Antimicrobial composition with colloidal silver nanoparticles, production method and uses of same

Abstract

The present invention relates to a bioactive scaffold comprising a silver-loaded antimicrobial composition, which acts as a therapeutic device, especially a scaffold, for wound healing and other applications related to the field of tissue engineering. The antimicrobial composition of the invention mainly comprises at least one absorbable matrix for cell-matrix and cell-cell interactions (a natural polymer), a calcium cross-linking component for forming a fibrin gel in situ, and a nanoemulsion containing colloidal silver nanoparticles as a bioactive compound to help control biofilm and infections in the wound. The structures comprising the antimicrobial composition act as dressings and means for aiding the healing of chronic wounds by assisting in infection management and / or improving the biological activity in chronic wounds.

Description

[0001] ANTIMICROBIAL COMPOSITION WITH COLLOIDAL SILVER NANOPARTICLES, METHOD OF PRODUCTION AND USES THEREOF Type of Invention

[0002] The present invention relates to the field of tissue engineering. Specifically, it relates to the field of antimicrobial compositions used in bioactive scaffolds that act as dressings, methods for producing such compositions, and their use in aiding the healing process of chronic wounds by assisting in the management of infections and / or enhancing their biological activity.

[0003] Background of the Invention

[0004] Polymeric scaffolds have been very successful in the field of wound healing in recent years by being able to provide biochemical signals that alter the wound environment, thereby accelerating and enhancing the healing processes.

[0005] In the field of the present invention, which belongs to tissue engineering, scaffolds should be understood as three-dimensional structures designed to support and guide the growth of cells and tissues. These scaffolds mimic a natural extracellular matrix to provide both physical support for healing agents and chemical signals in wounds, thereby enabling tissue regeneration or injury repair.

[0006] In particular, bioactive scaffolds, used as dressings, play a crucial role in the healing of chronic wounds, such as pressure ulcers and diabetic foot ulcers. These scaffolds combine physical support with functional characteristics, such as the ability to release bioactive substances, control infections, and stimulate biological activity.

[0007] One of the criteria a bioactive scaffold must meet is biocompatibility, meaning it must be compatible with the biological environment to which it will be applied to avoid adverse reactions such as inflammation or rejection. Secondly, it must have a porous structure that allows cell growth and the passage of nutrients and gases, in addition to providing mechanical support and guiding tissue regeneration.

[0008] Furthermore, these scaffolds must incorporate antimicrobial agents, growth factors, or other components that promote healing and minimize the risk of infection. It is also essential that they be biodegradable, so that they gradually degrade over time, allowing new tissue to replace them without the need for subsequent surgical removal.

[0009] Wound healing is a complex process of restoring damaged skin to preserve tissue homeostasis. This process involves the interaction of different cell types, growth hormones, cytokines, and a stable supply of metal ions, such as calcium, zinc, and magnesium. The four overlapping phases of normal wound healing can be summarized as: Coagulation, Inflammation, Proliferation, and Maturation.

[0010] When tissue injury occurs, the coagulation pathway is activated, forming a temporary fibrin matrix that allows cells to migrate to the injured site. Simultaneously, platelet-derived factors attract leukocytes, thereby activating the inflammatory response. Platelets and immune cells then secrete growth factors and cytokines, which promote wound re-epithelialization, extracellular matrix (ECM) deposition, and angiogenesis. Disruption of the normal wound-healing process leads to the formation of a chronic wound, which is much more difficult to heal.

[0011] On the other hand, according to a market research report by the business consultancy Grandview Research, the scaffolding technology market was valued at $1.76 billion in 2022 and is expected to grow at a compound annual growth rate (CAGR) of 13.87% from 2023 to 2030. This growth is partly attributed to the increasing prevalence of eye conditions in the population. The same report also mentions that one of the main reasons for the growth experienced and projected is the increase in the population affected by chronic and acute wounds who require such products, and that the demand for bioactive wound care products can range from newborns to the elderly.

[0012] The prior art related to the present invention, namely silver-loaded antimicrobial compositions for antimicrobial applications, presents disadvantages and complications such as a propensity for heat- and light-induced discoloration, solubility problems, and limited efficacy against resistant microorganisms. The technical problem solved by the present invention for promoting more efficient wound healing and recovery is achieved, unlike the prior art, through a scaffold incorporating silver nanoparticles that have superior stability and solubility, a broader antimicrobial spectrum, a lower risk of adverse effects, and greater ease of production.

[0013] Another important aspect of the medical and tissue engineering products reported in the prior art that have the purpose of wound healing is the incorporation of a calcium component, since calcium is essential for biological processes such as bone formation (osteogenesis) and blood clotting, making it especially valuable in products designed for contact with wounds or tissue regeneration areas.

[0014] While it is common for healthcare professionals to incorporate calcium in later stages of product preparation before applying the product to a wound, there are several reasons and technical challenges for adding calcium in the final stages of preparation, as reported in the prior art. First, to maintain chemical stability, avoiding unwanted reactions with other components that would occur if calcium were added from the beginning. Second, to have greater control over the amount and release of calcium, crucial for applications requiring a controlled effect, such as bone regeneration scaffolds. Third, to allow for formulation flexibility, enabling customization of the product to meet specific patient or treatment needs. However, these prior art strategies also have disadvantages.Incorporating calcium in later or final stages of matrix preparation makes it difficult to achieve uniform distribution in the product, which can affect its effectiveness. Furthermore, late incorporation complicates the manufacturing process, increasing production costs and time, and raising the risk of errors or contamination. Finally, strategies and technologies reported in the prior art that add calcium in the final stages of preparation result in less structural integration of the scaffold with other scaffold components, affecting product stability.

[0015] Therefore, there is still a need to solve that other technical problem of integrating the components into the scaffolding; which is solved by the present application through a process that allows the creation of a scaffolding which incorporates calcium without the need for additional steps, with the purpose of obtaining a safe, effective and efficient product.

[0016] Calcium chloride (CaCl2) has been used for crosslinking purposes in various applications, including the crosslinking of alginates. Some alginate scaffolds and hydrogels have been produced using CaCl2 due to its biocompatibility and coagulation potential. Polymeric scaffolds of chitosan and calcium alginate have shown evidence of positive effects on wound healing. On the other hand, CaCl2 has prothrombotic properties specifically due to its interactions with platelets in both blood and platelet-rich plasma. The crosslinking of gelatin and chitosan with CaCl2 is not currently considered due to its less compatible nature.

[0017] All of the above presents a relevant technical problem, since to this day there are no procoagulant and antimicrobial compositions that achieve high stability and solubility of silver and that at the same time have optimal functionalization with calcium and other divalent cations, without the need for additional steps or complex processes.

[0018] As previously mentioned, silver-laden antimicrobial compositions reported in the prior art present other disadvantages and complications, such as a propensity for heat- and light-induced discoloration, solubility problems, and limited efficacy against resistant microorganisms. This is the case with the prior art documents reported by the Mexican Institute of Industrial Property in its search report with file number IT / E / 2024 / 005176, issued on April 29, 2024, which reports the following background information:

[0019] Romanian patent application No. RO135509A2 discloses a manufacturing process for topical textile biomaterials with anti-inflammatory and antibacterial protective properties. The process described in the application comprises the following steps: creating a layer-by-layer polymer system consisting of two successive layers of sodium alginate and chitosan, respectively; successive immobilization and fixation of the two polymer layers to the surface of textile structures made of 100% worsted cotton yarns and 100% cotton yarns or a blend of 80% cotton yarns and 20% artificial cellulose fibers with zinc oxide content in the weave; and immobilization of bacitracin-type therapeutic agents and ZnO nanoparticles within the layer-by-layer polymer system fixed adjacent to the surface of the textile support.

[0020] On the other hand, Indian patent application No. IN201741017579A describes a wound dressing material, specifically a hydrogel dressing containing polysaccharides as a bioactive ingredient, with oatmeal being the key bioactive ingredient in hydrogels. This type of dressing can be used to treat various dermal wounds, including chronic ulcers. The hydrogel dressing extends its moist, anti-inflammatory microenvironment to the wound area and is believed to accelerate the healing process.

[0021] Regarding the international application No. WO2014147638A1, it comprises a multifunctional natural wound-healing matrix. This matrix consists of a wound base made of hydrophilic cotton fabric coated with low molecular weight zwitterionic chitosan on one side and embedded with biosynthesized silver nanoparticles on the other. Curcumin particles and tulsi extracts are used to further enhance its properties with herbal medicinal values ​​and to provide a synergistic effect, with all ingredients working together to deliver healthier healing results.These scaffolds may optionally contain aloe vera gel, collagen, and gelatin individually or in combination. The matrix is ​​a tissue, bandage, or fiber with an adhesive backing on the inner side containing activated carbon, glycerin, and lanolin, along with additional curcumin nanoparticles on the back, resembling wound bed tissue. Additionally, scaffolds were manufactured by Hiu, L et al. (2004) by mixing, freezing, and lyophilizing a gelatin-chitosan-hyaluronic acid mixture. This product was originally crosslinked with the chemical agents MES, EDC, and NHS (2-(N-morpholino)ethanesulfonate, 1-(3-D-methylaminopropyl)-3-ethylcarbodiimide, and N-hydroxysuccinimide). Its potential as a skin substitute was immediately recognized due to its favorable water retention capacity and successful preliminary studies of cell proliferation and biocompatibility.Some products based on this invention have already been developed, one of the most influential being Inbioderm+C, an autologous cell treatment for wound healing.

