Kit for the reconstitution of a cell-free biomedical device for use in regenerative medicine, biomedical device thus reconstituted and related synthesis process

Abstract

Kit for the reconstitution of a cell-free biomedical device for use in regenerative medicine, characterised in that it comprises a biomaterial in lyophilised form based on hyaluronic acid and heparin that is rehydrated by a secretome containing growth factors and chemokines, collected from cultured human mesenchymal stromal cells (MSC).

Description

[0001] KIT FOR THE RECONSTITUTION OF A CELL-FREE BIOMEDICAL DEVICE FOR USE IN

[0002] REGENERATIVE MEDICINE, BIOMEDICAL DEVICE THUS RECONSTITUTED AND RELATED SYNTHESIS PROCESS

[0003] DESCRIPTION

[0004] The present invention refers to a kit for the reconstitution of a cell-free biomedical device for use in regenerative medicine, to a cell-free biomedical device for use in regenerative medicine, and to a process for the synthesis of a cell-free biomedical device for use in regenerative medicine.

[0005] Prior art

[0006] For about a decade we have been hearing a lot about cell-free therapy based on a secretome for the repair of damaged organs and tissues. Secretome means the set of soluble factors secreted by the cells. In particular, the secretome of mesenchymal stromal cells (MSC) is considered a booster of regenerative medicine, a mixture of bioactive factors capable of stimulating endogenous repair processes (Mesenchymal Stem Cells: Time to Change the Name! - Stem Cells Translational Medicine, Volume 6, Issue 6, June 2017, Pages 1445-1451; Mesenchymal Stem Cell Secretome: Toward Cell-Free Therapeutic Strategies in Regenerative Medicine” - Int. J. Mol. Sci. 2017, 18(9), 1852). The secretome-based approach is considered safer than conventional cell transplantation because it would limit the potential risks related to the administration of cells (tumorigenicity, infections, immune reactions). The advantages of cell-free therapy include greater versatility of the secretome which can be produced in large quantities well in advance, stored and divided into ready-to-use batches (“Stem cells as drug delivery methods: Application of stem cell secretome for regeneration ” - Advanced Drug Delivery Reviews Volumes 82-83, March 2015, pages 1-11). At present, secretome-based therapy is in the experimental phase and comprises numerous registered clinical trials.

[0007] Critical aspects of the secretome-based therapy concern the stability and half-life of the bioactive factors contained therein (“Stem cell secretome, regeneration, and clinical translation: a narrative review ” - Ann Transl Med. 2021 Jan; 9(1): 70) on which the therapeutic efficacy of the secretome itself depends. Furthermore, since it is a soluble therapeutic agent, its administration requires some precautions that determine the way in which the soluble factors are “presented” to the damaged tissue, influencing the effectiveness of the treatment (“The stem cell secretome and its role in brain repair” - Biochimie Volume 95, Issue 12, December 2013, Pages 2271-22855). For example, the presence of a support or release system that guarantees the prolonged or controlled release of the secretome by increasing the contact time with the damaged tissue / organ. At the same time, the release system guarantees “stability” of the secretome by offering protection from clearance and in vivo enzymatic degradation (Polymers for Drug Delivery Systems - Annual Review of Chemical and Biomolecular Engineering Vol. 1:149-173 (August 2010); Hydrogels for protein delivery in tissue engineering - J Control Release 2012 Jul 20; 161 (2) .680-92).

[0008] Known release systems include hydrogels, three-dimensional hydrophilic materials with great affinity for water but which are insoluble due to the presence of intermolecular bonds that influence their physical and chemical properties (Growth factor delivery from hydrogel particle aggregates to promote tubular regeneration after acute kidney injury - J Control Release 2013 May 10;167(3):248-55,‘ Heparin desulfation modulates VEGF release and angiogenesis in diabetic wounds - J Control Release - 2015 Dec 28; 220 (PtA): 79-88).

[0009] Due to their biocompatibility and swelling capacity, hydrogels find wide application in regenerative medicine and drug delivery. Generally, hydrogels based on natural polymers are preferred over hydrogels based on synthetic materials and are currently used in the treatment of skin wounds (wound healing) (Functional Hydrogels as Wound Dressing to Enhance Wound Healing - ACS Nano. 2021 Aug 24;15(8):12687-12722; Local injection of high-molecular hyaluronan promotes wound healing in old rats by increasing angiogenesis - Oncotarget. 2018; 9:824 l-8252)and in cartilage regeneration (In situ forming hydrogels of new amino hyaluronic acid / benzoyl-cysteine derivatives as potential scaffolds for cartilage regeneration - Soft Matter Issue 18, 2012; In situ forming hydrogels of hyaluronic acid and inulin derivatives for cartilage regeneration - Carbohydr Polym. 2015 May 20; 122: 408-16).

[0010] Natural polymers include glycosaminoglycans (GAGs), known for their contribution to the regulation of the biological functions of the extracellular matrix (ECM), including the physical and mechanical regulation of connective tissues and cellular activity. Through specific epitopes, GAGS interact with growth factors and chemokines in the tissue microenvironment, controlling their accumulation and diffusion within cells and maintaining their concentration gradients in the ECM (Regulation of protein function by glycosaminoglycans— as exemplified by chemokines - Annu Rev Biochem - 2005;74:385-410). Hyaluronic acid (HA) is a non-sulfurised GAG and one of the major constituents of the ECM. HA offers a multitude of sites available for chemical crosslinking procedures that create covalent bonds among the macromolecules by stabilizing the structure of the HA itself. Chemical cross-linking was used to reduce the in vivo enzymatic degradation of an HA hydrogel and slow the diffusion of bioactive molecules loaded into the hydrogel itself to control its release (Hyaluronic acid-based hydrogels: from a natural polysaccaride to complex networks - Soft Matter, Volume 8, Issue 12), March 2012, Pages 3280- 3294). Due to its versatility, HA is widely used in tissue engineering and regenerative medicine applications (Molecular engineering of glycosaminoglycan chemistry for biomolecule delivery - Acta Biomaterialia, Volume 10, Issue 4, April 2014, Pages 1705-1719). Along with HA, the sulfurised GAG eparan sulphate (ES) is also known for its use in regenerative medicine thanks to its ability to mimic characteristics of the ECM (Materials Science and Design Principles of Growth Factor Delivery Systems in Tissue Engineering and Regenerative Medicine - Advanced Healthcare Materials - 06.11.2018- Citations: 108). Heparin (EP) is a poorly abundant constituent of the ECM and is considered an analogue of the ES. In fact, despite having structural differences (different molecular mass, differences in uronic acid residues), EP and ES have a similar ability to interact with various soluble proteins, including growth factors and chemokines, influencing numerous pathophysiological processes. In this regard, more than 22 proteins belonging to the fibroblast growth factor (FGF) family classified as “heparin binding proteins” (HBP) have been identified.

