Wound dressings

Abstract

The invention relates to wound dressings in the form of tissue scaffolds, gels and powders. The wound dressing comprising at least one polymer material and a coating agent coating the at least one polymer material, wherein the wound dressing is incorporated with an effective amount of an antimicrobial agent. The invention also extends to methods of making the wound dressings and also extends to wound dressings produced in accordance with the methods of the invention.

Description

[0001] WOUND DRESSINGS

[0002] FIELD OF INVENTION

[0003] THIS INVENTION is in the field of wound dressings, in particular scaffolds, more in particular permanent tissue scaffolds for use in skin and tissue regeneration. The invention extends to a method of making the wound dressing, in particular the scaffold. The invention further extends to the use of the wound dressing in healing a wound. The invention further extends to the wound dressing for use in a method of treating a wound.

[0004] BACKGROUND TO THE INVENTION

[0005] Recent developments in advanced technology and nanotechnology have proven to be promising solutions in therapeutics, including wound care management. Advanced wound dressings in the form of hydrocolloids, films, foams, hydrogels and scaffolds have been designed and used to solve health problems underlying wound care treatments. These wound dressings are designed to meet ideal requirements which include the ability to maintain a balanced moist environment, adherence to the wound, allow the exchange of oxygen and prevent bacterial infection. The requirements ensure the acceleration of wound healing and reduce the complications that are associated with the process. Wound dressings are designed as either temporary or permanent dressings. Temporary wound dressings provide, and control wound exudate absorption and protect them until closure. They also prevent or protect wounds from bacterial infection provided they contain an antimicrobial agent. These dressings are most suitable for superficial wounds, and they require to be changed to avoid bacterial contamination. Permanent dressings on the other hand tend to integrate with the skin and accelerate the tissue regeneration process. These dressings are designed in the form of tissue scaffolds that influence cell migration, adhesion, and proliferation to promote epidermal regeneration and reduce scarring.

[0006] Tissue scaffolds are suitable for deep chronic wounds that are too complicated to heal through conventional treatments. They are usually designed from natural and / or synthetic polymers. Only biocompatible and biodegradable polymers are used for the fabrication of tissue scaffolds. This is because the scaffolds are embedded in the wound and integrate with the skin tissue, therefore their toxicity must be thoroughly controlled. Synthetic polymers that have proven to be the best candidates for skin tissue scaffolds include poly(glycolic acid) (PGA), polyethylene glycol (PEG), poly(caprolactone) (PCL), poly(lactic-co-glycolic) acid (PLGA), polyvinylpyrrolidone (PVP) and polyurethane (Pll). These polymers are biocompatible, nontoxic, structurally stable, and biodegradable which makes them suitable to be applied in skin tissue regeneration. Natural polymers are derived from natural sources like plants and animals. By nature, they are biocompatible and biodegradable which makes them even more suitable for designing skin tissue scaffolds. The most used natural polymers in skin tissue scaffolds include alginate, dextran, chitosan, collagen, and gelatine.

[0007] One of the main requirements for skin tissue scaffolds is the ability to prevent bacterial infection. However, some natural and synthetic polymers are not antimicrobial active which is one of the drawbacks when it comes to wound healing. Bacterial infection is still a formidable issue in the wound healing process. Various approaches to dealing with bacterial infection in the wound-healing process have been reported. These include incorporating antibiotics and plant extracts, or antimicrobial active metal nanoparticles (NPs) on the scaffolds.

[0008] It is the object of the present invention to develop active tissue scaffolds that can be applied in the treatment of deep chronic wounds. It is particularly an object of the present invention to prepare a permanent nanocomposite scaffold for use in wound healing.

[0009] SUMMARY OF THE INVENTION

[0010] IN ACCORDANCE WITH A FIRST ASPECT OF THE INVENTION, THERE IS PROVIDED a wound dressing, in particular a tissue scaffold, the wound dressing comprising at least one polymer material and a coating agent coating the at least one polymer material, wherein the wound dressing is incorporated with an effective amount of an antimicrobial agent.

[0011] The at least one polymer material may be selected from synthetic polymers, natural polymers such as organic polymers, and combinations thereof.

[0012] The at least one polymer material may comprise a first polymer material and / or a second polymer material.

[0013] The first polymer material may be bonded, in particular crosslinked, with the second polymer material.

[0014] The first polymer material may be a polysaccharide, such as, chitosan. The second polymer material may be a protein-based polymer material, such as, gelatin.

[0015] The first and second polymer materials may be bonded by cross-linking.

[0016] The coating agent may be a biopolymer material, such as, polydopamine (PDA).

[0017] The antimicrobial agent may be silver or copper nanoparticles.

[0018] The wound dressing may incorporate pores designed to absorb wound exudate. These pores may be on a surface of the wound dressing and may partially or fully traverse or extend into the wound dressing.

[0019] The pores may be found dispersed across the surface of the wound dressing. Specifically, the pores may be a heterogeneous distributed on the surface of the wound dressing.

[0020] The pores may have a diameter ranging between 50 and 300 microns.

[0021] In one preferred embodiment, the wound dressing may be a 2D scaffold comprising a first polymer material coated with the coating agent and incorporating a therapeutically effective amount of the antimicrobial agent. The wound dressing may comprise about 93.2 % (w / w) of chitosan, about 6.2 % (w / w) of polydopamine, and about 0.6 % (w / w) of silver nanoparticles.

[0022] In another preferred embodiment, the wound dressing may be a 2D scaffold comprising the first polymer material that is cross-linked with the second polymer material and coated with the coating agent, and the wound dressing may incorporate a therapeutically effective amount of the antimicrobial agent. The wound dressing may comprise about 58 % (w / w) of chitosan, about 3.8 % (w / w) of polydopamine, about 38 % (w / w) gelatine and about 0.2 % (w / w) of silver nanoparticles.

[0023] In another preferred embodiment, the wound dressing may be a 3D scaffold comprising the first and second polymer which may be cross-linked and coated with the coating agent, and the wound dressing may comprise a therapeutically effective amount of the antimicrobial agent. In this other preferred embodiment, the wound dressing may comprise about 58% (w / w) of chitosan, about 3.8% (w / w) of polydopamine, about 38% (w / w) gelatine and about 0.2% (w / w) of silver nanoparticles.

[0024] It will be appreciated that the wound dressing may be in powder form or gel form. The gel may be the product before drying or a redispersion of the 2D / 3D scaffold in water. The powder is formed by mechanically crushing the 2D or 3D scaffold into fine particles.