[0022] However, one of the major drawbacks of the aforementioned polymer is its crosslinking through the MES-EDC-NHS combination, as this reduces the bioavailability of the administered compounds. This can occur because the formation of bonds between the polymer chains can restrict the movement of the encapsulated or linked molecules, hindering their release or availability to interact with other compounds or cells. Consequently, there remains a demand for wound care products that offer high stability, excellent solubility of silver nanoparticles, and functionalization with calcium and other divalent cations for tissue engineering and cell therapy applications, without the need for additional steps or complex processes.These formulations must also address issues such as susceptibility to heat- and light-induced discoloration, limited water solubility, and reduced efficacy against resistant microorganisms. Furthermore, they must consider the application of an effective dose with a lower risk of side effects, while simultaneously providing a broad antimicrobial spectrum, and utilizing natural polymers that offer technical advantages such as reabsorption in the specific applications of the present invention.

[0023] Summary of the Invention

[0024] To overcome the deficiencies of the prior art, the different aspects of the present invention relate to a silver-loaded antimicrobial composition that acts as a therapeutic device (scaffold) for wound healing and tissue engineering applications, which differs from the prior art because it mainly comprises at least one absorbent matrix for cell-matrix and cell-cell interactions based on at least one natural polymer, a calcium crosslinking component for in-situ fibrin gel formation, and a nanoemulsion with colloidal silver nanoparticles as a bioactive compound to help control biofilm and infections in a wound.

[0025] Some additional advantages of the present invention lie in its ability to harness the potent antimicrobial activity of silver, especially in its nanoparticle form, offering a broad spectrum of action against Gram-negative and Gram-positive fungi and bacteria, while minimizing the risk of antimicrobial resistance and reducing cytotoxicity to normal tissues when used at specific concentrations. Unlike conventional silver scaffolds, which tend to be prone to heat- and light-induced discoloration and often present solubility problems, the silver nanoparticle technology of the present invention is more robust and stable, allowing its use in a variety of devices and compositions.Furthermore, by requiring only 1 / 24 of the concentration used in other antimicrobials available on the market, the antimicrobial composition of the scaffold of the present invention can achieve higher performance, reducing the incidence of undesirable side effects such as discoloration and staining, which can improve the production and shelf life of the final products.

[0026] The addition of silver nanoparticles can also improve the physicochemical properties of the biomaterial, increasing its biocompatibility and biodegradability, and maximizing cell adhesion and cell-cell and cell-matrix interactions. Furthermore, the incorporation of these nanoparticles increases the product's shelf life, reducing supply chain costs. The antimicrobial activity of the nanoparticles and their potential application in nursing homes (in addition to clinical settings) also has the capacity to significantly reduce operating costs in the treatment of chronic wounds in clinics and hospitals.

[0027] In another aspect of this application, a process for obtaining the antimicrobial composition that is the subject of this invention and a scaffold comprising said antimicrobial composition is also described.

[0028] These and other objectives and advantages will be evident to the person skilled in the art from the following description of the figures and the detailed description of the invention; and the appended claims.

[0029] Brief Description of the Fiauras

[0030] Figure 1 shows the distribution capacity of different chemical elements a) - m) in the scaffold of the present invention by means of energy dispersive X-ray spectroscopy (EDS).

[0031] Figure 2 shows the distribution of silver specifically in the scaffolding that is the subject of the present invention by means of EDS.

[0032] Figure 3 shows the EDS spectrum of the scaffold that is the subject of the present invention with quantification values ​​for each chemical element tested.

[0033] Figure 4 shows calcium (in green) and silver (in purple) in EDS.

[0034] Figure 5 shows a scanning electron microscopy (SEM) image of the scaffold that is the subject of the present invention with a magnification of 100x.

[0035] Figure 6 shows a scanning electron microscopy (SEM) image of the scaffold that is the subject of the present invention showing its porosity, which averages 179.7 .m.

[0036] Figure 7 shows an SEM + EDS image showing a silver nanoparticle on the scaffold.

[0037] Figure 8 shows vahas silver nanoparticles in the scaffold that is the subject of the present invention.

[0038] Figure 9 shows the metabolic activity of cells on the scaffold vs. a control monolayer.

[0039] Figures 10A and 10B show images and a pore size distribution graph with different concentrations of additives at 10Ox.

[0040] Figures 11A and 11B show the cell adhesion results for WST-1 in a 7-day study in different groups with incremental concentrations of silver nanoparticles.

[0041] Figure 12 shows the cell fluorescence of a) the control monolayer on day 1, b) the scaffold of the present invention on day 1, c) the control monolayer on day 3 and d) the scaffold of the present invention on day 3. Figure 13 shows the degradation in 7 days of the scaffold of the present invention with respect to variations in calcium lactate concentration.

[0042] Detailed Description of the Invention

[0043] Some aspects of the present invention will now be described in more detail, also using reference to the accompanying drawings which show some embodiments and advantages of the present invention.

[0044] It will be evident to a person skilled in the art that various embodiments of the invention can be expressed in many different ways and should not be interpreted as being limited to the embodiments described herein; rather, these exemplary embodiments are provided to make this invention clear and complete and to fully convey the scope of the invention to those skilled in the art. For example, unless otherwise indicated, something described as first, second, or similar should not be interpreted as a particular order. As used in the description and in the appended claims, the singular forms "a," "an," "the," and "a" include plural referents unless the context clearly indicates otherwise.

[0045] The different aspects of the present invention relate to a silver-loaded antimicrobial composition, the scaffold comprising said antimicrobial composition and acting as a therapeutic device for wound healing and tissue engineering application, the method of manufacturing said products, and the uses thereof.

[0046] In one particular embodiment, the antimicrobial composition of the present invention comprises a nanoemulsion with silver nanoparticles, a reducing agent, stabilizers, dispersing agents, pH agents, and surfactants.

[0047] In another modality, the reducing agent is any of the group comprising ascorbic acid, sodium citrate, sodium borohydride (NaBH4), gallic acid, combinations thereof or similar.

[0048] In another modality, the stabilizer is any of the group comprising Polyvinylpyrrolidone (PVP), citrate, Polyethylene glycol (PEG), Tannic Acid, combinations thereof or similar.

[0049] Even in another modality, the dispersion medium is distilled water or similar.

[0050] In another modality, the pH agent is chosen from the group comprising hydrochloric acid (HCl) or sodium hydroxide (NaOH), nitric acid (HNO3), acetic acid (CH3COOH) or similar.

[0051] In another embodiment, the suitable surfactant according to the context of the present invention is selected from the group comprising Tween 20, Tween 80, SDS, CTAB, Triton X-100, combinations thereof or similar.

[0052] Now, “nanoemulsion,” “nanoemulsion with colloidal silver nanoparticles,” or any of its variations in the context of the present invention shall be understood to mean an emulsion in which one liquid phase is dispersed in another liquid phase in the form of very small droplets; generally, but not limited to, in the range of 10 to 900 nanometers in diameter. Containing colloidal silver nanoparticles, this nanoemulsion includes minute particles of metallic silver dispersed homogeneously at the nanoscale. Nanoemulsions are created to stabilize components that would otherwise be immiscible. The incorporation of colloidal silver nanoparticles adds antimicrobial and antibacterial properties to the nanoemulsion described in this application.

[0053] In another particular form, the nanoparticles are Silver Nitrate (AgNO3) nanoparticles.

[0054] In one particular modality, the size of the colloidal silver nanoparticles is in a range from 60 to 600 nm.

[0055] In some forms, colloidal silver has the following characteristics: (a) it is silver suspended in distilled water and produced by dispersion according to published guidelines (NIST, 2012) or by electrical methods with silver electrodes; (b) it has an atomic mass of 107.868 g / mol; (c) it has a melting point of 960.5°C; (d) it has a boiling point of 2000°C; (e) it has a density at 15°C of 10.49 g / mL; (f) it is not attacked by water or atmospheric oxygen; (g) it is darkened by ozone and hydrogen sulfide; (h) it is inert to many acids and reacts readily with dilute nitric acid and hot sulfuric acid; and (i) it is not light-sensitive in its metallic form.

[0056] In some embodiments, the composition of silver nanoparticles comprises: 73-95% water, 0.1-20% colloidal silver, 0.5-5.5% methyl vinyl ether copolymer, 0.01-15.00% African palm vegetable oil, 0.05-0.2% polyoxyethylene octylphenyl ether, 0.02-0.09% triethanolamine, 0.02-0.05% sodium hydroxide, and 0.005-0.015% sodium benzoate.

[0057] In some embodiments, the composition of silver nanoparticles comprises: 90.56% water, 5.00% colloidal silver, 3.50% methyl vinyl ether copolymer, 0.745% African palm vegetable oil, 0.099% polyoxyethylene octylphenyl ether, 0.052% triethanolamine, 0.035% sodium hydroxide, and 0.009% sodium benzoate.

[0058] In some formulations, the water is distilled or deionized. In some formulations, African palm oil is characterized by: a specific gravity of 0.925–0.935 g / ml; a melting point of 19–26°C; a refractive index at 40°C of 1.45–1.452; a saponification point of 239–257 mg / g; and an iodine content of 12–18 g per 100 g of the composition. In some formulations, sodium hydroxide is used to neutralize the African palm oil.

[0059] In some forms, colloidal silver has the following characteristics: (a) it is silver suspended in distilled water and produced by dispersion according to published guidelines (NIST, 2012) or by electrical methods using silver electrodes; (b) it has an atomic mass of 107.868 g / mol; (c) it has a melting point of 960.5°C; (d) it has a boiling point of 2000°C; (e) it has a density at 15°C of 10.49 g / ml; (f) it is not attacked by water or atmospheric oxygen; (g) it is darkened by ozone and hydrogen sulfide; (h) it is inert to many acids and reacts readily with dilute nitric acid and hot sulfuric acid; and (i) it is not sensitive to light in its metallic form.

[0060] In some formulations, all colloidal silver particles have an average size between approximately 60 nm and approximately 140 nm. In some formulations, at least 50% of the colloidal silver particles have a size between approximately 60 nm and approximately 140 nm. In some formulations, at least 90% of the colloidal silver particles have a size between approximately 60 nm and approximately 140 nm.