[0011] The use of biomaterials in cell delivery (Comparison of lignin derivatives as substrates for laccase-catalysed scavenging of oxygen in coatings and films - J Bio Eng. 2014 Jan 2, 8(l):l)and for tissue engineering applications is widely documented (Mesenchymal Stem Cells in Combination with Hyaluronic Acid for Articular Cartilage Defects- Scientific Reports, Article number: 9900 (2018); Combinations of Hydrogels and Mesenchymal Stromal Cells (MSCs) for Cartilage Tissue Engineering-A Review of the Literature - Gels. 2021 Nov 16;7(4):217). Conversely, the integration of biomaterials with secretome to ensure a slow release of the secretome itself is a rather recent approach that dates back to recent years. HA-based hydrogels integrated with MSC secretome for regenerative medicine applications are described in the literature (Hyaluronic Acid Hydrogel Integrated with Mesenchymal Stem Cell-Secretome to Treat Endometrial Injury in a Rat Model of Asherman's Syndrome — Advanced Healthcare materials Volume 8, issue 14, 25 / 07 / 2019; A nanocomposite hydrogel delivery system for mesenchymal stromal cell secretome - Stem Cell Research & Therapy - Article number: 205 (2020); Hyaluronic Acid Hydrogel Microspheres for Slow Release Stem Cell Delivery- ACS Biomater Sci Eng. 2021 Aug 9;7(8):3754-3763).

[0012] However, some critical aspects of secretome-based therapy remain unresolved, especially with regard to the stability and half-life of the bioactive factors contained therein, on which the therapeutic efficacy of the secretome itself depends.

[0013] We therefore feel the need to have a support for the secretome that allows to modulate the release of the bioactive factors contained therein so that the treatment of the damaged tissue can be more targeted and accurate, depending also on the type of tissue to be treated. Chronic skin wounds represent a major medical problem. In the US alone, about 6.5 million patients are affected at a cost of about $25 billion per year. In general, chronic wounds arise as a result of pathologies such as diabetes or vasculitis, or as complications of surgical wounds. To date, there are three treatments for skin wound healing approved by the FDA, two of which consist of the skin substitutes Apligraf and Dermagraft, while the third one is represented by Regranex, based on becaplermin or human platelet lysate (PDGF-BB)(£ fecAs of Topical Application of CHF6467, a Mutated Form of Human Nerve Growth Factor, on Skin Wound Healing in Diabetic Mice - Journal of Pharmacology and Experimental Therapeutics November 2020, 375 (2) 317- 331).

[0014] In general, most of the commercially available skin substitutes are elaborate and expensive, often failing to attach well to the wound or breaking. Furthermore, they can be potentially infected and are capable of triggering rejection reactions.

[0015] Document “ Preparation and characterization of a chitosan / galactosylated hyaluronic acid / heparin scaffold for hepatic tissue engineering" (Fan Jinyong et al. Journal of Biomaterials Science. Polymer edition, vol.28, no.6) describes a process which involves immersing the chitosan (CS) / galactosylated hyaluronic acid (GHA) / heparin scaffolds in an epidermal growth factor (EGF) solution, followed by freezing and lyophilization. Upon reconstitution of the dried biomaterial, the subsequent freezing and lyophilization steps could potentially impact the stability of EGF, due the significant temperature fluctuations and exposure to freeze-drying conditions. This susceptibility may have a more pronounced effect on a multifactorial product, such as the secretome, compared to a single factor-based system.

[0016] Document “Aerogel sponges of silk fibroin, hyaluronic acid and heparin for soft tissue engineering: composition-properties relationship" (Najberg Mathie et al; Carbohydrate polymers, applied science publishers, LTD Barking, GB, vol.237) discloses frozen sponges which were rehydrated with PBS, and excess liquid was removed with paper. Subsequently, the hydrated sponges underwent sterilization before adding the chemokine SDF-1 alpha. This process raises significant concerns regarding the stability of the material, as the hydrogel was formed prior to sterilization. Indeed rehydrating the sponge to form a hydrogel before sterilization could potentially increase the risk of contamination.

[0017] The task of the present invention is thus to provide a kit for the reconstitution of a cell-free biomedical device for use in regenerative medicine, to provide a cell-free biomedical device for use in regenerative medicine, and to provide a process for the synthesis of a cell-free biomedical device for use in regenerative medicine that enable the described drawbacks of the prior art to be overcome.

[0018] Within the scope of this technical task, an object of the present invention is to provide a kit for the reconstitution of a cell-free biomedical device that is ready-to-use and easy to manage.

[0019] Another object of the present invention is to provide a kit for the reconstitution of a cell-free biomedical device that allows it to be preserved and stored until it is used.

[0020] A further object of the present invention is to provide a biomedical device that is easy to apply and inexpensive.

[0021] Another object of the present invention is to provide a biomedical device that is safe.

[0022] Yet another object of the invention is to provide a process for the synthesis of a cell- free biomedical device that allows the release of the bioactive factors contained in the secretome to be effectively modulated based on the type of treatment to be made.

[0023] These and other objects of the present invention are achieved by means of the realization of a kit for the reconstitution of a cell-free biomedical device for use in regenerative medicine in accordance with claim 1.

[0024] Advantageously, in the present invention as claimed, comparing with the teachings of the prior art mentioned above “ Preparation and characterization of a chitosan / galactosylated hyaluronic acid / heparin scaffold for hepatic tissue engineering”, the secretome is separately harvested, processed and stored for immediate use, avoiding any additional processing that could increase risks of instability and denaturation. Subsequently, the secretome is added to the dried biomaterial just before administration, thereby avoiding the need for additional manipulation.

[0025] Preferably the biomaterial in lyophilised form and the secretome are sterilized.

[0026] This is a great advantage comparing the present invention with the prior art, for instance the teachings disclosed in document "'’Aerogel sponges of silk fibroin, hyaluronic acid and heparin for soft tissue engineering: composition-properties relationship”. Indeed according to the present invention the kit formulation comprises a sterile and dried biomaterial and a sterile secretome which can be reconstituted just before their application on a patient.

[0027] The innovative aspect of the present invention lies in the ability to prepare the reconstituted formulation extemporaneously. Packaging the biomaterial in a dried state, which could even be sterilized in its final packaging, and reconstituting the SECR-hydrogel just before application, represents a novelty in the field of regenerative therapies based on secretome-laden biomaterials, and offers an advantage over the existing examples.