[0025] IN ACCORDANCE WITH A SECOND ASPECT OF THE INVENTION, THERE IS PROVIDED a method of making a wound dressing, in particular a scaffold, more in particular a permanent scaffold, the method including: contacting at least one polymer with a coating agent and optionally with an effective amount of an antimicrobial agent, thus obtaining a mixture containing the at least one polymer, coating agent and optional antimicrobial agent; and drying the mixture containing the at least one polymer, coating agent and optional antimicrobial agent, thus obtaining the wound dressing.

[0026] In one embodiment in which the making of the wound dressing results in a 3D scaffold: The at least one polymer may be comprised in a solution comprising a first solution containing a first polymer that is contacted with a second solution containing a second polymer.

[0027] The contacting of the first solution comprising the first polymer with the second solution comprising the second polymer may comprise mixing the first and second solutions comprising the first and second polymers respectively, for a predefined period, of up to 2 hours, thus obtaining the solution comprising the at least one polymer.

[0028] In contacting the first solution comprising the first polymer with the second solution comprising the second polymer, a bonding agent may be added to one of the first and second solutions, preferably the first solution, prior to contacting the first solution with the second solution.

[0029] The bonding agent may be a crosslinking agent that is arranged to link, in particular facilitate, promote, and / or cause the crosslinking of the first polymer with the second polymer.

[0030] The bonding agent may be a natural crosslinking agent, such as, genipin.

[0031] The solution containing the first polymer may comprise of the first polymer dissolved in a solvent for the first polymer.

[0032] The solvent for the first polymer may be an acid, in particular an organic acid.

[0033] The organic acid may be a weak acid such as, acetic acid, more in particular glacial acetic acid. The first polymer may be a biomaterial, in particular a polysaccharide based material, such as alginate or chitosan, preferably chitosan.

[0034] The second solution comprising the second polymer may comprise of the second polymer dissolved in a solvent for the second polymer.

[0035] The solvent for the second polymer may be a polar solvent, such as, water.

[0036] The second polymer may be a protein-based polymer, such as gelatine.

[0037] The coating agent, that is contacted with the solution comprising the at least one polymer, may initially be in the form of a precursor of the coating agent.

[0038] The precursor of the coating agent may be comprised in a solution containing the precursor of the coating agent.

[0039] The precursor of the coating agent may be dopamine. The solvent for dopamine may be water.

[0040] The step of contacting the coating agent with the solution comprising the at least one polymer may be preceded by the step of contacting the precursor of the coating agent with the solution comprising the at least one polymer (i.e. , the first and second polymers), and the method may comprise the step of polymerizing the precursor of the coating agent, i.e., dopamine into the coating agent, i.e., polydopamine.

[0041] In polymerizing the dopamine into polydopamine, the solution comprising the crosslinked first and second polymers and the precursor of the coating agent may be contacted with a polymerizing agent, such as a base solution, that is arranged to cause the dopamine to self-polymerize into polydopamine. In other words, the method may comprise the step of exposing the solution comprising the first and second polymers to polymerizing conditions, such as adding a base solution to the solution comprising the first and second polymers, thus causing the precursor of the coating agent to self-polymerize.

[0042] The base solution may be an aqueous solution of ammonia, i.e. , NH4OH.

[0043] In another version, the solution comprising the first and second polymers may be contacted with pre-synthesised polydopamine.

[0044] The optional step of contacting the antimicrobial agent with the solution comprising the crosslinked first and second polymers and the coating agent may not be optional, and accordingly the antimicrobial agent may be contacted with the solution comprising the coating agent and the crosslinked first and second polymers.

[0045] The antimicrobial agent may be comprised in a solution. The solution comprising the antimicrobial agent may be a copper or silver nitrate containing solution.

[0046] In contacting the solution comprising the antimicrobial agent with the solution comprising the coating agent and the crosslinked first and second polymers, the method may include mixing the mixture of the antimicrobial agent, coating agent and the crosslinked first and second polymers.

[0047] The coating agent may have reducing agents arranged to reduce the ions of copper or silver in the copper or silver nitrate solutions into metallic copper or silver.

[0048] The reducing agents may be inherent to the coating agent and may include functional groups of, for example, catechol and amine functional groups.

[0049] The antimicrobial agent may be incorporated within the polymer matrix of the scaffold. The drying step may be a freeze-drying step, in particular a lyophilization step, which may comprise subjecting the resultant mixture comprising the coating agent, first and second polymers and the optional antimicrobial agent to a lyophilization process.

[0050] In a second embodiment in which the making of the wound dressing results in a 2D scaffold comprising one polymer, coating agent and silver nanoparticles:

[0051] The at least one polymer may be comprised in a solution comprising a first solution containing a first polymer.

[0052] The solution containing the first polymer may comprise of the first polymer dissolved in a solvent for the first polymer.

[0053] The solvent for the first polymer may be an acid, in particular an organic acid.

[0054] The organic acid may be a weak acid such as, acetic acid, more in particular glacial acetic acid.

[0055] The first polymer may be a biomaterial, in particular a polysaccharide based material, such as alginate or chitosan, preferably chitosan.

[0056] In one version, the coating agent, that is contacted with the solution comprising the first polymer, may initially be in the form of a precursor of the coating agent.

[0057] The precursor of the coating agent may be comprised in a solution containing the precursor of the coating agent.

[0058] The precursor of the coating agent may be dopamine. The solvent for dopamine may be water. The step of contacting the coating agent with the solution comprising the first polymer may be preceded by the step of contacting the precursor of the coating agent with the solution comprising the first polymer, and the method may comprise the step of polymerizing the precursor of the coating agent, i.e. , dopamine into the coating agent, i.e., polydopamine.

[0059] In polymerizing the dopamine into polydopamine, the solution comprising the first polymer and the precursor of the coating agent may be contacted with a polymerizing agent, such as a base solution, that is arranged to cause the dopamine to self-polymerize into polydopamine.

[0060] The base solution may be an aqueous solution of ammonia, i.e., NH4OH.

[0061] In another version, the solution comprising the first polymer may be contacted with pre-synthesised polydopamine.

[0062] The optional step of contacting the antimicrobial agent with the solution comprising the first polymer and the coating agent (either pre-synthesized or polymerized as mentioned above) may not be optional, and accordingly the antimicrobial agent may be contacted with the solution comprising the coating agent and the first polymer.