[0061] In one particular modality, colloidal silver nanoparticles are found in a concentration range from 1 to 5 ppm.

[0062] In one particular embodiment, the antimicrobial composition of the present invention is used in a silver-loaded antimicrobial scaffold that acts as a therapeutic device for wound healing and tissue engineering applications.

[0063] In one particular embodiment, the scaffold of the present invention comprises at least one absorbent matrix for cell-matrix and cell-cell interactions (a natural polymer), a calcium crosslinking component for in-situ fibrin gel formation, and a nanoemulsion with colloidal silver nanoparticles as described above, which acts as a bioactive compound to help control biofilm and wound infections.

[0064] In the context of this application, “absorbable matrix” refers to a three-dimensional structure that is capable of being gradually broken down and absorbed by the body over time. This matrix, generally composed of biodegradable and biocompatible materials (usually natural polymers), serves as a physical scaffold for cell growth and tissue regeneration. Its absorbable nature allows the scaffold to degrade as new tissue forms and matures, without requiring surgical removal. Within this matrix, cell-matrix interactions are essential for cells to adhere, proliferate, and form new tissue. The polymers of the absorbable matrix provide biocompatible surfaces that promote cell adhesion, creating an environment conducive to growth and regeneration.These interactions are essential for cells to begin integrating into the matrix and forming new tissues. Furthermore, cell-cell interactions also play a significant role; these interactions involve communication and adhesion between cells, enabling the formation of organized structures that mimic natural tissue. The aforementioned polymers help facilitate these interactions, promoting orderly tissue regeneration.

[0065] The term “natural polymer” shall mean at least one macromolecule consisting of repeating monomeric units linked by covalent bonds, which is biologically synthesized by living organisms such as plants, animals, bacteria, and other biological beings. In one embodiment, the at least one absorbable matrix is ​​a natural polymer selected from the group comprising gelatin, chitosan, hyaluronic acid, collagen, alginate, fibrin, silk fibroin, xanthan gum, combinations thereof, or similar materials.

[0066] In a preferred embodiment, the at least one absorbable matrix (natural polymer) is gelatin.

[0067] In another modality related to the above, gelatin is any selected from the group comprising Type A or B gelatin of bovine, porcine, piscino (derived from fish) or fungal origin, combinations of the same or similar.

[0068] In a specific modality related to the above, gelatin is found in a concentration range from 0.5 to 3%.

[0069] In another preferred form, the at least one absorbable matrix is ​​chitosan.

[0070] In another modality related to the above, chitosan is any selected from the group comprising chitosan (of low, medium and high molecular weight), trimethyl-chitosan, carboxymethyl-chitosan.

[0071] In a specific modality according to the above, chitosan is found in a concentration of 1 to 5%.

[0072] In another preferred modality, at least one absorbable matrix is ​​hyaluronic acid.

[0073] In another modality related to the above, hyaluronic acid is any selected from the group comprising hyaluronic acid / sodium hyaluronate (of different MW).

[0074] In a specific modality related to the above, sodium hyaluronate is found in a concentration range from 0.005 to 1%.

[0075] In one particular embodiment, the antimicrobial composition of the present invention comprises: 65%-71% w / w gelatin, 15%-21% chitosan and 5%-11% hyaluronic acid.

[0076] In one particular embodiment, the antimicrobial composition of the present invention comprises: 65%-71% w / w collagen, 15%-21% chitosan and 5%-11% sodium hyaluronate.

[0077] On the other hand, "calcium crosslinking component" shall be understood as an agent or substance that induces the formation of cross-links or crosslinking through the action of calcium ions. In the context of the present invention, this means that the component uses calcium to bind and stabilize polymeric structures, creating a solid, porous, three-dimensional matrix. Calcium crosslinking is common in polymers such as polysaccharides, where calcium ions can bind to these polymers through specific interactions, forming bonds that interlock the polymer chains and generate a more rigid and stable structure.

[0078] In another embodiment, the calcium crosslinking component is any selected from the group comprising calcium chloride (CaCh), calcium carbonate (CaCO3), calcium phosphate (Ca3(PO4)2), calcium lactate (CeH CaOe), sodium alginate, pectin, carrageenan, chitosan-CaCO3, calcium gluconate, calcium acetate, or combinations thereof. In a preferred embodiment, the calcium crosslinking component is calcium chloride (CaCl2).

[0079] In a preferred embodiment, the calcium crosslinking component is calcium carbonate (CaCO3).

[0080] In a preferred embodiment, the calcium crosslinking component is calcium phosphate (Ca3(PO4)2).

[0081] In a preferred embodiment, the calcium crosslinking component is calcium lactate (CeH CaOe).

[0082] In another particular modality related to the above, the calcium crosslinking component is found in a concentration range from 0.5 to 3%.

[0083] In another particular embodiment, the scaffolding that is the subject of the present invention contains a calcium concentration that is in the range of 0.018 M - 2 M.

[0084] In another particular embodiment, the scaffold of the present invention further comprises additional bioactive compounds to improve its functional properties.

[0085] In a particular embodiment related to the foregoing, the suitable bioactive compound according to the present invention is any selected from the group comprising growth factors, antimicrobials and antibiotics, antioxidants, combinations thereof or similar.

[0086] In a specific modality, the growth factor can be any selected from the group comprising Epidermal Growth Factor (EGF), Fibroblast Growth Factor (FGF), Platelet-Derived Growth Factor (PDGF), combinations thereof or similar.

[0087] In a specific modality, the antimicrobial / antibiotic can be any selected from the group comprising Chlorhexidine, Antibiotics such as Gentamicin or Vancomycin, combinations of the same or similar.

[0088] In a specific modality, the antioxidant can be any selected from the group comprising curcumin, resveratrol, combinations thereof or similar.

[0089] In one specific embodiment, the additional bioactive compound is icariin, a chemical compound that can be isolated from Epimedium, a traditional Chinese herbal medicine. In this regard, it is known in the field of the present invention that icariin improves keratinocyte migration and proliferation by 33% following topical application in wound healing.

[0090] Furthermore, in one particular embodiment, a wetting agent may be optionally added to the mixture, which may be any substance selected from the group comprising glycerol, propylene glycol, polypropylene glycol, urea, polyethylene glycol, and / or sodium lactate, or combinations thereof. In another particular embodiment, a viscosity-increasing agent may also be optionally added, which may be a synthetic clay mineral, a natural clay mineral, or a cellulose ether.

[0091] According to the above, the viscosity-increasing agent is any selected from the group comprising hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose and sodium carboxymethyl cellulose, polyacrylate, a natural gum, a chemically modified natural gum, a chemically modified cellulose ether with aliphatic chains, a synthetic gum, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylamide and a polyamino acid including one of polyaspartic and polyglutamic, combinations thereof or similar.

[0092] In another particular embodiment, the pharmaceutical presentation of the resulting product, according to the present invention, is designed in various forms of hemostatic materials; that is, different types of products or devices designed to stop or control bleeding (hemostasis) in a medical or surgical context. These hemostatic materials are used to help stop blood flow, facilitate coagulation, and provide a favorable environment for healing.

[0093] In one particular formulation, related to the above, the resulting product is any selected from the group comprising dry powder, sponge, foam, hydrogel, sheets, hydrated or dry microspheres, and gauze, or covered with a semipermeable porous dry gel containing a calcium component and the addition of colloidal silver nanoparticles as an enhancement compound. The aforementioned formulations are conceived and designed to regulate wound moisture under the dressing, with a permeability deliberately selected to maintain this moisture within an optimal therapeutic range, stimulating coagulation, facilitating cell-cell interactions, and preventing biofilm formation or expansion.

[0094] In a preferred embodiment, the antimicrobial composition of the present invention is used in the areas of cosmetics, orthopedic surgery (cartilage reconstruction and / or implants), bone reconstruction, mandibular reconstruction surgery, Mohs surgery, eye surgery, chronic wounds (radiation (dermatitis), pressure, vascular, diabetic), or similar.

[0095] Other important definitions and terms

[0096] The following terms are important for a complete understanding of the present invention, so that it is well defined and its scope is clear and concise.

[0097] Intertwining

[0098] In the context of the present invention, “crosslinking” means the process of creating chemical bonds between two atoms in a compound. In the context of wound healing and tissue engineering, crosslinking refers more specifically to the process of stabilizing collagen or collagen compounds within medical devices by creating new bonds between collagen strands. This process inhibits collagen degradation by proteases and prolongs its presence in the wound. The type of crosslinking process that occurs directly affects the durability of acellular matrices. Crosslinking methods involving chemical (e.g., aldehydes) or mechanical (e.g., heat or radiation) processes can result in little control over the degree of crosslinking and produce bonds between collagen strands that are short and inflexible.These short, inflexible crosslinks can inhibit cell migration and vascular regeneration, and residual crosslinking agents in the product can induce cytotoxicity or an inflammatory response, causing the matrix to be rapidly absorbed by the host tissue and potentially resulting in scar tissue formation. Newer crosslinking processes produce more elastic and flexible crosslinks between collagen strands and are less prone to enzymatic breakdown. These more flexible crosslinks, produced by newer processes, are thought to allow cells to migrate and proliferate in an organized manner similar to normal tissue repair.

[0099] Alginate

[0100] On the other hand, "alginate" refers to a biomaterial that has found numerous applications in biomedical sciences and engineering due to its favorable properties, including biocompatibility and ease of gelation. Alginate hydrogels have been particularly attractive in wound healing, drug delivery, and tissue engineering applications to date, as these gels retain structural similarity to the extracellular matrices in tissues and can be manipulated to perform various critical roles.