[0028] The present invention also refers to a process for the synthesis of a cell-free biomedical device for use in regenerative medicine comprising the following steps:

[0029] Synthesis of an intermediate product starting from at least one amino derivative of hyaluronic acid, with an average molecular weight comprised in the range 50-1000 kDa, with functionalisation in amino groups ranging from 25 to 50 mol% with respect to the repeating units of hyaluronic acid;

[0030] Cross-linking of said at least one amino derivative of hyaluronic acid after dispersion thereof in an aqueous medium to obtain a hydrogel;

[0031] Lyophilisation of said hydrogel;

[0032] Heparinisation of said lyophilisate until obtaining a biomaterial based on heparin and said amino derivative of hyaluronic acid; Integration of said lyophilised biomaterial with secretome containing growth factors and chemokines collected from cultured human mesenchymal stromal cells (MSC).

[0033] In accordance with a second embodiment of the invention, the process for the synthesis of a cell- free biomedical device for use in regenerative medicine comprises the following steps:

[0034] Synthesis of an intermediate product starting from at least one amino derivative of hyaluronic acid, with average molecular weight comprised between 50 and 1000 kDa with functionalisation in amino groups ranging from 25 to 50 mol% with respect to the repeating units of hyaluronic acid;

[0035] Cross-linking of said at least one amino derivative of hyaluronic acid after dispersion thereof in an aqueous medium in the presence of heparin to obtain a heparinised hydrogel;

[0036] Lyophilisation of said heparinised hydrogel until obtaining a biomaterial based on heparin and said amino derivative of hyaluronic acid;

[0037] Integration of said lyophilised biomaterial with secretome containing growth factors and chemokines collected from cultured human mesenchymal stromal cells (MSC).

[0038] Other salient aspects of the invention are set forth in the following dependent claims.

[0039] Further characteristics and advantages of the invention will more fully emerge from the description of a preferred but not exclusive embodiment of the kit for the reconstitution of a cell-free biomedical device for use in regenerative medicine, of the cell-free biomedical device and of the process for the synthesis of the cell-free biomedical device illustrated by way of non-limiting example in the appended drawings.

[0040] Description of the drawings Figure 1 shows a 12-well plate containing the biomaterial based on secretome-rehydrated hyaluronic acid (HA) and heparin (EP).

[0041] Figures 2 A, 2B and 2C respectively show the kinetics of the release from the hydrogel of the growth factors VEGF-A and HGF (Fig. 2A) and chemokines IL-6, IL-8, SDF-la (Fig. 2B), GRO- a and MCP-1 (Fig. 2C) contained in the secretome. Briefly, the concentrated secretome was loaded into the dehydrated biomaterial. The assay was carried out at 37 °C in a humidified atmosphere with 5% CO2. The amount of soluble factors released at each time-point quantified with Luminex and expressed in pg / ml as a function of time (mean ± SD, n = 3). Grey curve: release of the factors from the EPi% biomaterial; black curve: release of the factors from the EP2i% biomaterial.

[0042] Figure 3 shows the functionality of the secretome released from the hydrogel obtained after reconstitution of the heparinised biomaterials HA / EPi% and HA / EP21 % with the secretome. In vitro angiogenesis assay (tube formation assay). Representative images of HUVEC (human umbilical vein endothelial cells) plated on the Matrigel acquired after 6 hours in culture. (A) Positive control, HUVEC resuspended in the secretome before its integration into the biomaterial (score 4). (B) HUVEC resuspended in the secretome released from the HA / EPi% biomaterial on day 6 (early representative time-point) (score 4). (C) HUVEC resuspended in the secretome released from the HA / EPi % biomaterial on day 20 (late representative time-point) (score 0). (D) HUVEC resuspended in the secretome released from the HA / EP2i% biomaterial on day 6 (score 3). (E) HUVEC resuspended in the secretome released from the HA / EP2i% biomaterial on day 20 (score 0).

[0043] Figure 4 shows the functionality of the secretome released from the hydrogel. Real-time cell migration assay recorded with the XCELLigence for 7 hours. Secretome prior to addition to the biomaterial and serum-free alpha-MEM culture medium were used as a positive and negative control. (A) Fibroblast migration induced by the secretome released from thei% HA / EP biomaterial measured at different time-points. (B) Absence of fibroblast migration performed with a secretome released from the2i% HA / EP biomaterial. Abbreviations: Pos Ctrl, positive control; neg Ctrl, negative control; EP, heparin.

[0044] Figure 5A shows the lyophilisedi% HA / EP biomaterial prior to secretome addition.

[0045] Figure 5B shows the experimental plan with the treatments.

[0046] Figure 6 shows the treatment of the pressure ulcer. The images are representative of the ulcers on day 3, 12, 21 and 28 after the injury. On day 12, an evident reduction of erythema can be observed in the ulcers treated with hydrogel / secretome compared to hydrogel alone and vehicle groups.

[0047] Figure 7A shows a graph representing the residual open area on day 28 of the treatment referred to in figure 6.

[0048] Figure 7B shows a graph representing the residual de-epithelialised area on day 28 of the treatment referred to in figure 6.

[0049] Figure 8 shows an image of the residual open area of figure 7A and the residual de-epithelialised area of figure 7B.

[0050] Figure 9 shows the number of open ulcers at the end of the treatment (day 28) referred to in Figure 6. Abbreviations: seer, secretome; CO, umbilical cord; DF, foetal dermis. One-way ANOVA and Tukey's post-hoc treatment vs vehicle group, *p<0.05; **p<0.01; ***p<0.001; ns, not significant. Figure 10 A, 10 B, 10 C show the histological analysis of the treated ulcers made on day 28. (A) Representative photomicrographs of H&E stained ulcers taken at the equator of the lesion. The arrows in the vehicle group indicate the granulation step in the central area of the lesion. (B) The same images taken at a higher magnification. (C) Morphometric evaluation of the epidermal thickness measured at the end of the treatments. One-way ANOVA and Dunnet's post-hoc treatment vs vehicle group, *p <0.05; **p<0.01; ns, not significant. Magnification bars: 500 pm (A); 100 pm (B). Abbreviations: seer, secretome; CO, umbilical cord; DF, foetal dermis.