[0063] The antimicrobial agent may be comprised in a solution. The solution comprising the antimicrobial agent may be a silver nitrate containing solution.

[0064] In contacting the solution comprising the antimicrobial agent with the solution comprising the coating agent and the first polymer, the method may include mixing a mixture of the antimicrobial agent, coating agent and the first polymer. The coating agent may have reducing agents arranged to reduce the ions of silver in the silver nitrate solutions into metallic silver.

[0065] The reducing agents may be inherent to the coating agent and may include functional groups of, for example, catechol and amine functional groups.

[0066] The antimicrobial agent may be incorporated within the first polymer of the scaffold.

[0067] The drying step may comprise subjecting the mixture of the coating agent and the first polymer to thermal drying, in which the mixture of the coating agent and the first polymer is dried in a heated environment, such as an oven, thus obtaining a 2D film / scaffold wound dressing comprising chitosan, polydopamine and silver nanoparticles incorporated in the wound dressing.

[0068] In a third embodiment in which the making of the wound dressing results in a 2D scaffold comprising a first polymer, second polymer, a coating agent, and silver nanoparticles:

[0069] The at least one polymer may be comprised in a solution comprising a first solution containing a first polymer that is contacted with a second solution containing a second polymer.

[0070] The contacting of the first solution comprising the first polymer with the second solution comprising the second polymer may comprise mixing the first and second solutions comprising the first and second polymers respectively, for a predefined period, of up to 2 hours, thus obtaining the solution comprising the at least one polymer. In contacting the first solution comprising the first polymer with the second solution comprising the second polymer, a bonding agent may be added to one of the first and second solutions, preferably the first solution, prior to contacting the first solution with the second solution.

[0071] The bonding agent may be a crosslinking agent that is arranged to bond, in particular facilitate, promote, and / or cause the crosslinking of the first polymer with the second polymer.

[0072] The bonding agent may be a natural crosslinking agent, such as, genipin.

[0073] The solution containing the first polymer may comprise of the first polymer dissolved in a solvent for the first polymer.

[0074] The solvent for the first polymer may be an acid, in particular an organic acid.

[0075] The organic acid may be a weak acid such as, acetic acid, more in particular glacial acetic acid.

[0076] The first polymer may be a biomaterial, in particular a polysaccharide based material, such as, alginate or chitosan, preferably chitosan.

[0077] The second solution comprising the second polymer may comprise of the second polymer dissolved in a solvent for the second polymer.

[0078] The solvent for the second polymer may be a polar solvent, such as, water.

[0079] The second polymer may be a protein-based polymer, such as gelatine.

[0080] In one version, the coating agent, that is contacted with the solution comprising the at least one polymer, may initially be in the form of a precursor of the coating agent. The precursor of the coating agent may be comprised in a solution containing the precursor of the coating agent.

[0081] The precursor of the coating agent may be dopamine. The solvent for dopamine may be water.

[0082] The step of contacting the coating agent with the solution comprising the at least one polymer may be preceded by the step of contacting the precursor of the coating agent with the solution comprising the at least one polymer (i.e. , the first and second polymers), and the method may comprise the step of polymerizing the precursor of the coating agent, i.e., dopamine into the coating agent, i.e., polydopamine.

[0083] In polymerizing the dopamine into polydopamine, the solution comprising the (bonded) first and second polymers and the precursor of the coating agent may be contacted with a polymerizing agent, such as a base solution, that is arranged to cause the dopamine to self-polymerize into polydopamine.

[0084] The base solution may be an aqueous solution of ammonia, i.e., NH4OH.

[0085] In another version, the solution comprising the first and second polymers may be contacted with pre-synthesised polydopamine.

[0086] The optional step of contacting the antimicrobial agent with the solution comprising the bonded first and second polymers and the coating agent (which may be resynthesized or polymerized as mentioned above) may not be optional, and accordingly the antimicrobial agent may be contacted with the solution comprising the coating agent and the bonded first and second polymers. The antimicrobial agent may be comprised in a solution. The solution comprising the antimicrobial agent may be a silver nitrate containing solution.

[0087] In contacting the solution comprising the antimicrobial agent with the solution comprising the coating agent and the bonded first and second polymers, the method may include mixing the mixture of the antimicrobial agent, coating agent and the bonded first and second polymers.

[0088] The coating agent may have reducing agents arranged to reduce the ions of silver in the silver nitrate solutions into metallic silver.

[0089] The reducing agents may be inherent to the coating agent and may include functional groups of, for example, catechol and amine functional groups.

[0090] The antimicrobial agent may be incorporated within the polymer matrix of the scaffold.

[0091] The drying step may comprise subjecting the mixture of the coating agent, the first and second polymers to thermal drying, in which the mixture of the coating agent, the first and second polymers is dried in a heated environment, such as an oven, thus obtaining a 2D scaffold wound dressing comprising chitosan, gelatine, polydopamine and silver nanoparticles incorporated in the wound dressing.

[0092] IN ACCORDANCE WITH A THIRD ASPECT OF THE INVENTION, THERE IS

[0093] PROVIDED wound dressing(s) of the first aspect of the invention or the wound dressing(s) prepared in accordance with the second aspect of the invention for use in a method of treating a wound. The wound dressing(s) may be applied onto the wound and secured thereto by suitable securing means, such as, a gauze and an adhesive bandage.

[0094] IN ACCORDANCE WITH ANOTHER ASPECT OF THE INVENTION, THERE IS PROVIDED use of the wound dressing(s) of the first aspect of the invention or the wound dressing(s) prepared in accordance with the second aspect of the invention for treating a wound.

[0095] IN ACCORDANCE WITH YET ANOTHER ASPECT OF THE INVENTION, THERE IS PROVIDED a method of making a wound dressing, in particular a scaffold, more in particular a permanent scaffold, the method including: coating a scaffold comprising at least one polymer with a coating agent, wherein the scaffold and / or coating agent are optionally incorporated / impregnated with an antimicrobial agent.

[0096] The scaffold comprising the at least one polymer may comprise a first polymer that is bonded, in particular crosslinked, to a second polymer.

[0097] The first polymer may be a polysaccharide-based polymer, such as chitosan.

[0098] The second polymer may be a protein-based polymer, such as gelatine.

[0099] The antimicrobial agent may be an effective amount of metallic silver or copper nanoparticles.

[0100] The coating agent may be polydopamine.