[0101] Hydroqel

[0102] The word “hydrogey” is defined as a unique group of biocompatible, three-dimensional polymeric substances that can act as scaffolds and mimic the properties of tissues in the body. The mechanism involves the incorporation of cells into their structure while they eventually degrade, leaving only healthy tissue.

[0103] Polymers

[0104] The basic types of biomaterials used in tissue engineering can be classified into two main groups: synthetic polymers, which include relatively hydrophobic materials such as α-hydroxy acids (a family that includes poly(lactic-co-glycolic acid), PLGA), polyanhydrides, and others; and naturally occurring polymers, such as complex sugars (hyaluronan, chitin); and inorganic materials (hydroxyapatite). There are also functional or structural classifications, such as whether they are hydrogels (1), injectable (2), surface-modified (3,4), capable of drug release (5), by specific application, and so on. The breadth of materials used in tissue engineering stems from the multiplicity of anatomical locations, cell types, and specialized applications.For example, relatively strong mechanical properties may be required in situations where the device may be subjected to weight or tensile loads, or where it is necessary to maintain a specific cytoarchitectural structure. In other cases, looser networks may be needed or even preferred. The type of materials used also depends on the intended mode of application (open implantation vs. injection or minimally invasive procedure), the nature of the bioactive molecules that may be released, the need for surface functionalization, the porosity requirements of the cell types of interest, and other factors. Despite this broad spectrum of potential materials, certain generic properties are desirable.

[0105] Scaffolding

[0106] Cells, scaffolds, and growth-promoting signals are generally referred to as the tissue engineering triad, the key components of engineered tissues. Scaffolds, typically made of polymeric biomaterials, provide the structural support for cell adhesion and subsequent tissue development. Intuitively, the best scaffold for an engineered tissue should be the extracellular matrix (ECM) of the target tissue in its native state. However, the multiple functions, complex composition, and dynamic nature of the ECM in native tissues make it difficult to mimic exactly. Therefore, the contemporary concept of scaffolds in tissue engineering is to at least partially mimic the functions of the native ECM.As a result, the important roles that scaffolds play in engineered tissues, as reviewed elsewhere, are analogous to the functions of the extracellular matrix (ECM) in native tissues and are associated with their architectural, biological, and mechanical characteristics. In this regard, the following definitions related to these functions and characteristics are of great importance.

[0107] Scaffolding architecture

[0108] Scaffolds must provide space for vascularization, new tissue formation, and remodeling to facilitate host tissue integration after implantation. Biomaterials must be processed to produce a sufficiently porous structure for efficient transport of nutrients and metabolites without significantly compromising the scaffold's mechanical stability. Furthermore, the biomaterials must also be degradable after implantation at a rate that matches the rate of new matrix production by the developing tissue.

[0109] Cytological and tissue compatibility in scaffolds

[0110] Scaffolds must provide support for externally applied or endogenous cells to adhere, grow, and differentiate both during in vitro culture and in vivo implantation. The biomaterials used to fabricate the scaffolds must be compatible with the cellular components of the engineered tissues and the endogenous cells in the host tissue.

[0111] Bioactivity in scaffolding

[0112] Scaffolds can actively interact with the cellular components of designed tissues to facilitate and regulate their activities. Biomaterials can incorporate biological signals such as adhesive ligands to enhance adhesion or physical signals such as topography to influence cell morphology and alignment. The scaffold can also serve as a delivery vehicle or reservoir for exogenous growth signals to accelerate regeneration. In this regard, biomaterials must be compatible with biomolecules and capable of encapsulation for the controlled release of biomolecules without loss of bioactivity. For example, hydrogels synthesized through covalent or ionic crosslinking can trap proteins and release them through a mechanism controlled by hydrogel swelling.

[0113] Mechanical properties in scaffolding

[0114] Scaffolds provide mechanical and shape stability to the tissue defect. The intrinsic mechanical properties of the biomaterials used to fabricate scaffolds, or their post-processing properties, must match those of the host tissue. Recent studies in mechanobiology have highlighted the importance of a scaffold's mechanical properties on implanted cells. Cells exert tensile forces on a substrate; many types of mature cells, such as epithelial cells, fibroblasts, muscle cells, and neurons, sense substrate stiffness and exhibit dissimilar morphologies and adhesive characteristics. This mechanosensitivity has also been demonstrated in the differentiation of mesenchymal stem cells (MSCs), where the stiffness of the agarose gel determines the differentiation tendency. MSCs differentiate into neuronal, muscular, or bone cell lineages depending on whether the stiffness approximates that of brain, muscle, and bone tissues, respectively.

[0115] Synthetic scaffolding

[0116] Synthetic polymers offer advantages over natural polymers in several characteristics, such as adjustable properties, unlimited shapes, and established structures. The support provided by synthetic biomaterials can enable the restoration of damaged or diseased tissue structures and functions. The polymerization, interconnection, and functionality (modified by block structures, combinations, or copolymerization) of their molecular weight, molecular structure, and physical and chemical characteristics make them easier to synthesize compared to natural polymers. The disadvantages of synthetic biomaterials include the lack of cell adhesion sites and the need for chemical modifications to enhance cell adhesion. Many commercially available synthetic polymers exhibit physicochemical and mechanical properties similar to those of biological tissues.In biodegradable polymers, synthetic polymers are a major category and can be produced under controlled conditions. Across a broad spectrum, their mechanical and physical characteristics are predictable and reproducible, such as strength, Young's modulus, and degradation rate. Aliphatic polyesters, including PCL, PGA, PLA, and its copolymer PLGA, and polyethers, including PEO and PEG, PVA, and PU, are the most studied biodegradable synthetic materials. These are probably the most popular examples, although many other synthetic materials are currently being explored. These polymers have varying levels of biodegradability, biocompatibility, and mechanical properties, but no single polymer possesses all three critical properties at the optimum level. Organic scaffolds.

[0117] Organic or "natural" biopolymers have re-emerged in recent decades as primary bioactive substances used in medical materials applications. Based on their monomeric units and structure, biopolymers are roughly categorized into three classes:

[0118] Polypeptides and proteins: collagen, fibrin, fibrinogen, gelatin, silk, elastin, myosin, keratin and actin.

[0119] Polysaccharides: chitin, chitosan, alginate, hyaluronic acid, cellulose, agarose, dextran and glycosaminoglycans.

[0120] Polynucleotides: DNA, linear plasmid DNA and RNA.

[0121] These consist of long chains, including nucleotides, amino acids, or monosaccharides, made of repeating covalently bonded groups. Biofunctional molecules that ensure bioactivity, biomimetic nature, and natural restructuring are typically found in such polymers. Bioactivity, biocompatibility, 3D geometry, antigenicity, non-toxic biodegradation byproducts, and intrinsic structural similarity are the most important properties of natural polymers. Conversely, their main disadvantages, such as microbial contamination (e.g., endotoxins), reduced conformability, immunogenicity, uncontrollable degradation rate, and low mechanical strength, limit their application for hard tissue regeneration. Organic polymers make important contributions to tissue engineering, especially in the fabrication of scaffolds for the delivery of therapeutic agents.New, natural polymeric materials are poised to enhance various therapies due to their inherent bioactivity, biocompatibility, and bioresorptivity. Naturally derived polymers, including collagen, chitin, chitosan, gelatin, silk fibroma, soy, fibrinogen, fibrin, elastin, proteoglycan, hyaluronan, and laminin, have shown great potential in the biomedical field. However, they can be extremely difficult to scale up, especially on a global scale, and may cause allergic reactions in some cases, depending on the specific application.

[0122] Chitosan

[0123] In recent decades, chitosan has emerged as a promising candidate for tissue engineering applications. Derived from chitin, chitosan is a unique natural polysaccharide with exceptional properties, including excellent biodegradability, biocompatibility, and antimicrobial activity. Due to the presence of free amino groups in its backbone, chitosan can be chemically modified to possess additional functional properties useful for the development of various biomaterials in regenerative medicine.

[0124] Gelatin

[0125] Gelatin is a naturally occurring protein derived from the hydrolysis of collagen. Here, we present a state-of-the-art overview of gelatin-based compounds to overcome the limitations of this polymeric material and modulate the properties of formulations. Controlled release of bioactive molecules, formulations with conductive properties, or systems with enhanced mechanical properties can be achieved using gelatin-based compounds. Numerous studies have found that using calcium phosphate ceramics and various synthetic polymers in combination with gelatin improves the mechanical properties of the structures. Furthermore, polyaniline and carbon-based nanosubstrates are promising molecules for imparting conductive properties to gelatin-based systems, particularly for cardiac and nervous tissue engineering.Finally, this review provides an overview of the different types of gelatin-based structures, including nanoparticles, microparticles, 3D scaffolds, electrospun nanofibers, and in situ gelation formulations. Thanks to the significant progress already achieved, along with further future advancements, the safe and effective clinical implementation of gelatin-based products is expected to accelerate and expand in the near future.

[0126] Hyaluronic acid

[0127] Hyaluronic acid (HA) plays a vital role in cellular processes, and its contribution to physical and immunological barriers is considered an important property for the formulation of modern therapies. With the increasing demand for non-toxic, targeted therapies, HA-based materials could be used for biomedical applications due to their tendency to biomimic host cells. Furthermore, HA is a versatile compound in the fabrication of HA-based products, such as hydrogels, nanofibers, and 3D materials. These have been implemented in various medical fields, including bone and tissue regeneration, topical gels for wound healing, and cancer treatment using HA-loaded drug delivery approaches. Sodium hyaluronate is a solid form of HA.

[0128] Glacial Acetic Acid

[0129] Acetic acid, systematically called ethanoic acid, is a colorless, acidic, liquid organic compound with the chemical formula CH3COOH. Vinegar contains at least 4% acetic acid by volume, making it the main component of vinegar besides water.