[0051] Figures 11A and 11B show the neo- vascularization of the treated skin area analysed by immunofluorescence and anti-lamin-IR antibody. (A) Representative photomicrographs of the treated ulcers and the vehicle corresponding to the equator of the lesion. The arrows indicate capillaries. (B) Morphometric evaluation of the anti-laminin antibody positive area (immunoreactive area), expressed as a fraction of the immunoreactive area with respect to the total area considered (capillary density). One-way ANOVA and Dunnet's post-hoc treatment vs vehicle group, *p<0.05; ns, not significant. Abbreviations, seer: secretome; CO, umbilical cord; DF, foetal dermis.

[0052] DETAILED DESCRIPTION OF THE INVENTION

[0053] The present invention relates to a newkit for the reconstitution of a cell-free biomedical device for use in regenerative medicine, characterised in that it comprises a biomaterial in lyophilised form based on hyaluronic acid (HA) and heparin (EP) that is rehydrated by secretome containing growth factors and chemokines, collected from cultured human mesenchymal stromal cells (MSC).

[0054] For greater descriptive clarity, the term biomaterial means a lyophilised (dried) material, while the term hydrogel means a hydrated biomaterial.

[0055] Advantageously, the lyophilised biomaterial is frozen until the time of reconstitution.

[0056] Advantageously, the secretome is also frozen until the time of reconstitution.

[0057] Advantageously, the cell- free biomedical device is ready to use after reconstitution. Advantageously, the biomaterial in lyophilised form and the secretome are sterilized.

[0058] Preferably, the weight content of heparin in the lyophilised biomaterial is modulated for a controlled release of the growth factors and chemokines present in said secretome.

[0059] In particular, the heparin is contained in a concentration range of 1-37.5% by weight in the lyophilised biomaterial, more preferably in a concentration range of 1-21% by weight in the lyophilised biomaterial.

[0060] In particular, the hyaluronic acid is present in a concentration range of 62.5-99% by weight in the biomaterial, more preferably in a concentration range of 79-99% by weight in the lyophilised biomaterial. The present invention further relates to a cell-free biomedical device ready to use in regenerative medicine characterised in that it is reconstituted with the kit described above.

[0061] Finally, the present invention also relates to a process for the synthesis of a cell-free biomedical device ready to use in regenerative medicine.

[0062] Advantageously, the present invention provides two different processes for the synthesis of the cell-free biomedical device ready to use in regenerative medicine.

[0063] In particular, according to a first embodiment of the invention, the process for the synthesis of a cell-free biomedical device ready to use in regenerative medicine comprises the following steps:

[0064] Synthesis of an intermediate product starting from at least one amino derivative of hyaluronic acid, with an average molecular weight comprised in the range 50-1000 kDa, with functionalisation in amino groups ranging from 25 to 50 mol% with respect to the repeating units of hyaluronic acid;

[0065] Cross-linking of said intermediate product obtained from at least one amino derivative of hyaluronic acid after dispersion thereof in aqueous medium to obtain a hydrogel;

[0066] Lyophilisation of said hydrogel;

[0067] Heparinisation of said lyophilised hydrogel until obtaining a biomaterial based on heparin and said amino derivative of hyaluronic acid;

[0068] Integration of said biomaterial with a secretome containing growth factors and chemokines collected from cultured human mesenchymal stromal cells (MSC).

[0069] Advantageously, the process, according to the first embodiment, provides, between the step of heparinising said hydrogel and the step of integrating said biomaterial, for a further step of sterilizing said lyophilised biomaterial.

[0070] In accordance with a second embodiment of the invention, the process for the synthesis of a cell- free biomedical device ready to use in regenerative medicine comprises the following steps: Synthesis of an intermediate product starting from at least one amino derivative of hyaluronic acid, with average molecular weight comprised between 50 and 1000 kDa with functionalisation in amino groups ranging from 25 to 50 mol% with respect to the repeating units of hyaluronic acid;

[0071] Cross-linking of said intermediate product obtained from at least one amino derivative of hyaluronic acid after dispersion thereof in aqueous medium in the presence of heparin to obtain a heparinised hydrogel;

[0072] Lyophilisation of said heparinised hydrogel until obtaining a biomaterial based on heparin and said amino derivative of hyaluronic acid;

[0073] Integration of said lyophilised biomaterial with secretome containing growth factors and chemokines collected from cultured human mesenchymal stromal cells (MSC).

[0074] Advantageously, the process, also in the second embodiment, provides, between the step of heparinising said hydrogel and the step of integrating said biomaterial, for a further step of sterilizing said lyophilised biomaterial

[0075] The heparinisation step, according to the first embodiment of the synthesis process, is performed by modulating the content by weight of EP added to said intermediate product for a controlled release of the growth factors and chemokines present in said secretome.

[0076] The cross-linking step, in accordance with the second embodiment of the synthesis process, is performed by modulating the weight content of EP added to said intermediate product for a controlled release of the growth factors and chemokines present in said secretome.

[0077] The heparinisation step preferably comprises a step of lyophilising the heparinised biomaterial prior to the integration step.

[0078] According to the present invention, the biomedical device used in particular for regenerative medicine applications and consisting of a biomaterial based on HA and EP integrated with secretome, has been designed so that it can be reconstituted at the time of application by adding an appropriate volume of secretome to the biomaterial that will be in its lyophilised form. After reconstitution, the device will absorb the entire volume of secretome and can be placed on the tissue to be repaired.

[0079] Essentially, compared to existing methods, the innovation of the present invention lies in the disclosure of a ready-to-use secretome-loaded hydrogel, formulated prior to its application onto the wound bed. By reconstituting a dried HA-based biomaterial with an aqueous dispersion of secretome, we are able to overcome serious challenges related to the stability and sterility of the secretome during processing. With advancement, respect to prior art, holds the potential to expedite the translation of our technology to the clinic.

[0080] In particular, the cell-free biomedical device obtained by means of the kit is a single-use device that is reconstituted at the time of application by adding the right volume of secretome to the lyophilised biomaterial.

[0081] Particularly the secretome is in the form of an aqueous dispersion.

[0082] Once rehydrated, the biomaterial forms a semi-permanent hydrogel suitable for applications that do not require complete degradation of the biomaterial. The device applied to the wound acts as a hydrated patch capable of controlling / prolonging the release of the soluble proteins (growth factors and chemokines) of the secretome by exploiting the ability of the hydrogel to retain these soluble factors. This capacity will depend on the various parameters that influence the diffusion of the soluble proteins through the porous structure of the hydrogel itself, but above all on the affinity of the molecules for HA and EP.