[0101] In another version, the scaffold may comprise or consist of a first polymer only coated with the coating agent. BRIEF DESCRIPTION OF THE FIGURES

[0102] The invention will now be described in more detail by way of non-limiting example, with reference to worked experimental examples and to the accompanying drawings, in which:

[0103] Figure 1 shows a schematic diagram showing the synthetic route of 3D Ag NPs incorporated scaffolds;

[0104] Figure 2 shows photographed images of (1 ) CS, (2) Gel, (3) CS / Gel, (4) PDA / CS / Gel, (5) Ag@PDA / CS / Gel;

[0105] Figure 3 shows FTIR spectra of the synthesized scaffolds, showing their characteristic functional groups;

[0106] Figure 4 shows SEM micrographs (a) CS, (b) Gel, (c) CS / Gel, and (d)

[0107] PDA / CS / Gel;

[0108] Figure s shows (a) SEM micrograph, (b) magnified SEM micrograph, and (c) EDS spectrum of Ag@PDA / CS / Gel;

[0109] Figure 6 shows PXRD diffractograms of the synthesized scaffolds;

[0110] Figure 7 shows graphs showing (a) the fluid uptake (FU) and (b) the biodegradability of the scaffolds;

[0111] Figure 8 shows antimicrobial activity of (a, b) Ag scaffolds against E. coli and S. aureus. [ (A) CS, (B) Gel, (C) CS / Gel, (D) PDA / CS / Gel, (E & F) Ag@PDA / CS / Gel];

[0112] Figure 9 shows cell viability studies of the scaffolds towards BHK-21 cells; Figure 10 shows optical microscopy images (scale bar = 100 pm) of (a) untreated cells (control), (b) CS / Gel, (c) PDA / CS / Gel, (d) Ag@PDA / CS / Gel treated fibroblasts after 24 h and (B) 48 h of culture;

[0113] Figure 11 shows digital images depicting wound closure on (a) diabetic-induced and (b) non-diabetic rats: (Left wounds = no treatment; Right wounds = Scaffolds treated);

[0114] Figure 12 shows a graphical representation of wound healing kinetics;

[0115] Figure 13 shows digital images of the silicon ring placement (A) and the scaffold application (B) on the wounds; the wounds on the left are untreated and wounds on the right are treated with the 3D scaffolds;

[0116] Figure 14 shows digital images of the wound closure on the diabetes-induced rats; the wounds on the left were untreated and the wounds on the right were treated with the 3D scaffold;

[0117] Figure 15 shows a graphical representation of the wound healing kinetics of the 3D scaffold-treated wounds v untreated wounds;

[0118] Figure 16 shows histological H&E stained sections of wounds of diabetic rats obtained from skin tissue harvested on days 4, 8, 14 and 18 of the wound healing process;

[0119] Figure 17 shows digital images of the 2D scaffold placement on the wounds; the wounds on the left are untreated and the wounds on the right are treated with the 2D scaffolds;

[0120] Figure 18 shows digital images of the wound closure on the rats; the wounds on the left are untreated and the wounds on the right are treated with the 2D scaffold; and

[0121] Figure 19 shows a graphical representation of the wound healing kinetics of the 2D scaffold-treated wounds v untreated wounds. EXAMPLES

[0122] FEATURES OF THE INVENTION as described above will be apparent from the worked experimental examples that follow.

[0123] PART 1 : SYNTHESIS OF 3D SCAFFOLDS, EXPERIMENTS AND DISCUSSION

[0124] Synthesis of 3D scaffolds

[0125] The synthesis of PDA-coated chitosan / gelatine 3D scaffold decorated with Ag nanoparticles (NPs) (i.e., having nanoparticles of Ag dispersed on and incorporated in the scaffold) was conducted following the reaction scheme shown in Figure 1.

[0126] 1.5 g of chitosan (CS), in powder / particulate format, was dissolved in 2% (v / v) glacial acetic acid. To the CS solution, 1 % (w / v) of genipin (cross-linker) was added, and the mixture was stirred for 2 h. This was then followed by the addition of 1 g of gelatine (Gel), in powder form which was pre-dissolved in water. The mixture was stirred for 2 more hours at room temperature to crosslink the two polymers. The colour of the blend changed from clear to light pink, confirming the crosslinking of the two polymers. To coat CS / Gel with polydopamine (PDA), 0.1 g of dopamine, in powder form, dissolved in distilled water was added to the solution of the crosslinked CS / Gel mixture. This was followed by the addition of 0.1 mL of base (NH4OH) solution to create alkaline conditions for dopamine to self-polymerize. The reaction was carried out for 24 h. The colour of the mixture changed from light pink to black indicating the formation of PDA. CS / Gel and PDA / CS / Gel solutions were immediately refrigerated at -80 °C for 24 h. The frozen polymers were then lyophilized for 24 h to form CS / Gel and PDA / CS / Gel 3-dimensional (3D) scaffolds (in other words, the polymers are first frozen then they are placed into a freeze dryer for the solvent to be sucked out of the frozen polymers under vacuum). For application purposes, the scaffold may be used as it is in 3D shape or pulverized into fine powder. The scaffold can also be converted to a hydrogel by adding a predefined amount of ultrapure / sterile water.

[0127] To incorporate Ag NPs within the PDA / CS / Gel structure, in situ reduction of AgNOs was conducted. A solution of AgNOs (1 or 0.5 mmol) was added in PDA / CS / Gel solution. The mixture was stirred for 30 min at room temperature. Catechol and amine groups from PDA were able to completely reduce Ag ions to form Ag@PDA / CS / Gel. Upon reaction completion, the final solutions were refrigerated at - 80 °C for 24 h, followed by lyophilization for another 24 h to obtain Ag1 @PDA / CS / Gel (0.5 mmol AgNOs), Ag2@PDA / CS / Gel (1 mmol AgNOs) 3D scaffolds. In the context of the specification, “Ag1” and “Ag2” shall mean concentrations of 0.5mmol and 1 mmol of AgNOs respectively, and shall mean “inside or within the scaffold matrix”.

[0128] Properties of the materials used to synthesize the scaffolds

[0129] Ag and Cu NPs are known to possess a fast and broad spectrum of antimicrobial activity against viruses, fungi and gram-(positive and negative) bacteria. Additionally, these Ag NPs are also non-toxic to mammalian cells at regulated concentrations, rendering them suitable for wound healing applications. Incorporating Ag NPs on the surface of biocompatible polymers such as polydopamine (PDA), gelatine, and chitosan will further improve their applicability in wound healing.