[0130] Calcium chloride (CaCk)

[0131] Calcium chloride (CaCk) has been widely used as a preservative and firming agent in the fruit and vegetable industry, for both whole and fresh-cut produce. The application of calcium can reduce over-ripening of fruit, as it binds and cross-links with free carboxyl groups of polygalacturonic acid in pectins, improving fruit firmness. Pre-storage treatment with calcium in apples has been shown to reduce the incidence of physiological disorders, softening rates, and fungal decay. Calcium infiltration significantly increased the sensory firmness and overall acceptability of the apples. Similarly, papaya treated with a 2% CaCk solution maintained cell wall integrity due to the influx of calcium, which may have aided in the formation of calcium pectate within the cell wall, thus extending shelf life.Calcium carbonate (CaCOs).

[0132] The high calcium carbonate content can effectively recruit mesenchymal stem cells (MSCs) near the defect area, induce their osteogenic differentiation, and ultimately accelerate the bone regeneration process. Considering its ease of preparation and excellent osteogenicity, the chitosan-calcium carbonate scaffold has high potential for the treatment of massive bone defects.

[0133] Calcium lactate (CeHioCaOfí)

[0134] Calcium lactate hydrate is a salt of lactic acid, a common source of calcium in food, and has also been used as a firming agent and food additive. Furthermore, the tensile strength and water-holding capacity of biocomposite films can be improved by incorporating calcium lactate. In another study, nylon 6 carpets coated with calcium lactate were reported to have improved cell adhesion, growth, and proliferation. However, this process is laborious and tedious, as lactic acid-coated nylon 6 nanofibers must be prepared and then neutralized with calcium hydroxide to generate calcium lactate within the nanofibers. Therefore, a more reliable and simpler process for forming composite nanofibers could involve the direct incorporation of calcium lactate into the polymer solution.

[0135] Calcium phosphate

[0136] Calcium phosphate, also known as apatite or CaP, has been widely used in bone regeneration applications because it exhibits osteoconductive and, in some cases, osteoinductive properties. The release of calcium and phosphorus ions regulates the activation of osteoblasts and osteoclasts to facilitate bone regeneration. Controlling the surface properties and porosity of calcium phosphate affects cell / protein adhesion and growth, and regulates bone mineral formation. The properties that affect bioactivity vary depending on the type of calcium phosphate, such as HAP and TCP, and can be used in various applications due to differences in ion release, solubility, stability, and mechanical strength. To leverage these properties, different calcium phosphates have been used together or blended with other materials to compensate for their weaknesses and highlight their advantages.Calcium phosphate has been used to enhance bone regeneration in various ways, such as by increasing osteoconductivity for bone growth, improving osteoinductivity for bone mineralization with controlled ion release, and encapsulating drugs or growth factors. It also has broad applications in wound healing due to its potential to stimulate cell adhesion.

[0137] Collagen molecules self-assemble into fibrils with a periodicity of 67 nm and 40 nm inter-end spaces. Within these spaces, biological apatite settles and grows as tiny, plate-like nanoscale crystals with a specific crystalline orientation along the c-axes, parallel to the long axes of the collagen fibrils. This in vivo process of biological apatite formation is called biological mineralization or biomineralization. During biomineralization, organized assemblies of organic macromolecules regulate the nucleation, growth, and morphology of the inorganic crystals.

[0138] Agitation

[0139] Mechanical agitation can be used to suspend particles in a liquid to promote mass transport or a chemical reaction. The liquids used in such applications generally have low viscosity, and the particles will settle when the agitation stops. There are also times when a relatively homogeneous suspension needs to be achieved in a mixing vessel, especially when preparing materials for further processing.

[0140] Icariin (derived from Epidmedium)

[0141] Icariin (ICRN), a key bioactive flavonoid from the Epimedium plant, has a wide range of applications in scaffold enhancement as a stable, non-immunogenic material, and in stimulating cell growth, chondrocyte differentiation, and the differentiation of embryonic stem cells into cardiomyocytes. Furthermore, the fusion of ICRN into hydrogel scaffolds or chemical crosslinking can increase the secretion of collagen matrix and proteoglycans in bone and cartilage tissue engineering. In various types of cancer cells, ICRN plays a crucial role by increasing cytochrome c secretion, the Bax / Bcl2 ratio, poly(ADP-HBo) polymerase, and caspase stimulation. Remarkably, ICRN can induce apoptosis, reduce viability and inhibit the proliferation of cancer cells, and repress tumorigenesis and metastasis.Furthermore, cancer cells cease to grow by arresting the cell cycle at two checkpoints, G0 / G1 and G2 / M, through the inhibition of NF-κB by ICRN. It also mitigates nephrotoxicity caused by cisplatin and prevents multidrug resistance, which are other applications of this biomaterial.

[0142] Functionalization

[0143] The inert nature of most commercial polymers and nanomaterials results in application limitations in various industrial fields. This can be overcome through surface modifications to improve physicochemical and biological properties, such as adhesion, printability, wettability, and biocompatibility. Polymer functionalization allows for the grafting of specific groups and the conjugation of molecules that enhance material performance. In recent decades, several approaches have been developed in industry and academia for grafting functional groups onto surfaces.

[0144] Calcium and its role in wound healing

[0145] In addition to being a critical clotting factor during hemostasis, the calcium ion has been shown to act as a fundamental signaling molecule, directing the cellular functions of different cell types during wound healing. Calcium plays a vital role as an extracellular signaling molecule and intracellular second messenger for keratinocytes and fibroblasts. Previous studies have explored the impact of calcium concentrations on keratinocyte proliferation and differentiation. However, the effects of calcium on dermal fibroblasts have not yet been fully elucidated. A modest number of studies have demonstrated that calcium influences fibroblast morphology, proliferation, and collagen deposition.

[0146] Nanoparticles

[0147] Nanoparticles are submicron materials that often possess different properties than the bulk material of the same type. Nanoparticles have been studied for use in many fields, including diagnostic and therapeutic applications in the life sciences. Due to their small size and unique properties, nanoparticles often have improved distribution in the body compared to larger particles. Furthermore, they can be specifically targeted to particular sites in the body by attaching one or more components to the nanoparticle surface (i.e., functionalization). Functionalizing a nanoparticle with a component that has an affinity for a specific target in the body, such as collagen, can direct the nanoparticle to tissues containing the target molecule.

[0148] Homeostasis

[0149] Hemostasis begins the moment an injury occurs, as tissue is disturbed and blood vessels rupture. The processes that occur during hemostasis (from the Greek words κος, "blood," and δος, "stasis") aim to temporarily seal the damaged tissue and stop the associated bleeding by initiating vasoconstriction, platelet plug formation, and blood clotting. Vasoconstriction is the first response to injury. Vasoconstrictor factors, such as serotonin and thromboxane A2, act to limit the amount of blood flow through the damaged area. Platelets play a crucial role in hemostasis, adhering to and aggregating with the damaged endothelium. The platelets form a transient plug to prevent local blood loss.Then, the platelets themselves provide a surface for clotting factors such as fibrinogen, which in turn is converted into fibrin (through the intrinsic and extrinsic coagulation cascade), forming a robust blood clot.

[0150] The blood clot acts as a scaffold, allowing a temporary extracellular matrix (ECM) to form around it in the wound area. The ECM is rich in fibrous proteins such as collagen, fibronectin, and fibrin, as well as liquid proteoglycans like hyaluronic acid. Importantly, the ECM contributes to wound repair not only structurally but also through signaling, attracting different cells to itself. A key ECM component involved in wound healing is fibronectin. Fibronectin is a large adhesion glycoprotein in the ECM that interacts with integrin receptors to stimulate the migration and adhesion of fibroblasts, keratinocytes, and endothelial cells, as well as promoting angiogenesis (the physiological process by which new blood vessels are generated from pre-existing vessels). The temporary matrix envelops platelets as part of the matrix itself.Platelets in the matrix attract and activate numerous cells, such as endothelial cells, fibroblasts, neutrophils, and macrophages. Thus, in addition to acting as a "static" plug, platelets also pave the way for subsequent wound healing steps by releasing various pro-inflammatory factors (including prostaglandins, prostacyclin, thromboxane, and histamine), and several cytokines and growth factors, including platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-β), and vascular endothelial growth factor (VEGF).

[0151] Homeostasis has become the central unifying concept in physiology and is defined as a self-regulating process by which an organism can maintain internal stability while adjusting to changing external conditions. Homeostasis is not static and immutable; it is a dynamic process that can change internal conditions as needed to survive external challenges. It is also important to note that homeostatic regulation is not simply the product of a single negative feedback loop, but rather reflects the complex interaction of multiple feedback systems that can be modified by higher control centers. This hierarchical control and feedback redundancy result in finer control and greater flexibility, allowing the organism to adapt to changing environmental conditions.It can be said that the health and vitality of the organism are the end result of homeostatic regulation. Normal physiology cannot be understood without an appreciation of this concept. In turn, it follows that the disruption of homeostatic mechanisms leads to disease, and effective therapy must be directed toward restoring these homeostatic conditions.

[0152] Cytokines

[0153] Cytokines are small proteins secreted by cells that have a specific effect on cell-cell interactions and communication. "Cytokine" is a general term; other names include "lymphokine" (cytokines produced by lymphocytes), "monokine" (cytokines produced by monocytes), "chemokine" (cytokines with chemotactic activities), and "interleukin" (cytokines produced by a leukocyte that act on other leukocytes). Cytokines can act on the cells that secrete them (autocrine action), on nearby cells (paracrine action), or, in some cases, on distant cells (endocrine action). There are pro-inflammatory and anti-inflammatory cytokines. Significant evidence shows that certain cytokines / chemokines are involved not only in the initiation but also in the persistence of pathological pain by directly activating nociceptive sensory neurons.Certain inflammatory cytokines are also involved in central sensitization induced by nerve injury or inflammation, and are related to the development of contralateral hyperalgesia / allodynia.