[0083] In fact, thanks to its peculiar composition, the device is capable of controlling the release over time of the bioactive factors contained in the secretome. In particular, thanks to the different biological affinity of HA and EP for the growth factors and chemokines contained in the secretome, it is possible to regulate the relative amount of HA and EP and therefore modulate the affinity of the secretome for the biomaterial. In particular, since EP, compared to HA, has a higher affinity towards most of the soluble proteins of the secretome, it will be possible to control the release of the secretome by modulating the percentage of EP contained in the biomaterial. In this way, a controlled release of soluble factors will be obtained at the damaged tissue. It is therefore clear that the efficiency of the biomaterial to retain the soluble molecules of the secretome depends in particular on the percentage of EP contained in the biomaterial itself.

[0084] In particular, the biomaterial based on HA and EP is produced by a chemical cross-linking reaction starting from an amino derivative of HA to which an amount by weight of EP comprised between 1 and 37.5% with respect to the total weight of the lyophilised biomaterial is bound.

[0085] According to some specific solutions of the invention, the secretome integrated into the HA / EP composite biomaterial is collected from cultured human MSCs.

[0086] In particular, the human MSCs used herein are MSCs derived from foetal dermis (DF-MSC) and from umbilical cord (CO-MSC).

[0087] The present invention will now be further illustrated by the following examples, which are not intended to be limiting.

[0088] EXAMPLES

[0089] Example 1 : hyaluronic acid / heparin (HA / EP) device synthesis procedures

[0090] Synthesis of the amino derivative of HA

[0091] 1 g of HA (with an average molecular weight comprised between 50-1000 kDa) is solubilised in 50 ml of distilled water and ethylenediamine (2.5 ml or 5.0 ml) is added to the solution obtained. The pH of the solution is brought to 6.8, then 1.4 g of hydroxybenzotriazole (HOBt) (solubilised in 10 ml of water: dimethylsulfoxide 1:1 mixture) and 1.6 g of l-ethyl-3-[3- (dimethylamino)propyl] -carbodiimide (EDC) are added. The reaction is carried out at a temperature of 25 °C or 40 °C for 24 or 72 hours while maintaining the pH at 6.8. The product is purified by dialysis (cut-off 12-14 kDa) against a 5% w / v NaCl solution for 48 hours, then against water for a further 72 hours and isolated by lyophilisation. The variation in temperature and reaction time allows different derivatives to be obtained with a functionalisation in amino groups ranging from 25 to 50 mol% with respect to the HA repeating units.

[0092] First embodiment of the process for producing the biomedical device HA / EP

[0093] 200 mg of the lyophilised HA-amine derivative (25 or 50 mol% functionalisation) and 20 mg of 1000 kDa HA (HMW) are mixed and dispersed in 4 ml of MES buffer pH 5.5 at 40 °C for at least 24 hours. EDC and NHS in varying amounts depending on the amino derivative used (Table 1) are added to the dispersion obtained and the same is placed in a Petri dish and incubated at 37 °C for 24 hours. The obtained hydrogel is frozen at -80 °C and lyophilised to obtain a spongy structure which is rehydrated, washed with water and dried again by lyophilisation.

[0094] Table 1. Amount range of 25% and 50 mol % HA, HA-HMW, EDC and NHS (in 4 ml of water) used for the production of the hydrogel.

[0095] For the heparinisation of the biomaterial, the EP is solubilised in MES buffer pH 5.5 at a concentration of 2% w / v in the presence of EDC / NHS (Table 2). After 2 hours of incubation at 37 °C, the solution obtained is used to rehydrate the biomaterial based on the amine derivative of HA. After 24 hours of incubation at 37 °C, the biomaterial is washed with water and lyophilised. The amount of bound EP will vary depending on the amine derivative used for the preparation of the biomaterial, the degree of cross-linking of the biomaterial and the degree of heparinisation to be obtained. Table 2 shows the amounts of EP used to treat 100 mg of biomaterial obtained starting from the two amino derivatives of HA. Table 2. Amount of EP, EDC and NHS used for the heparinisation of the biomaterial based on the amine derivative of HA

[0096] Second embodiment of the process for producing the biomedical device HA / EP 100 mg of the lyophilised HA-amine derivative (25 or 50 mol% functionalisation) and 10 mg of

[0097] 1000 kDa HA (HA-HMW) are mixed and dispersed in 2 ml of MES buffer pH 5.5 at 40 °C for at least 24 hours. The dispersion is then mixed with a solution of EP and EDC / NHS in MES pH 5.5. gelling is carried out at 40 °C for at least 24 hours. The various compositions of the hydrogelforming dispersions are shown in Table 3. The hydrogel obtained is frozen, freeze-dried, washed with water and dried again.

[0098] Table 3. Composition of the hydrogel- forming dispersions in the presence of EP

[0099] Example 2: HA / EP biomaterial production with 21% EP on total weight of the device

[0100] 100 mg of 25 mol% functionalised HA 200 kDa amine derivative and 10 mg of HA 1000 kDa

[0101] (HA-HMW) are dispersed in 1.5 mL of 0.5M MES buffer (pH 5.5). The dispersion is placed in an orbiting incubator at 37 °C overnight before adding 30 mg of heparin solubilised in 200 pL of 0.5M MES buffer (pH 5.5). 24 mg of EDC in 500 pL of 0.5M MES buffer (pH 5.5) and 14 mg of NHS are added to the polysaccharide dispersion and the same is placed in a Petri dish at room temperature for 12 hours to complete the gelling process. The obtained hydrogel is lyophilised to obtain a spongy structure which is rehydrated, washed with distilled water and dried again by lyophilisation.

[0102] Example 3: HA / EP biomaterial production with 1% EP with respect to the total weight of the device

[0103] 100 mg of 25mol % functionalised HA 200 kDa amine derivative and 10 mg of HA 1000 kDa (HA-HMW) are dispersed in 1.5 mL of 0.5M MES buffer (pH 5.5). The dispersion is placed in an orbiting incubator at 37 °C overnight before adding 1.3 mg of heparin solubilised in 200 pL of 0.5M MES buffer (pH 5.5). 24 mg of EDC in 500 pL of 0.5M MES buffer (pH 5.5) and 14 mg of NHS are added to the polysaccharide dispersion and the same is placed in a Petri dish at room temperature for 12 hours to complete the gelling process. The obtained hydrogel is lyophilised to obtain a spongy structure which is rehydrated, washed with distilled water and dried again by lyophilisation.