[0130] PDA has been widely used in biomedical applications, including wound healing and as an antioxidant, due to its biocompatibility and non-toxic properties. It can also adhere to various kinds of surfaces due to the presence of catechol and amine functional groups.

[0131] Chitosan has been reported to be an effective component in wound healing, especially at the beginning due to its haemostatic effects.

[0132] Gelatine on the other hand is composed of peptides sequence which is very important for cell adhesion. In addition, gelatine can easily absorb exudates and control a moist environment thus promoting the wound-healing process.

[0133] Therefore, scaffolds comprising of the aforementioned materials will not only prevent bacterial infection but also promote the development of new skin tissue and promote a fast-healing process.

[0134] To determine the fluid uptake (Fll) of the 3D scaffolds, swelling studies were performed in phosphate buffered saline (PBS) (7.4%) and ultra-pure water at 37 °C. 100 mg (Wd) of each scaffold was added in separate glass vials followed by the addition of 2 Ml of PBS solution. The vials were then incubated at 37 °C. The Fll was recorded every 20 min for 2 h, and then after 24 h and 48 h. Before recording the Fll, the surface-absorbed PBS was removed by filter paper. The wet weight (Ww) of the scaffolds was recorded and the % Fll was determined using the equation below:

[0135] / M -

[0136] % FU = x 100

[0137] V J where %FU stands for percentage fluid uptake, Ww for wet weight, and Wd for dry weight as stated above.

[0138] Degradation studies The degree of biodegradability of the 3D scaffolds was determined over 14 days. Initial weight (Wo) of 100 mg of each scaffold was added to a glass vial followed by 2 mL of PBS. One vial was removed each day and the remaining scaffolds were dried over 72 h and their final (Wf) weights were recorded. The degradability of the scaffolds was then evaluated using the equation below: 100

[0139] Where Wo is the initial weight and Wf is the final weight.

[0140] Cell viability studies

[0141] The toxicity of the 3D scaffolds was determined using the Baby Hamster Kidney (BHK-21) cell line. The cell viability was assessed using the 3-(4,5-dimethylthiazol-2- yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell proliferation assay. The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillinstreptomycin antibiotic solution. The cells were grown in a 37 °C incubator with a humidified atmosphere of 5% CO2.

[0142] For the assay, the BHK-21 cells were seeded at a density of 1x106cells / mL in 96- well tissue culture plates. The cells were incubated at 37 °C under 5% CO2 for 48 h to allow for stabilization. The scaffolds’ releasing media was prepared in DMEM solution and stored in sterile tubes at 4°C for later use. Two-fold serial dilutions of the scaffolds’ releasing media were then introduced into the wells containing the cells, with final concentrations on the plates ranging between 7.8 - 1000 pg / mL. The plates were then incubated under standard culture conditions (37 °C and 5% CO2) for 72 h, after which the viability of the cells was determined using the MTS dye. The MTS solution, at a volume of 20 pL / well, was added and the plates were incubated at 37 °C for an additional 4 h. The optical density (OD) was then determined at 490 nm using a plate reader and untreated BHK-21 cells (cells without scaffolds) were used as control. The percentage of viable cells (%Viability) was calculated using the following equation:

[0143] Absorbance of Sample — Absorbance of Medium %Viability = — - - - - - - - - - - - - - - X 100

[0144] Absorbance of Control — Absorbance of Medium

[0145] Antimicrobial activity tests

[0146] Well diffusion method

[0147] The antimicrobial activity of the 3D scaffolds was tested using a conventional welldiffusion method against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Typically, Lysogenic Broth (LB) was prepared using ultra-pure water followed by autoclaving at 121 °C for 15 min. The bacterial strains (both E. coli and S. aureus) were each inoculated separately in the LB and cultured at 37 °C on a rotary shaker at 250 rpm overnight. Bacteria strains were then aseptically spread on the surface of Tryptic Soy Agar (TSA) plates, followed by the puncture of wells on the agar using a cork borer. 100 pL of scaffold release media (i.e., scaffold dissolved in a buffer) were introduced into the wells and the plates were incubated at 37 °C for 18-24 h. Following incubation, the clear zones around the wells (zones of inhibition) were measured using a ruler. The release media of the scaffolds was prepared by completely dissolving 4 mg of each scaffold in 4 mL of LB.

[0148] Minimum inhibitory concentration (MIC) and bacteriostatic studies

[0149] The MIC test was conducted using the standard microdilution method. Eight sterilized test tubes were used to conduct the tests. The concentration of bacteria in the tubes was adjusted to 1*106CFU / mL using the McFarland method. Two-fold dilutions of the 3D scaffold release media were prepared in 2 mL LB inoculated with bacterial cells. The concentration range of the scaffolds was 300 to 2 mg / mL from the first to the last tube. The test tubes were then closed and incubated at 37 °C for 24 h. The clear tube with the least concentration of the scaffold release media was then considered the MIC. The milky colour in other tubes was observed due to the growth of bacterial cells.

[0150] In vivo studies using 3D scaffolds: Study A

[0151] Six (6) male Sprague Dawley rats were acquired. The six four(4)-week-old rats were individually placed in cages, and the rodent food pallets and water were made readily available for the rats to access as they required. The room temperature was kept at ±25 °C and maintained under 12 h light:dark cycle. The food and water intake were monitored and their weight was recorded ad libitum. The rats were monitored for 3 more weeks until they were seven (7) weeks old. Of the six (6) rats, three (3) were induced with diabetes and the other three (3) were used as control.

[0152] Diabetic induction

[0153] Before diabetes induction, all six (6) rats were fasted overnight. The following morning, glucose level was tested in all rats prior to induction. Diabetes was then induced in three (3) of the six (6) rats using streptozotocin (STZ). 110 mg / kg of nicotinamide was administered to the three (3) rats followed by 55 mg / kg of STZ after 15 min. Nicotinamide was used to circumvent the severity of STZ. After 72 h the glucose level was recorded again, following overnight fasting. The glucose level was measured again after seven (7) days and there was no change which indicated that the rats were not diabetic. This was attributed to the nicotinamide overprotecting the beta cell of the pancreas which hindered the STZ effect. Another 55 mg / kg of STZ was administered again in 3 fasted rats without the co-administration of the nicotinamide. The glucose level was measured again after 72 h. The rats were found to be diabetic since the glucose level raised from an average of 4.5 mmol / dl to an average of 13 mmol / dl. The glucose of the control rats was still normal (around 4.5 mmol / dl) indicating that they were not diabetic. The glucose level was measured again after seven (7) days, and the STZ-administered rats were still diabetic and the control rats were not. The rats were then prepared for surgery / wound creation.