[0154] Ions

[0155] Recently, the role of inorganic ions in wound healing applications has been explored. Ions play a key role in normal skin function, including epidermal barrier properties, maintenance of redox balance, enzymatic activities, tissue remodeling, and more. Chronic wound care is a concern, and new, cost-effective therapeutic strategies that modulate the wound microenvironment and cell behavior are needed. Specific signaling is required to initiate, modulate, and complete the molecular mechanisms that occur during the healing process. Extracellular matrix proteins, growth factors, and cytokines present in the wound bed are fundamental activators of blood coagulation.They also contribute to keratinocyte activation, modifying the function and expression of adhesion molecules that play a key role in inflammation and keratinocyte migration. Thus, certain biological markers and biochemical pathways are associated with each stage of wound healing physiology. However, dysfunction of these mechanisms can occur, resulting in fibrosis and chronic, non-healing ulcers, which are associated with morbidity and mortality due to tissue inflammation and infection.

[0156] Metal ions are essential catalytic and / or structural elements for many proteins, enzymes, and transcription factors. They can also modulate the expression and activity levels of various proteins through the activation of metal-sensitive transcription factors and conformational changes. Data from both older and more recent literature show that the action of many factors involved in the wound-healing machinery is modulated by interaction with copper ions. Copper plays a complex role in various cells, modulating several mechanisms of action of cytokines and growth factors, and is essentially involved in all stages of the wound-healing process.

[0157] Stages of wound healing

[0158] Wound healing is a natural physiological response to tissue injury. However, it is not a simple phenomenon, but rather involves a complex interaction between numerous cell types, cytokines, mediators, and the vascular system. The initial cascade of vasoconstriction and platelet aggregation is designed to stop the bleeding. This is followed by the arrival of a variety of inflammatory cells, beginning with neutrophils. These inflammatory cells, in turn, release a variety of mediators and cytokines to promote angiogenesis, thrombosis, and re-epithelialization. Fibroblasts, meanwhile, deposit extracellular components that will serve as scaffolding.

[0159] The inflammatory phase is characterized by hemostasis, chemotaxis, and increased vascular permeability, limiting further damage, closing the wound, removing cellular debris and bacteria, and promoting cell migration. The inflammatory phase typically lasts several days.

[0160] The proliferative phase is characterized by the formation of granulation tissue, re-epithelialization, and neovascularization. This phase can last several weeks.

[0161] The maturation and remodeling phase is where the wound reaches its maximum strength as it matures. Fibrin

[0162] Fibrin is a fibrous, non-globular protein involved in blood clotting. It is formed by the action of the protease thrombin on fibrinogen, causing it to polymerize. Polymerized fibrin, along with platelets, forms a hemostatic plug or clot over a wound site. Fibrin clots are open, porous networks, which is important for their role in hemostasis, fibrinolysis, and wound healing. The mechanical properties of fibrin are unique, as it is a viscoelastic polymer, meaning it has both reversible elastic characteristics and irreversible plastic or viscous properties. It also undergoes strain hardening, or increased stiffness under high stress, which helps prevent damage under severe conditions, such as arterial transection. Furthermore, fibrin clots possess extreme extensibility and compressibility, allowing them to deform considerably without rupturing.The properties of the fibrin network can be significantly modulated by a wide variety of environmental factors.

[0163] It has become increasingly clear that fibrin is essential for hemostasis, a key factor in thrombosis, and plays an important biological role in infection, inflammation, immunology, and wound healing. Furthermore, fibrinogen replacement therapy has become an important and preferred treatment for severe bleeding in trauma and surgical settings. Finally, fibrin is a unique biomaterial used as a sealant or adhesive, as a cell matrix, as a scaffold for tissue engineering, and as a carrier and / or vector for the delivery of specific drugs.

[0164] thrombocyte

[0165] A small, disc-shaped cell found in the blood and spleen. Thrombocytes are fragments of very large cells in the bone marrow called megacancells. They help form blood clots to slow or stop bleeding and to help wounds heal. Having too many or too few thrombocytes, or having thrombocytes that don't function properly, can cause problems. Checking the number of thrombocytes in the blood can help diagnose certain diseases or conditions. They are also called platelets.

[0166] Leukocytes

[0167] A type of blood cell produced in the bone marrow and found in the blood and lymphatic tissue. Leukocytes are part of the body's immune system. They help the body fight infections and other diseases. The types of leukocytes are granulocytes (neutrophils, eosinophils, and basophils), monocytes, and lymphocytes (T cells and B cells). Checking the number of leukocytes in the blood is usually part of a complete blood count (CBC). It can be used to look for conditions such as infections, inflammation, allergies, and leukemia. They are also called white blood cells. Inflammatory response

[0168] Inflammation is a biological response of the immune system that can be triggered by a variety of factors, including pathogens, damaged cells, and toxic compounds. These factors can induce acute and / or chronic inflammatory responses in the heart, pancreas, liver, kidney, lung, brain, intestinal tract, and reproductive system, potentially leading to tissue damage or disease. Both infectious and non-infectious agents, as well as cell damage, activate inflammatory cells and trigger inflammatory signaling pathways, commonly the NF-κB, MAPK, and JAK-STAT pathways.

[0169] Extracellular matrix (EMO)

[0170] The extracellular matrix (ECM) is the non-cellular component present in all tissues and organs. It provides not only an essential physical scaffold for cellular components but also initiates crucial biochemical and biomechanical signals required for tissue morphogenesis, differentiation, and homeostasis. The importance of the ECM is vividly illustrated by the wide range of syndromes, from minor to severe, that arise from genetic abnormalities in ECM proteins (Jarvelainen et al., 2009). Although fundamentally composed of water, proteins, and polysaccharides, each tissue has an ECM with a unique composition and topology that is generated during tissue development through a dynamic and reciprocal biochemical and biophysical dialogue between the various cellular components (e.g., epithelium, fibroblasts, adipocytes, endothelial elements) and the evolving cellular and protein microenvironment.In fact, the physical, topological, and biochemical composition of the ECM is not only tissue-specific but also remarkably heterogeneous. Cell adhesion to the ECM is mediated by ECM receptors, such as integrins, discoidin domain receptors, and syndecans. Adhesion mediates the docking of the cytoskeleton to the ECM and is involved in cell migration across the ECM. Furthermore, the ECM is a highly dynamic structure that is constantly being remodeled, both enzymatically and non-enzymatically, and its molecular components are subject to a myriad of post-translational modifications. Through these physical and biochemical characteristics, the ECM generates the biochemical and mechanical properties of each organ, such as its tensile and compressive strength and elasticity, and also mediates protection through a cushioning action that maintains extracellular homeostasis and water retention.Furthermore, the ECM directs essential morphological organization and physiological function by binding growth factors (GFs) and interacting with cell surface receptors to induce signal transduction and regulate gene transcription. The biochemical, biomechanical, protective, and organizational properties of the ECM in a given tissue can vary enormously from one tissue to another (e.g., lungs versus skin versus bone) and even within the same tissue (e.g., renal cortex versus renal medulla), as well as from one physiological state to another (normal versus cancerous). Ankylosis.

[0171] Angiogenesis is the growth of blood vessels from existing vasculature. It occurs throughout life, in both health and disease, beginning in utero and continuing into old age. No metabolically active tissue in the body is more than a few hundred micrometers from a blood capillary, which is formed by the process of angiogenesis. Capillaries are needed in all tissues for the diffusion of nutrients and metabolites. Changes in metabolic activity lead to proportional changes in angiogenesis and, therefore, to proportional changes in capillarity. Oxygen plays a fundamental role in this regulation. Hemodynamic factors are critical for the survival of vascular networks and for the structural adaptations of vascular walls.

[0172] Growth Factors

[0173] Growth factors are endogenous signaling molecules that regulate cellular responses necessary for wound healing processes, such as migration, proliferation, and differentiation. However, the exogenous application of growth factors has limited efficacy in clinical settings due to their low in vivo stability, restricted absorption through the skin around lesions, elimination by exudation before reaching the wound area, and other undesirable side effects. Sophisticated systems are required to control the spatiotemporal release of growth factors for their effective and safe use as regenerative treatments in clinical practice, such as biomaterial-based drug delivery systems (DDS).

[0174] Heparin

[0175] Heparin, a widely used anticoagulant for cardiac patients, has been studied for its effects on wound healing. The role of heparin in wound healing has been demonstrated in both in vitro and in vivo studies. In cell culture studies, heparin and growth factors are associated with rapid and effective endothelial cell repair. In clinical studies, burn patients and those with diabetic foot ulcers showed increased capillary circulation and decreased healing time. In contrast, heparin may not be beneficial in populations with ischemia, malnutrition, and vascular problems, although research in these populations is limited. However, heparin continues to offer therapeutic advantages for wound healing in carefully selected patients.

[0176] Betacellulin

[0177] Betacellulin (BTC) belongs to the epidermal growth factor (EGF) peptide ligand family, characterized by a six-cysteine ​​consensus motif that forms three intramolecular disulfide bonds, crucial for binding to the ErbB receptor family. BTC was initially described, purified, and cloned from a mouse insulinoma cell line. BTC is proteolytically processed from a membrane-anchored precursor and is a potent mitogen for a wide variety of cell types. BTC binds to and activates ErbB-1 and ErbB-4 homodimers and is further characterized by its unique ability to activate all possible heterodimeric ErbB receptors. BTC is widely expressed in most tissues and body fluids, including milk. Expression is particularly high in the pancreas, where it is thought to play a role in pancreatic beta cell differentiation.Although much is known about BTC's ErbB receptor binding characteristics and its effect on various cells cultured under different conditions, the challenge lies in determining BTC's role in vivo. This review will focus on the structure of BTC and the various biological effects attributed to this member of the EGF family.