[0104] Example 4: cell source; secretome collection, concentration, quantification and storage

[0105] The MSCs used in the study are derived from the cell bank of the “Fondazione Ri.MED and IRCCS ISMETT” and were isolated from the foetal dermis (DF-MSC) of skin biopsies resulting from therapeutic abortions according to a protocol approved by the Institutional Research Review Board of IRCCS ISMETT (IRRB / 00 / 15) and following an informed consent signed by the donor (Small Extracellular Vesicles from Human Fetal Dermal Cells and Their MicroRNA Cargo: KEGG Signaling Pathways Associated with Angiogenesis and Wound Healing. Stem Cells Int. 2020 Aug 13;2020:888937). Similarly, umbilical cord MSCs (CO-MSC) were obtained according to a protocol approved by the Institutional Research Review Board of IRCCS ISMETT (IRRB / 18 / 14) and following informed consent signed by the donors (Extracellular Vesicle-Derived microRNAs of Human Wharton 's Jelly Mesenchymal Stromal Cells May Activate Endogenous VEGF-A to Promote Angiogenesis. Int J Mol Sci. 2021 Feb 19;22(4):2045). The MSCs were cultured in DMEM medium with 10% foetal bovine serum (FBS) and used at established passages (p2-p8). Immunophenotypic characterization of the MSCs, performed by flow cytometry, confirmed the typical immunophenotype of MSCs (CD90, CD 105 and CD73 positive, CD34, CD45 and HLA- DR negative) (data not shown). At 85% cell confluence, the culture medium was eliminated and the cells were rinsed with phosphate buffer (PBS) before adding the strictly serum-free secretome collection medium (alpha-MEM). The secretome was collected in the form of conditioned medium after 24 hours, centrifuged at 2000 x g to remove debris, aliquoted and stored at -80 °C. The soluble factors were dosed by Luminex xMAP technology (simultaneous detection of multiple analytes) and considering a panel of growth factors and chemokines with a role in wound healing including vascular endothelial growth factor (VEGF)-A, hepatocyte growth factor (HGF), interleukin-6 (IL- 6), interleukin-8 (IL-8), derived stromal factor (SDF)-l alpha, growth-related oncogene (GRO)- alpha and monocyte chemoattractant protein (MCP-1). The concentration of soluble factors was calculated with the software coupled to the Luminex 200 instrument and expressed in pg / ml. Secretomes from different samples having a similar concentration of growth factors and chemokines were pooled and subsequently concentrated 5-fold by volume by ultrafiltration (Amicon Ultra 15 ml and 3 kDa cutoff filters). Concentrated secretome batches were stored at - 80 °C until the time of use. Table 4 shows the composition of the non-concentrated and 5-fold concentrated secretome measured with Luminex considering a panel of growth factors and chemokines associated with angiogenesis and wound healing. A significant enrichment of the concentration of the various factors considered can be noted.

[0106] Table 4. Luminex quantification of the DF-MSC secretome prior to its addition to the HA / EP biomaterial The values reported (mean ± SD) represent the concentration of the bioactive factors in the nonconcentrated secretome (n = 6) compared to the concentration in the 5-fold concentrated secretome

[0107] (n = 6); *p < 0.05; **p < 0.009 (Student's t-test). Abbreviations: DF: Foetal dermis.

[0108] Example 5: kinetics of the release of the secretome from the HA / EPi% and HA / EP2i% materials For the release kinetic studies, the dried devices EPi% and EP2i% were placed on sterile 12-well plastic plates and loaded with 200 pl of concentrated secretome, while 600 pl of PBS were added to the outside (Fig. 1). Once reconstituted with secretome, the device was maintained at 37 °C in a humidified atmosphere with 5% CO2. The secretome released was collected at different timepoints (24 hours, 48 hours and then every 3 days for a total of 30 days). To ensure the same volume, the secretome collected at each time-point was immediately restored with an equal volume of PBS.

[0109] Finally, the various secretome time-points released were quantified with Luminex. To calculate the cumulative release of each soluble factor, the various time-points were added and the value obtained was compared with the amount of the factor present in the secretome before being added to the biomaterial. As represented in Figures 2A and 2B, the kinetics of the release of the secretome from the HA / EP1% biomaterial indicated a gradual release of the bioactive factors with a peak release after 6 days for VEGF-A and after 10 days for HGF (Fig. 2A), after 18 days for GRO-a, after 16 days for MCP-1 and after 12 days for SDF-la, IL-6 and IL-8 (Fig. 2B, 2C). The kinetics of the release of the secretome from the HA / EP22% biomaterial revealed a general retention of soluble factors. In particular, a marked reduction in the release profile was observed for all the chemokines considered (Fig. 2B, 2C). As regards the growth factors, the retention by the biomaterial was lower (VEGF-A) or even zero (HGF) (Fig. 2A).

[0110] Example 6: functionality of the secretome released from the HA / EPi% and HA / EPzi% biomaterials

[0111] The secretome released from the hydrogel was tested in vitro for functionality (ability to induce biological responses in the target cells). The individual aliquots released at each time-point were tested in an angiogenesis assay (“tube formation assay”) using human umbilical vein endothelial cells (HUVEC) as target cells. In short, about 10,000 HUVEC were kept (cultured in the absence of serum) for 3 hours, plated on the Matrigel and maintained at 37 °C in a humidified atmosphere with 5% CO2 (Small Extracellular Vesicles from Human Fetal Dermal Cells and Their MicroRNA Cargo: KEGG Signaling Pathways Associated with Angiogenesis and Wound Healing. Stem Cells Int. 2020 Aug 13;2020:888937). The formation of the “tubes” (capillary-like structures) induced by the secretome was monitored under the inverted microscope by assigning a score from 0 to 5 to each pattern according to the specifications of the assay (Millipore). In short, the positive control (HUVEC resuspended in the secretome before being integrated into the hydrogel reached the “close polygons” pattern (score 4) (Fig. 3A), same as the pattern reached by the HUVECs resuspended in the secretome released from thei% HA / EP biomaterial at early time points (Fig. 3B, representative time-point day 6). The “individual cells, well separated” pattern (score 0, absence of angiogenesis) was observed for all late time-points (Fig. 3C, representative time-point day 20). For the HA / EP2i% biomaterial, the maximum score reached at early time-points was score 3, corresponding to the “sprout of new capillary tubes” pattern (Fig. 3D), while the “individual cells, well separated” pattern (score 0) can be found at the late time-points indicating the absence of angiogenesis (Fig. 3E).