[0154] Surgery / Wound creation

[0155] A biopsy punch was used to create wounds in the rats 30 min before surgery, 0.1 mL of morphine was administered through intraperitoneal injection. The rats were then anaesthetized with isoflurane gas to maintain anaesthesia. Hair over the dorsal area was shaved off the skin and the area was cleaned for incision before the biopsy. Two full-depth wounds (2 mm) with a diameter of ±6 mm was created on the left and right side (Figure 1 ) using the biopsy punch. Forceps was used to lift the incised skin and scissors to cut the skin tissue from the body. The skin tissues were stored in 10% formalin buffer for analysis. The rats were treated for pain for three days post-wound creation.

[0156] Wounds treatment

[0157] The wounds on the left of the rats were only covered with sterile gauze and the wounds on the right were filled with scaffolds. Each scaffold on each of the wounds on the right was enclosed with dry sterile gauze and fixed with an adhesive bandage. The wound healing process was monitored by measuring the wound contraction over fourteen (14) days. The wound contraction / size was measured on days 0, 3, 7, 11 , and 14. At the end of the experiment, the rats were euthanized using sodium phenobarbital. The skin tissues from the healed wound area were harvested for further analysis. The degree of wound healing was also determined using the equation below: 100

[0158] In vivo studies using 3D scaffolds: Study B

[0159] Thirty (30) male Sprague Dawley rats aged between seven (7) to eight (8) weeks and weighing between 250 and 280g were individually placed in cages. The rodent food pallets and water were readily available for the rats to access as required. The room temperature was kept at ±25 °C and maintained under 12h light: dark cycle. The food and water intake was monitored and the weight was recorded according to the Wits Research Animal Facility (WRAF) protocols and study requirements.

[0160] Diabetes induction

[0161] The method of diabetes induction method adopted in study A resulted in the death of some of the diabetic animals. It has been previously reported that the mortality rate could reach up to 45% in STZ-treated rats. Therefore, a modified diabetes induction method was adopted for study B to avoid fatalities.

[0162] Prior to diabetes induction, all rats were weighed and fasted overnight. The following morning, glucose levels were tested in all rats before induction. Diabetes was then induced in all rats using streptozotocin (STZ). Nicotinamide at a dose of 25 mg / kg was administered first, followed by STZ at a dose of 25 mg / kg after a 15-m inute interval. Nicotinamide was dissolved in sterile water and STZ was dissolved in citrate buffer with a pH of 4.5. The STZ solution was injected within 5 minutes after dissolution to avoid degradation. After 3 days of the initial injection, no noticeable changes in glucose levels were observed in all animals. Additional doses of STZ were administered every 3 days until the animals were diabetic. The onset of diabetes in the rats occurred on various days, with only 1 successful diabetes induction observed after the 2 doses. After 3 doses, about 2 animals were diabetic and 3 were on the diabetes onset. After 4 injections 12 animals were diabetic, 6 were on the diabetes onset and 1 was non-diabetic. No further injections were administered after 4 doses. The animals were kept stable for a week and then all taken for surgery. It is important to note that before every administration of STZ, the rats fasted overnight, and glucose levels were measured on the morning of the STZ injection to confirm if the animals were diabetic.

[0163] Surqery / wound creation

[0164] Wound healing in humans primarily occurs via re-epithelialization, while in rats it occurs via muscle contraction. In study B, the splinted excisional wound model was used to ensure that the skin around the wound does not move / contract. This was done to minimize the wound contraction healing mechanism and allow for a more qualitative investigation of the molecular mechanisms involved in the wound healing process with the scaffold.

[0165] A biopsy punch (6 mm) was used to create wounds in the rats. One hour before surgery, 0.1 mL of morphine was administered through intraperitoneal injection. 1 hour after the morphine injection the rats were anaesthetized with isoflurane gas.

[0166] Hair over the dorsal area was shaved off the skin and the area was cleaned for incision before the biopsy. Two full-depth wounds (2 mm) with a diameter of ±6 mm were created on the left and right sides of the dorsal area using the biopsy punch. Forceps were used to lift the incised skin and scissors to cut the skin tissue from the body. 10 mm silicon rings were fixed onto the skin around the wound using surgical- grade superglue. The silicon rings were placed to manipulate the wound-healing mechanism by minimizing wound contraction and ensuring that wound healing by epithelialization is dominating. The animals were treated for pain for three days postwound creation. Additionally, after surgery, the rats were offered assisted feeding by crushing and wetting the food to ensure that they did not have difficulty feeding.

[0167] Wounds treatment

[0168] The wounds on the left were not treated while the wounds on the right were filled with the scaffold material. After the application of the scaffold material, the wounds were enclosed with Tegaderm tape and fixed with an adhesive bandage. The Tegaderm tape and bandages were changed on days 4, 8, and 14. The wound healing process was monitored by imaging the wounds over 18 days and measuring the wound contraction from the images.

[0169] To measure the wound closure, the bandages from at least three animals were removed, followed by the careful removal of the Tegaderm tape and the scaffold. The wounds were then imaged, the three animals were terminated / euthanized using sodium phenobarbital, and the wounded skin tissue was harvested for histological analysis. This was done on days 4, 8,14 and 18. The images were analysed for wound contraction measurements using Image J software to measure the area of the wound. The degree of wound closure was determined using the equation below: ... , .... Wound area day 0 -Wound area day t .. ■ ■■

[0170] Wound Closure (%) = - - -

[0171] Wound area day 0 — * lOOResults and discussion

[0172] Characterization of the scaffolds

[0173] The bioactive scaffolds were obtained by coating the cross-linked CS / Gel with PDA followed by in situ decoration with Ag NPs. The optical images of the scaffolds are shown in Figure 2. The pristine CS and Gel scaffolds were characterized by white colour, while the crosslinked CS / Gel appeared to be light pink. The pink colour was a confirmation of the successful crosslinking of CS and Gel. The coating of CS / Gel resulted in the change in colour to black which was an indication of the successful formation of PDA coating the CS / Gel scaffolds. The incorporation of the NPs did not have any influence on the colour of the scaffolds.