[0178] Mesenchymal Stem Cells (MSCs)

[0179] Stem cells have two key characteristics: the ability to differentiate into different lineages and the capacity for self-renewal. Two main types of stem cells have been described: embryonic stem cells and adult stem cells. Embryonic stem cells (ESCs) are derived from the inner cell mass of the blastocyst and are associated with tumorigenesis. The use of human ESCs raises ethical and legal concerns. The use of adult mesenchymal stem cells presents fewer issues in this regard. Mesenchymal stem cells (MSCs) are stromal cells that have the capacity for self-renewal and also exhibit multilineage differentiation. MSCs can be isolated from a variety of tissues, including umbilical cord blood, endometrial polyps, menstrual blood, bone marrow, and adipose tissue.The ease of obtaining and the available quantity make these sources the most practical for potential experimental and clinical applications. Recently, MSCs have been found in new sources, such as menstrual blood and endometrium. There are likely more sources of MSCs waiting to be discovered, and these may be good candidates for future experimental or clinical applications.

[0180] Fibroblast

[0181] A fibroblast is a type of cell that contributes to the formation of connective tissue, a fibrous cellular material that supports and connects other tissues or organs in the body. Fibroblasts secrete collagen proteins that help maintain tissue structure.

[0182] Keratinocyte

[0183] As the most dominant cell type in the skin, keratinocytes play critical roles in wound repair, not only as structural cells but also by exerting important immune functions. This review focuses on the communication between keratinocytes and immune cells in wound healing, which is mediated by various cytokines, chemokines, and extracellular vesicles. Keratinocytes can also interact directly with T cells through antigen presentation. In addition, keratinocytes produce antimicrobial peptides that can directly kill invading pathogens and contribute to wound repair in many ways. We also review known epigenetic mechanisms that regulate keratinocyte immune functions, including histone modifications, non-coding RNAs (e.g., microRNAs and long non-coding RNAs), and chromatin dynamics.

[0184] Furthermore, the present invention also relates to a process for obtaining the antimicrobial composition and the scaffold described above, which comprises said antimicrobial composition. This process can be generally divided into five main phases involving: 1) Solution preparation, 2) Mixing, 3) Pouring, 4) Gradual cooling, and 5) Lyophilization; and where, in general, the following steps are carried out: i) Preparing individual solutions of a first natural polymer that will form the absorbable matrix, a calcium crosslinking component (calcium salts), a second natural polymer, and a third natural polymer;

[0185] i) Mix the solution of the second natural polymer from the previous step with the solution of the third natural polymer from the previous step in a suitable ratio and allow to stir; iii) Add the aforementioned calcium crosslinking component to the mixture from the previous step and allow to stir; iv) Add the solution of the first natural polymer from step i) in a first proportion to the solution of the second and third natural polymers from step i) to finish with a second proportion, and allow to stir; v) Add the silver nanoparticles at a controlled pH to obtain the antimicrobial composition of the present invention; vi) Pour the solution resulting from the previous step into 80 mm or 60 mm Petri dishes; vii) Allow the Petri dishes containing the base polymer solution to cool; viii) Peel the samples from the edges with a spatula once the time indicated in step vii has elapsed;ix) Place the Petri dishes with the polymer solution in a container with thermal insulating properties and freeze; x) Transfer the Petri dishes with the polymer solution from step ix) to an ultra-low temperature freezer; and xi) Remove the dishes from the ultra-low temperature freezer and place them inside the freeze dryer for the vacuum freeze-drying process.

[0186] In one particular modality, and by way of example without this implying a limitation to the type of natural polymer used, for the preparation of the solutions of step i) chitosan is used as the first natural polymer in a concentration that is in a range of 1 to 5%, calcium chloride (CaCh) in a concentration from 0.5 to 3%, gelatin as the second natural polymer in a concentration range from 0.5 to 3% and sodium hyaluronate as the third natural polymer in a concentration range from 0.005 to 1%.

[0187] In a particular modality, the mixing conditions of step i) are: gelatin as the second natural polymer and sodium hyaluronic acid as the third natural polymer in a proportion range from 5:1 to 10:1, and where the agitation also comprises a range from 30 min to 2 hours.

[0188] In another particular modality, the agitation of step iii) is carried out for a time range from 15 min to 1 h.

[0189] In one particular formulation, in step iv), the first natural polymer from step i) is chitosan, and the second and third natural polymers from step i) are gelatin and sodium hyaluronate, respectively. Furthermore, the first ratio ranges from 1:8 to 4:8, and the second ratio is 7:2:1. Finally, stirring is carried out for a period of time ranging from 2 to 4 hours.

[0190] In one particular modality, the silver nanoparticles of step v) are found in a concentration range from 1 to 10 ppm; and where the controlled pH range is also from 7 to 8.

[0191] In another particular modality, the temperature range of step vii) is from 2 to 10 o C, while the cooling time is at least 8 hours but no more than 28 hours.

[0192] In one particular modality the temperature of step ix) is in a range from -10 to -30°C, and the freezing time is from 12 hours to 48 hours.

[0193] In one particular mode, the appropriate temperature in step x) is in a range from -60 to -90°C and the freezing time in a range from 12 to 48 hours.

[0194] In another particular modality, the freeze-drying conditions of step x¡) are: a vacuum in a range from 0.600 to 0.040 mBar, a temperature ranging from -30 to -80°C and a time in a range of 12 to 48 hours.

[0195] To provide a better understanding of the invention described herein, the following examples are shown. It should be understood that these examples are for illustrative purposes only. Therefore, they should not limit the scope of this invention in any way.

[0196] EXAMPLES

[0197] Example 1. Preparation of the antimicrobial composition

[0198] 1. Preparation of solutions a) 2% chitosan solution A 2% w / v high molecular weight chitosan solution was prepared. This was achieved by adding acetic acid in a 1:100 ratio to water to obtain a 1%–1.5% w / v solution. Chitosan was then added in a 2:100 ratio, resulting in a 2% w / v chitosan solution. This solution was stirred overnight at a temperature of 55°C. e C (131 e F). b) 1.1% CaCh solution

[0199] A 1.1% w / v CaCh solution was prepared by adding CaCh at a ratio of 1.1:100 to the aforementioned HEPES solution. It was mixed until the salt was completely dissolved. c) 1% gelatin solution

[0200] A 1% w / v type B gelatin solution was prepared. To achieve this, type B gelatin powder was added to water at a ratio of 1:100. The solution was stirred at 55 e C (131 eF) until the gelatin was completely dissolved. c) 0.01% sodium hyaluronate solution

[0201] A 0.01% w / v sodium hyaluronate solution was prepared by adding a 1.6% w / v sodium hyaluronate solution in a 0.64:100 ratio to water to obtain a 0.01% w / v sodium hyaluronate solution. This solution was then heated to 35 e C (95 e F).

[0202] 2. Mixing

[0203] The gelatin solution was mixed with the sodium hyaluronate solution in a 7:1 ratio and stirred for 1 hour. After one hour, either calcium salt was added to the mixture and stirred for 30 minutes. Then, the chitosan solution was added in a 2:8 ratio to the gelatin and sodium hyaluronate solution, resulting in a 7:2:1 ratio of gelatin, calcium, and chitosan, respectively, and stirred for 3 hours. This step is optimal for the addition of silver nanoparticles at a concentration range of 1–10 ppm. The mixture was prepared at a pH of 7.2.

[0204] The final result of this step was the basic polymer solution for the scaffolding described in this application.

[0205] 3. Spill

[0206] Once the final solution was prepared, it was poured into 80 mm or 60 mm Petñ plates. 20 mL of solution were poured into each 80 mm plate to achieve a thickness of 3 mm, or 10 mL into each 60 mm plate to obtain the same thickness of 3 mm.

[0207] 4. Gradual cooling

[0208] The Petri dishes with the base polymer solution were cooled in a refrigerator to 4 e C (39.2 e F) for at least 12 hours but not for more than 24 hours. After 24 hours, the samples were loosened from the edges of the Petri dish with a spatula, and then the Petri dishes with the base polymer solution were placed in a polystyrene cooler and put in a freezer at -20 e C (-4 e F) for 24 hours. After this, the ice chest with the Petri dishes was transferred to an ultra-low temperature freezer at -80 e C (-112 eF) for at least 24 hours. In this sense, the polystyrene cooler was used to generate a gradual change in the temperature of the samples, which impacts the freezing time, and in turn, has a direct impact on the size of the ice crystals.

[0209] 5. Lyophilization

[0210] The Petri dishes were removed from the ultra-low temperature freezer and placed inside a freeze dryer. The freeze-drying parameters were a vacuum ranging from 0.750 mBar to 0.040 mBar, at -50 e C, and the process lasted 24 hours. Lyophilization in this step removed water from the samples and resulted in a basic polymer.

[0211] Example 2. Tests of incorporation of organic elements in the scaffolding of the present invention made from natural polymers.

[0212] The incorporation of elements into the polymer scaffold was verified using SEM and EDS, comparing the results of a polymer without additives and one synthesized with silver nanoparticles and calcium lactate. To conduct these experiments, samples were sectioned using a sharp blade to obtain 1 cm squares, cutting them from the central part of the polymer to accurately represent the microstructure.

[0213] Samples with increasing concentrations of silver nanoparticles were selected, including 1, 5, 10, and 150 ppm, in addition to 0.35 M calcium lactate in combination with 5 ppm of nanoparticles. Measurements were made with and without palladium coating to avoid erroneous measurements due to the proximity of silver and palladium on the periodic table, and all samples were observed at 100x, 500x, and 1500x magnification.

[0214] As can be seen in Figures 1 to 4, the scaffolds subject to this application were able to incorporate different organic elements into their structure. Energy-dispersive X-ray spectroscopy (EDS) was used to verify the capacity of the scaffolds, made from natural polymers, to incorporate the different organic elements tested into their base structures.