[0112] The functionality of the secretome was also assayed by means of cell migration measured with the xCELLigence Real-Time Cell Analyzer (RTCA) (Small Extracellular Vesicles from Human Fetal Dermal Cells and Their MicroRNA Cargo: KEGG Signaling Pathways Associated with Angiogenesis and Wound Healing - Stems Cells Int. 2020 Aug 13; 2020:8889379)and using fibroblasts as target cells. The system allows to record in real-time the active passage of cells (chemotaxis) from the upper chamber to the lower chamber which are separated from each other by an electrode (all three constituting the CIM plate) that detects the electrical impedance generated by the passage of cells. Briefly, the secretome (chemoattractant) was loaded in the lower chamber, while 30,000 starved fibroblasts were resuspended in serum-free medium and loaded in the upper chamber. The CIM plate was assembled to the instrument and maintained at 37 °C in a humidified atmosphere with 5% CO2 and cell migration was recorded for 7 hours. The analysis was performed by means of the RTCA Software 1.2 coupled with the xCELLigence and the results obtained expressed as a cell index (CI), figure 4A shows that the aliquots of secretome released from thei% HA / EP biomaterial from 1 hour to day 6 (early time-points) induce a migratory response reaching CI values similar to the positive control (secretome-induced migration before being added to the biomaterial). Lower CI values were recorded for the late time-points (starting from day 9 onwards), until they reached values similar to the negative control (serum- free culture medium loaded in the lower chamber) indicating the absence of migration (Fig. 4A). The CI values obtained by assaying aliquots of secretome released from the2i% HA / EP biomaterial indicated the absence of cell migration for all the time-points considered (Fig. 4B) Example 7: topical application of the device consisting ofi% HA / EP biomaterial integrated with secretome for the treatment of diabetic ulcers: in vivo proof-of-concept

[0113] The functionality of the medical device was tested in an efficacy proof-of-concept on a mouse model of pressure ulcer. Lyophilised biomaterial discs and secretome batches were prepared in advance and frozen until the time of shipment to the IRET Foundation (Tecnopolo in Bologna) to which the study was commissioned. The study was conducted in accordance with the directives of the European Community Council (2010 / 63 / EU) and approved by the Ministry of Health (authorisation no. 391 / 2017-PR). The mouse model of diabetic ulcer is represented by diabetic mice (C57BL / KsJ-m+ / +Leprdb db / db) with a pressure ulcer caused by a fold of the skin placed between two magnets (12 mm in diameter and 5 mm thick) generating a pressure (hence the definition of pressure ulcer) necessary to cause local ischemia (Effects of Topical Application of CHF6467, a Mutated Form of Human Nerve Growth Factor, on Skin Wound Healing in Diabetic Mice. J Pharmacol Exp Ther. 2020 Nov;375(2):317-331; Development of a simple, noninvasive, clinically relevant model of pressure ulcers in the mouse-J Invest Surg. 2004 Jul-Aug;17(4):221- 7; Molecular mechanisms of skin wound healing in non-diabetic and diabetic mice in excision and pressure experimental wounds- Cell Tissue Res. 2022 Jun;388(3):595-613). In total, three ischemia-reperfusion (I / R) cycles of 12 hours each were caused, interspersed with 12 hours of rest. Diabetic mice exhibited blood glucose levels of >350mg / dL for the entire duration of the experiment with no difference in body weight between the various groups (data not shown). The following experimental groups were included in the study (n = 8 animals per group based on power analysis and calculation of sample size):

[0114] • control, intact animals

[0115] • dbdb mice + vehicle (untreated ulcer)

[0116] • dbdb mice + hydrogel alone

[0117] • dbdb mice + hydrogel + DF-MSC secretome • dbdb mice + hydrogel + CO-MSC secretome

[0118] Each mouse had two dorsal ulcers (n= 16 ulcers), one untreated (vehicle) and one treated. The topical application of the devices (diameter lxl cm2and thickness comprised between 0.3 and 0.5 cm) (Fig. 5A) started on day 3 after the last I / R cycle and was performed every 9 days for a total of three applications (Fig. 5B). The frequency of the treatments was established on the basis of the release kinetic experiments (example 3). The treatment started after curettage and the ulcer was covered with Tegaderm. The ulcers were inspected daily for the entire duration of the experiment (28 days). Ulcer images were taken on days 3, 12, 21 and 28 (Nikon C-Leds camera and X-Entry Alexasoft software) and the ulcer area measured with the Nis-Elements AR 3.2 software.

[0119] At the end of the treatments (day 28) the mice were killed, the left ulcers were collected for future molecular biology analyses and the right ulcers were collected for morphological analysis, half ulcer for histology and half for immunohistochemistry. For histology, the samples were fixed in 4% paraformaldehyde, included in paraffin and sections (4 pm) stained with haematoxylin eosin (H&E). The inspection of the ulcers that had taken place at different time-points (day 3, 12, 21 and 28) showed an evident reduction in skin erythema as early as 12 days after the start of the treatment and only in ulcers treated with secretome compared to hydrogel alone and vehicle groups (Fig. 6). At day 28, all treatments, including hydrogel alone, induced a significant reduction in the open wound area compared to the vehicle; however, the reduction was greater in ulcers treated with DF- MSC secretome (Fig. 6). The residual de-epithelialized area of the ulcers treated with DF-MSC secretome was significantly smaller than the vehicle group (Fig. 7A, 7B). The evaluation of the ulcers remained open at the end of the treatments (day 28) highlighted 5 open ulcers out of 16 in the mice treated with DF-MSC and CO-MSC secretome, 7 open ulcers out of 16 in the mice treated with hydrogel alone and 15 open ulcers out of 16 in the vehicle group (Fig. 9).

[0120] Effects of the treatment on the epidermal thickness The samples were fixed as above. The samples were fixed as above and the thickness of the epidermis was determined at the equator of the lesion by H&E staining. For the statistical analyses, the mean of five measurements / section and three sections / animal were considered. The data were expressed as the mean ± SEM. The Student's t-test or one-way ANOVA were used for data analysis and the results considered significant at p <0.05. Histological analysis of the ulcers at the end of the treatments (day 28) highlighted the presence of granulation tissue in the central area (arrows) in the ulcers of the vehicle and hydrogel alone groups (Fig. 10a, B), while in the ulcers treated with the secretome of both MSCs the granulation tissue was present to a lesser extent. Histological analysis revealed significantly greater epidermal thickness in the secretome-treated ulcers of both MSCs compared to the other two groups (Fig. 10C).