[0174] The chemical structures of the pristine (CS and Gel), cross-linked CS / Gel, PDA coated (PDA / CS / Gel), and the NPs’ incorporated scaffolds were studied using FT-IR spectroscopy. Figure 3 shows the FT-IR spectra of the scaffolds with their characteristic functional groups. The FT-IR spectrum of CS displayed a broad and a small vibrational band around 3250 cm’1and 2900 cm’1, attributed to the overlap of amines (-NH) and the hydroxyl (-OH) stretching modes, and the aliphatic C-H stretch, respectively. The spectrum also displayed peaks around 1634 and 1555 cm’1which correspond to the amide I and amide II vibrational modes (-CH3-C=O). The band around 1446 cm’1was attributed to the symmetrical deformation mode of CH3, while the one at 1246 cm’1was attributed to -OH groups. The vibrational modes associated with the saccharide units of CS are represented by bands around 891 and 1147 cm’1. The bands around 1073 cm’1and 1023 cm’1were attributed to C-N and C-0 stretching modes. The FT-IR spectrum of the Gel also displayed vibrational bands around 3250 cm-1which corresponds to the overlap of -NH and -OH vibrational modes. The band around 2900 cm’1was also attributed to the CH asymmetric stretch. The carbonyl and amino vibrational bands around 1637 cm’1and 1527 cm’1correspond to amide I and amide II, respectively. The vibration band around 1238 cm’1was attributed to amide III due to C-N stretching and -NH in-plain bending vibrations

[0054] , The vibrational bands around 1320 cm’1and 1437 cm’1were attributed to the wagging vibrations of proline side chains confirming the presence of type-l Gel. The FT-IR spectrum of CS / Gel shows all the characteristic functional groups that were present in the spectrum of the individual polymers. The increase in the intensity of the vibrational bands around 1025 cm’1was observed, indicating the interaction between the CS and Gel. Furthermore, the presence of a band around 942 cm’1also confirms the interaction between the two polymers. The vibrational band at 942 cm’1was absent from the Gel spectrum and was observed from the CS spectrum at lower energy (891 cm’1). These observations further confirm the interaction between the CS and Gel. The intensity of all the vibrational bands got reduced upon coating with PDA. PDA also has -OH and -NH functional groups which are also present in CS and Gel structures. These will cause the overlap of these vibrational bands at around 3250 cm’1. The chemical structure of the CS / Gel was maintained after coating. This shows that PDA did not chemically interact with CS / Gel but through hydrogen bonding. The intensity of the vibrational bands decreased further upon incorporation with both Ag NPs. A slight band shift towards low energies was observed. This could be due to the physical interaction between the NPs and polymers. 3D scaffolds with good porosity have added advantages in the wound healing process. They can control wound exudates, promote cell migration and guide vascular infiltration, making them suitable dermal substitutes for skin tissue regeneration. To study the scaffolds’ morphology / structure and the nature of their pores, SEM was used. Shown in Figure 4 are the SEM micrographs of (a) CS, (b) Gel, (c) CS / Gel, and (d) PDA / CS / Gel scaffold. CS showed porous interconnected circular-like 3D structures. Gel depicted irregular flake-like morphology which is also porous and interconnected. The pore sizes of the CS scaffold were between 300 pm and 600 pm. The pore structure of CS was slightly disturbed upon blending with Gel. CS / Gel appeared to have small pores compared to the individual polymers. In addition, the pores of CS / Gel were not well arranged, and the surface looked rougher compared to that of individual CS. The loss in structures and change in the surface texture of individual polymers (CS and Gel) upon blending could be an indication of a successful cross-linking. The structural features of CS / Gel were not affected upon coating with PDA. PDA / CS / Gel scaffold was structurally similar to the CS / Gel scaffold with the pore sizes ranging from 50 pm to 300 pm. The reason PDA coating could not affect the structural features of CS / Gel could be the fact that the interaction between PDA and CS / Gel was physical. These observations were also made during the FT-IR analysis, wherein introducing PDA in the CS / Gel scaffold did not affect their chemical structure.

[0175] SEM micrograph of Ag NPs decorated scaffold (Ag@PDA / CS / Gel) and its corresponding magnified micrograph are shown in Figure 5(a and b). The incorporation of NPs within the scaffold matrix could not disturb their structural features. The pores and the sizes of the decorated scaffolds were still like those of PDA / CS / Gel scaffolds. The magnified micrographs revealed the presence of Ag NPs within the matrix of the scaffolds. In other words, the Ag NPs were incorporated on the surface of the scaffold and were also dispersed within the scaffolds and dispersed on the crosslinked CS / Gel polymers. This was further confirmed by energy-dispersive X-ray spectroscopy (EDS) in Figure 5(c). These findings prove that PDA successfully coated the surface of the scaffolds. This is because there was no reducing agent used to reduce the Ag ion / salt into metallic Ag NPs. The reduction of Ag ions was facilitated by the presence of catechol and amine functional groups inherent in PDA.

[0176] The crystallinity and the phase structure of pristine polymers (CS and Gel), CS / Gel, PDA / CS / Gel, and Ag@PDA / CS / Gel were elucidated using PXRD. The diffractograms of pristine polymers, CS / Gel, and PDA / CS / Gel scaffolds are shown in Figure 6(a). A broad diffraction peak was observed between diffraction angles of 10° and 30° from the diffractograms of scaffolds without NPs. This diffraction peak was attributed to the amorphous nature of the polymers. Figure 6(b) shows the diffractogram of Ag@PDA / CS / Gel. The diffractogram of Ag@PDA / CS / Gel was characterized by two diffraction peaks: a highly intense broad peak between 15° and 25° and the other one at a 39° diffraction angle. The broad diffraction peak was attributed to the amorphous nature of the polymers as was also observed from the diffractograms of the pristine scaffolds. The diffraction peak at 39° was attributed to the (111) lattice plane of the face-centered cubic (FCC) crystal phase of pure metallic Ag (PDF 01-087-0720). The intensity of the Ag lattice plain was suppressed, and other diffraction peaks could not be observed. This could be due to the low concentration of the metal ions used relative to that of the polymers, which could also be the reason why other peaks were not present. The (111) plain is densely packed, meaning that it has more scatterers compared to other crystal plains, hence a pronounced peak. There were no other impurities observed from the scaffolds. The data obtained from the PXRD analysis confirmed the successful in-situ incorporation of the NPs within the polymer’s matrix. This further confirms that Ag species observed from the EDS were in a metallic rather than ionic state.