[0215] Figure 3 specifically shows the EDS spectrum of the scaffold subject to the present invention with quantification values ​​of each chemical element tested where the presence of silver can be seen at different levels, demonstrating a homogeneous dispersion of the same and a size variation of each nanoparticle.

[0216] In Figure 4 specifically, the presence of a first element, calcium (in green), and a second element, silver (in purple), can also be observed in EDS, demonstrating the capacity of the scaffolds subject to this application to simultaneously incorporate mixtures of different additives of interest for the intended therapeutic applications, such as antimicrobial elements (silver) and procoagulants (calcium). Example 3. Pore size of the scaffolds.

[0217] As explained above, one of the critical parameters for the success of the scaffolding subject to this application in solving the technical problem posed is the pore size of the scaffolding, since the cells need an appropriate physical space to be able to proliferate in the scaffolding.

[0218] Figures 5 to 8 show scanning electron microscopy (SEM) images of the scaffold that is the subject of the present invention, showing its porosity, which averages 179.7 µm; which is optimal for cell growth and vascularization; between 120 µm and 200 µm according to what has been reported in the state of the art and as proven by the inventors of the present application.

[0219] Example 5. Metabolic Activity of cells contained in the scaffold.

[0220] The WST-1 test is an assay used to measure cell proliferation and viability. To evaluate the effect of scaffolds on cell populations, samples from all experimental groups were first sterilized. These groups included scaffolds with calcium lactate at concentrations of 0.01, 0.25, 0.35, and 0.42 M, as well as scaffolds with silver nanoparticles at concentrations of 0.1, 5, and 10 ppm, with replicates for days 1, 3, 5, and 7. Sterilization was achieved by immersing the scaffolds in 70% ethanol for two hours. The residue was then removed, and the scaffolds were dried in a laminar flow hood. The sterile samples were immersed in cell culture medium overnight, after which the residue was removed. Cells were seeded without culture medium to promote adhesion to the scaffold. After one hour, culture medium was added, and the WST-1 reagent was added as needed for the measurement days.

[0221] The capacity of the scaffolds subject to this application to support the metabolic activity of the test cells was evaluated. The total number of cells living on the scaffold (control scaffold) was measured using a WST1 test compared to a control monolayer (without scaffold) on day 1 and day 3. The results shown in Figure 9 demonstrate the cell support capacity of the scaffolds, as the cell count increased significantly at the end of the test period.

[0222] Example 6. Impact on the 3D structure of the scaffolding.

[0223] The 3D structure of the scaffolding was analyzed using SEM images obtained at 100x magnification. ImageJ was used to trace the outline of at least 100 pores to obtain a list of individual areas. The equivalent diameter was then calculated from these results, and a statistical analysis was performed.

[0224] Tests were performed on the scaffolding subject to this application, analyzing more than 100 pores in groups containing different additives. The results obtained, shown in Figures 10A and 10B, demonstrate the scaffolding's ability to distribute material homogeneously, even when incorporating combinations of additives.

[0225] Example 7. Cell adhesion tests on cells contained within the scaffold of the present application.

[0226] Cell adhesion was assessed on the first day of WST-1. After seeding the cells onto the scaffold, they were incubated in culture medium for 24 hours. Once this period was complete, all samples were removed and transferred to a new plate, so that only the cells adhering to the scaffold were in the new plate, and all remaining cells were in the original plate. Cell activity was measured in both plates to compare cell activity.

[0227] Figures 11A and 11B show the results of an experimental protocol in which cells were seeded on the scaffolds of the present invention and left on them for one day, after which the percentage of adhesion of the seeded cells was determined.

[0228] A WST-1 reagent was used to collect data over a 7-day period in test groups with incremental concentrations of silver nanoparticles.

[0229] As shown in the results obtained and shown in Figures 11 A and 11 B, a significant increase in the adhesion of the test cells to the scaffolds subject to this request was obtained, as well as trends that demonstrate cellular activity.

[0230] The above demonstrates the ability of the scaffolds subject to this application to also promote the adhesion of the test cells.

[0231] Figure 12, on the other hand, is a fluorescent image of a monolayer mesenchymal stem cell culture that demonstrates the growth of the cells contained in the pores after three days compared to the controls.

[0232] Example 8. Scaffold degradation tests.

[0233] Degradation of the scaffold was observed by immersing the entire polymer in cell culture medium and leaving it at 37°C for 7 days, taking daily weight measurements and comparing them to the initial polymer weight. The percentage of degradation is given by the equation: Initial weight of sample ~ Final weight

[0234] % degradation: - - - — — - 100

[0235] Weight starts!

[0236] Figure 13 shows the degradation of the scaffold of the present invention over 7 days with respect to variations in calcium lactate concentration. The photographs in Figure 13 show the degradation after 5 days under different calcium concentration variations, thus demonstrating the degradability control exhibited by the scaffolds of this application, as well as their ability to withstand varying levels of coagulant and nanoparticles.

[0237] Many modifications and other embodiments of the invention will occur to a person skilled in the art to which the invention belongs, having benefited from the teachings presented in the preceding descriptions and associated drawings. It should therefore be understood that the invention is not to be limited to the specific embodiments and examples described, but that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are used herein, they are used only in a generic and descriptive sense and not for limiting purposes. It should also be understood that the raw materials from which the various components comprising the invention described herein may be manufactured, and other elements, may vary without departing from the scope and spirit of the invention, and therefore the embodiments referred to should not be considered limiting.

Claims

CLAIMS 1. A bioactive scaffold, characterized in that it comprises i) an absorbable matrix made from at least one natural polymer and at least one calcium crosslinking component and i) a bioactive compound in the form of a nanoemulsion with silver nanoparticles which in turn comprises a reducing agent, stabilizing agents, dispersing agents, pH agents and surfactants.

2. The bioactive scaffold according to claim 1, wherein the absorbable matrix is ​​made of at least one natural polymer selected from the group comprising gelatin, chitosan, hyaluronic acid, collagen, alginate, fibrin, silk fibroin, xanthan gum, gelatin or any combination thereof.

3. The bioactive scaffold according to claim 1, wherein the average size of at least 50% of the silver nanoparticles is between approximately 60 nm and approximately 140 nm.

4. The bioactive scaffold according to claim 1, wherein the calcium crosslinking component is any selected from the group comprising calcium chloride (CaCh), calcium carbonate (CaCO3), calcium phosphate (Ca3(PO4)2), calcium lactate (CeH CaOe), sodium alginate, pectin, carrageenan, chitosan-CaCO3, calcium gluconate, calcium acetate, and combinations thereof 5. The bioactive scaffold according to claim 1, wherein the colloidal silver nanoparticles are in a concentration range from 1 to 5 ppm.

6. The bioactive scaffold according to claim 1, wherein the reducing agent is selected from any of the group comprising ascorbic acid, sodium citrate, sodium borohydride (NaBF), gallic acid, combinations thereof or similar.

7. The bioactive scaffold according to claim 1, wherein the stabilizing agent is any of the group comprising Polyvinylpyrrolidone (PVP), citrate, Polyethylene glycol (PEG), Tannic acid, combinations thereof or similar.

8. The bioactive scaffold according to claim 1, wherein the dispersion medium is distilled water or similar.

9. The bioactive scaffold according to claim 1, wherein the pH agent is selected from the group comprising hydrochloric acid (HCl) or sodium hydroxide (NaOH), nitric acid (HNO3), acetic acid (CH3COOH), or the like. The bioactive scaffold according to claim 1, wherein the suitable surfactant, in the context of the present invention, is selected from the group comprising Tween 20, Tween 80, SDS, CTAB, Triton X-100, and any combination thereof.

10. A method for manufacturing a bioactive scaffold as claimed in any of claims 1 to 9, the method comprising: i) Prepare a solution comprising at least one first natural polymer; i) Prepare a solution comprising at least one calcium crosslinking component; iii) Prepare a solution comprising at least one second natural polymer; iv) Prepare a solution comprising at least one third natural polymer; v) Mix the solution containing the at least one second natural polymer from step iii) with the solution comprising the at least one third natural polymer from step iv) and stir this mixture; vi) Add the solution comprising the at least one calcium crosslinking component to the mixture from the previous step and stir; vii) Add the solution comprising the at least one natural polymer from step i) to the solution resulting from step vi) and continue stirring; viii) Add silver nanoparticles at a controlled pH to the previous mixture; ix) Pour the solution resulting from the previous step into a container and allow to cool; x) Freeze the previously cooled solution;and xi) Freeze-dry the frozen solution under vacuum to form the scaffold.; 11. The method according to claim 10, wherein the at least first, second and third natural polymers are selected from the group comprising gelatin, chitosan, hyaluronic acid, collagen, alginate, fibrin, silk fibroin, xanthan gum, gelatin or any combination thereof.

12. The method according to claim 10 wherein the calcium crosslinking component is any selected from the group comprising calcium chloride (CaCh), calcium carbonate (CaCO3), calcium phosphate (Cas(PO4)2), calcium lactate (CeH CaOe), sodium alginate, pectin, carrageenan, chitosan-CaCO3, calcium gluconate, calcium acetate, and combinations thereof 13. The method according to claim 10, wherein the colloidal silver nanoparticles are in a concentration range from 1 to 5 ppm.

14. The method according to claim 10, wherein the silver nanoparticles are in a concentration range from 1 to 10 ppm.

15. The method according to claim 10, wherein the controlled pH range is from 7 to 8.

16. A bioactive scaffold as claimed in any of claims 1 to 9, for use in healing wounds.

17. The use of a bioactive scaffold as claimed in any of claims 1 to 9, for manufacturing a pharmaceutical product for healing wounds.

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