[0121] Effects of the treatment on capillary microcirculation (angiogenesis)

[0122] For immunofluorescence, the samples were fixed as above and cryostat sections were prepared as previously described (Effects of Topical Application of CHF6467, a Mutated Form of Human Nerve Growth Factor, on Skin Wound Healing in Diabetic Mice - Journal of Pharmacology and Experimental Therapeutics November 2020, 375 (2) 317-331). The sections were incubated with primary antibody (anti-laminin, diluted 1:150) and with secondary antibody (Cy2 Donkey antiRabbit IgG, diluted 1:100) and fitted in glycerol containing 1,4-phenylenediamine. The fluorescence images were acquired with a microscope (Nikon Eclipse E600) coupled with a camera (Q Imaging Retiga-2000RV digital CCD). The analysis was carried out with the Nis- Elements AR 3.2 software, by applying the same procedure to all the images considered. The immunoreactive area was calculated as area / fraction (percentage of the laminin-positive area considered on a total area of 400 x 300 pm). The analyses were performed blindly. Immunofluorescence with anti-laminin antibody showed a significant increase in capillary density in the ulcers treated with hydrogel / secretome of both MSCs compared to hydrogel alone and vehicle groups (Fig. 11 A). The result was confirmed by the relative quantification of the immunoreactive area (laminin-positive area) (Fig. 1 IB).

[0123] The results of the proof-of-concept show the therapeutic effect of the hydrogel / secretome medical device as a topical treatment for skin ulcers. Both treatments, hydrogel / secretome and hydrogel alone accelerated the closure of the ulcer (wound closure or re-epithelialization) with respect to the vehicle. However, the presence of the secretome would seemingly potentiate the therapeutic effect of the hydrogel. The significant reduction in erythema observed at mid-treatment (day 12) only in the ulcers treated with hydrogel / secretome compared to the hydrogel alone or vehicle suggests the anti-inflammatory properties of the secretome. Moreover, the hydrogel / secretome treatment induced a thicker epidermis and a greater vascularization of the treated area compared to the hydrogel alone or to the vehicle. In general, the results support the use of the proposed biomedical device as a pro-healing agent of diabetic skin ulcers. The device was well tolerated by the animals. The semi-permanent consistency of the hydrogel and the possibility of easily removing it from the wound bed facilitated serial applications (three applications in total).

[0124] The kit for the reconstitution of a cell- free biomedical device for use in regenerative medicine, the cell-free biomedical device and the processes for the synthesis of the cell-free biomedical device thus conceived, are susceptible of numerous modifications and variants all falling within the scope of the inventive concept described and claimed.

Claims

CLAIMS1. A kit for the reconstitution of a cell-free biomedical device for use in regenerative medicine, characterised in that it comprises a biomaterial in lyophilised form based on hyaluronic acid and heparin that is rehydrated by a secretome containing growth factors and chemokines, collected from cultured human mesenchymal stromal cells (MSC), said lyophilised biomaterial and said secretome being frozen until the time of reconstitution of said cell-free biomedical device, wherein said reconstituted biomedical device is ready to use.

2. The kit for the reconstitution of a cell-free biomedical device for use in regenerative medicine according to the preceding claim, characterised in that said biomaterial in lyophilised form and said secretome are sterilized.

3. The kit for the reconstitution of a cell-free biomedical device for use in regenerative medicine according to any preceding claim, characterised in that the weight content of heparin in the lyophilised biomaterial is modulated for a controlled release of the growth factors and chemokines present in said secretome.

4. The kit for the reconstitution of a cell-free biomedical device for use in regenerative medicine according to the preceding claim characterised in that the heparin is contained in a concentration range of 1-37.5% by weight in the lyophilised biomaterial, preferably in a concentration range of 1-21% by weight in the lyophilised biomaterial.

5. The kit for the reconstitution of a cell-free biomedical device for use in regenerative medicine according to any preceding claim, characterised in that the hyaluronic acid is present in a concentration range of 62.5-99% by weight in the biomaterial, preferably in a concentration range of 79-99% by weight in the lyophilised biomaterial.

6. A cell-free biomedical device ready to use in regenerative medicine characterised in that it is reconstituted with a kit in compliance with any preceding claim.

7. A process for the synthesis of a cell-free biomedical device ready to use in regenerative medicine comprising the following steps:Synthesis of an intermediate product starting from at least one amino derivative of hyaluronic acid, with an average molecular weight comprised in the range 50-1000 kDa, with functionalisation in amino groups ranging from 25 to 50 mol% with respect to the repeating units of hyaluronic acid;Cross-linking of said at least one amino derivative of hyaluronic acid after dispersion thereof in an aqueous medium to obtain a hydrogel;Lyophilisation of said hydrogel;Heparinisation of said lyophilisate until obtaining a biomaterial based on heparin and said amino derivative of hyaluronic acid;Integration of said lyophilised biomaterial with secretome containing growth factors and chemokines collected from cultured human mesenchymal stromal cells (MSC).

8. The process for the synthesis of a cell-free biomedical device ready to use in regenerative medicine according to claim 7, characterised in that it provides, between said step of heparinising said lyophilisate and said step of integrating said biomaterial, a further step of sterilising said lyophilised biomaterial.

9. The process for the synthesis of a cell-free biomedical device ready to use in regenerative medicine comprising the following steps:Synthesis of an intermediate product starting from at least one amino derivative of hyaluronic acid, with average molecular weight comprised between 50 and 1000 kDa with functionalisation in amino groups ranging from 25 to 50 mol% with respect to the repeating units of hyaluronic acid;Cross-linking of said at least one amino derivative of hyaluronic acid after dispersion thereof in an aqueous medium in the presence of heparin to obtain a heparinised hydrogel;Lyophilisation of said heparinised hydrogel until obtaining a biomaterial based on heparin and said amino derivative of hyaluronic acidIntegration of said lyophilised biomaterial with secretome containing growth factors and chemokines collected from cultured human mesenchymal stromal cells (MSC).

10. The process for the synthesis of a cell-free biomedical device ready to use in regenerative medicine according to claim 9, characterised in that, between said step of lyophilising said hydrogel and said step of integrating said biomaterial, a step of sterilizing said lyophilised biomaterial is provided.

11. The process for the synthesis of a cell-free biomedical device ready to use in regenerative medicine according to claim 7 characterised in that said heparinisation step is carried out by modulating the weight content of heparin added to said intermediate product for a controlled release of the growth factors and chemokines present in said secretome.

12. The process for the synthesis of a cell-free biomedical device ready to use in regenerative medicine according to claim 8 characterised in that said cross-linking step is carried out by modulating the weight content of heparin added to said intermediate product for a controlled release of the growth factors and chemokines present in said secretome.

13. The process for the synthesis of a cell-free biomedical device ready to use in regenerative medicine according to claim 7 characterised in that said heparinisation step comprises a step of lyophilising the heparinised biomaterial before the integration step.

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