types significantly increases the number of both cell types as well as their viability in the matrix ( [Fig.11]).

[0241] Very interestingly, the results show that the association between ASCs and different types of macrophages induces increased IL-10 production and an explosive production of IL-6, two cytokines that play a central role in the resolution of inflammation and in tissue repair. In the presence of LPS, the induction of IL-β production by matrix macrophages is strongly inhibited by co-seeding with ASCs ([Fig. 12]).

[0242] All these results demonstrate the benefit of associating macrophages and ASCs in the matrix to increase their viability and promote a pro-resolving and repairing environment.

[0243] 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

[0244] Following the results obtained previously (CE), a second in vivo murine protocol was carried out to evaluate the effect of the association of M2 Macrophages with ASCs in the matrix on wound healing in a context of type 2 diabetes. Thanks to the non-invasive experimental device used previously, wound closure was monitored daily by taking standardized photographs, from the creation of the wound on J0 until the sacrifice of the mice on J14.

[0245] From a quantitative perspective, wound closure was determined by daily measurement of the wound surface area for each mouse. The results are presented as the percentage of wound closure at day 14 (sacrifice day) relative to the wound surface area on the day the wound was created. As expected ([Fig. 9]), the application of 3D matrices seeded with M2 macrophages improved wound closure compared to the control group. Interestingly, the combination of M2 macrophages with ASCs significantly increased the percentage of wound closure at day 14 compared to untreated mice.

[0246] Furthermore, the quantification of the filling tissue clearly shows a greater amount of filling tissue in the group that received the matrix containing M2 macrophages and ASCs compared to the other groups.

[0247] 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 combination of PEC 40 / 60 matrices with pro-resolving macrophages and ASCs allows for faster wound filling and closure.< / e> < / e> < / e> < / e> < / e> < / e> < / e> < / e> < / e> < / e> < / e> < / e> < / e>

Claims

Demands

1. A three-dimensional biomaterial comprising a matrix of polyelectrolyte complexes (PECs) based on alginate and chitosan, modified or unmodified, and macrophages, said matrix having an interconnected open macroporosity in which the alginate / chitosan mass ratio is between 20 / 80 and 80 / 20, and in which alginate has an M / G ratio between 1.4 and 2.7, a molecular weight (Mw) between 150,000 and 250,000, and a polydispersity index (PI) less than 2, and in which chitosan has a degree of deacetylation (DDA) between 75% and 90%, an Mw between 130,000 and 400,000, and a polydispersity index less than 2.

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

3. Biomaterial according to claim 1 or 2, wherein the alginate has an M / G ratio of 2 + / -0.2, and a polydispersity index (PI) preferably of about 1.

5.

4. Biomaterial according to any one of claims 2 or 3, wherein the chitosan has a degree of deacetylation (DDA) between 75 and 85%, an Mw preferably between 200,000 and 400,000, and a polydispersity index preferably of about 1.

8.

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

6. Hybrid biomaterial according to claim 5, wherein the pro-healing macrophages are pro-resolving macrophages.

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

8. A method for preparing a hybrid biomaterial according to any one of claims 1 to 7, comprising the steps: (a) culturing monocytes and / or macrophages under conditions enabling the production of pro-healing macrophages; (b) preparing a polyelectrolyte complex (PEC) matrix 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) seed the PEC matrix with pro-ci-healing macrophages obtained in step (a) possibly combined with MSCs.

9. A method for preparing a hybrid biomaterial according to claim 8, 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.

10. Skin dressing for regenerative medicine comprising a three-dimensional biomaterial according to any one of claims 1 to 7.

11. Pro-healing macrophages, optionally combined with MSCs, for use in regenerative medicine in a subject, characterized in that said pro-healing macrophages and the optional MSCs are in a form suitable for administration to said subject by means of a three-dimensional biomaterial according to any one of claims 1 to 7.

12. Pro-healing macrophages, possibly combined with MSCs, for their use according to claim 11, for the treatment of a wound, in particular a chronic wound.

13. Pro-healing macrophages, possibly combined with MSCs, for their use according to claim 12, characterized in that the subject has diabetes and / or is an elderly subject.

14. Pro-healing macrophages, optionally combined with MSCs, for use according to any one of claims 11 to 13, characterized in that the three-dimensional biomaterial is in the form of a dressing, a patch, or an implantable matrix.

15. Pro-healing macrophages, optionally combined with MSCs, for use according to any one of claims 11 to 14, characterized in that said pro-healing macrophages and the optional MSCs were previously obtained from cells of said subject among bone marrow cells, iPS cells or blood monocytes, and ASCs from adipose tissue.

16. 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 based on alginate and chitosan, said matrix having an interconnected open macroporosity in which the alginate / chitosan mass ratio is between 20 / 80 and 80 / 20, preferably 40 / 60; - a culture medium suitable for the culture of monocytes or macrophages pro-healing and / or a culture medium adapted to the differentiation of bone marrow cells into macrophages of pro-healing phenotype.

17. Kit according to claim 16, wherein the PEC matrix is ​​sterilized.

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