[0177] Swelling and Biodegradable studies

[0178] The swelling ability and the biodegradability of the scaffolds are very crucial in skin tissue engineering. The perfect scaffold for skin tissue engineering should be able to absorb wound exudate, keep the skin environment moist, and be able to degrade with time. An added advantage of the skin tissue scaffold would be its ability to unravel bacterial infections since it affects tissue regeneration and compromises the healing process. The swelling ability was investigated by determining the Fll (the amount of fluid the scaffolds absorb) with time. In this case, PBS at Ph 7.4 was used as the fluid. The amounts of Fll in % form were determined in the intervals of 20 min for 2 h and the last two values were recorded after 24 h and 48 h and the data is presented in Figure 7a together with the plot’s legend in Figure 7c. All scaffolds showed rapid swelling within the first 20 min. The Fll for pristine CS and Gel was relatively low compared to the other scaffolds but continued to increase until 80 min before it plateaued for the rest of the experimental time. CS showed the least Fll, and this can be attributed to the fact that it is slightly insoluble in water. Chitosan requires slightly acidic media (Ph < 6.5) to dissolve, and the PBS used in the present invention had a Ph of 7.4. CS / Gel scaffold has shown an improved swelling ability as indicated by a Fll of over 1400% within the first 20 min. The Fll of CS / Gel remained almost the same as time passed by, proving that it had reached equilibrium. The PDA / CS / Gel together with Ag@PDA / CS / Gel have shown similar Fll as CS / Gel for the period of the experiment (i.e., 2880 min / 2 days). The high Fll of CS / Gel, PDA / CS / Gel, and Ag@PDA / CS / Gel scaffolds can be attributed to the crosslinking of the two polymers (CS and Gel) which improve their mechanical properties.

[0179] As time passed by, the texture of the crosslinked (CS / Gel) scaffolds changed from being soft sponge-like to a stretch thick hydrocolloid mimicking the texture of skin tissue. This change in texture is very crucial in the wound healing process since it may facilitate the contact of the scaffolds with the skin tissue in the wounded area. The texture of PDA / CS / Gel, and Ag@PDA / CS / Gel scaffolds did not completely change, or they were rather showing a slow change. This could be attributed to the improved physical properties brought about by coating with PDA. This change in the scaffold’s texture when in contact with wound exudates is advantageous in the wound healing process and this can reduce the frequent change of wound dressing or eliminate it, consequently reducing the treatment cost.

[0180] The other important feature / property of the scaffolds for skin tissue engineering is the ability to maintain their structural integrity and be able to be degraded by body fluids or processes with time. The biodegradability study of the scaffolds was conducted over 7 days and the data is shown in Figure 7b with the corresponding legend in Figure 7c. The pristine Gel scaffold appeared to degrade faster than the other scaffolds, which completely degrade within 2 days. Unlike Gel, the pristine CS scaffold showed the least degradation rate compared to other scaffolds. These findings align clearly with the observations made on the FU studies. CS does not have a good swelling ability, which means it does not have good FU hence a slow degradation. The cross-linked CS / Gel scaffold on the other hand showed a low biodegradation rate compared to Gel, but a fast one compared to CS. The rate of biodegradation of the crosslinked CS / Gel started to slow down after two (2) days with 30% of the content remaining. The slow biodegradation continued for the duration of the experiment with biodegradation of almost 80% after 7 days. This controlled biodegradation of CS / Gel with time was anticipated due to crosslinking that improves the mechanical strength of the scaffolds.. The biodegradability of PDA coated, and NPs incorporated scaffolds (PDA / CS / Gel and Ag@PDA / CS / Gel, respectively) appeared to be increasing with time in a well-controlled manner compared to the rest of the scaffolds. This indicates the ability of PDA to control the biodegradability of the scaffolds with time, which is highly recommended in skin tissue engineering. Moreover, these scaffolds managed to maintain almost perfect structural integrity during the degradation period, which may help in terms of controlling the wound exudate while keeping a moist environment and providing a barrier against infection.

[0181] Antimicrobial activity of the scaffolds

[0182] The antimicrobial activity of the scaffolds was investigated against gram-negative (E. coli) and gram-positive (S. aureus) bacteria using a standard well-diffusion method. The ability of the scaffolds to inhibit bacterial growth was assessed by observing the clear zones of bacterial inhibition on the agar plates. Figures 8(a and b) show the photographs of agar plates with the bacterial inhibition zones due to pristine and Ag- based scaffolds against E. coli and S. aureus. All the scaffolds showed to have a certain level of antimicrobial activity. The antimicrobial activity of CS and Gel was very low as indicated by small zones of inhibition. It appears that crosslinking the two polymers and coating them could not have any significant effect on the antimicrobial activity of the polymers. This can be seen by more or less the same sizes of zones of inhibition of CS, Gel, CS / Gel, and PDA / CS / Gel scaffolds which were found to be between 9 mm and 10 mm against both strains as shown in table 1. The minimum inhibitory concentrations (MIC) of these scaffolds were also found to be the same (150 pg / ml). The MICs of these scaffolds were determined to investigate their antimicrobial activity, quantitatively, through the serial dilution method and are also shown in Table 1. Incorporating Ag NPs within the scaffolds significantly improved their antimicrobial activity. This is due to the wide spectrum of antimicrobial activity that Ag NPs possess. Incorporating these Ag NPs within the scaffolds brings about a synergistic effect toward antimicrobial activity. The zones of inhibition due to Ag NPs incorporated scaffolds appeared big / large compared to the other scaffolds which were not incorporated with Ag NPs. The zones of inhibition became even bigger / larger when the concentration of AgNOs was increased. This can be seen from the zones labelled F, in Figures 8(a and b), which represents Ag@PDA / CS / Gel with a high concentration of Ag. The improved antimicrobial activity of the scaffolds due to Ag NPs incorporation was further supported by the values of MIC obtained as shown in table 1. The MIC values decreased from 150 pg / ml to 37.5 pg / ml for Ag@PDA / CS / Gel. This decrease in MIC values is an indication of an improved antimicrobial activity of the scaffolds upon incorporation with Ag NPs.

[0183] Table 1 : Zones of inhibition and the MIC values of the scaffolds against E. coli and S. aureus. [ (A) CS, (B) Gel, (C) CS / Gel, (D) PDA / CS / Gel, (E& F) Ag2@PDA / CS / Gel],

[0184] Inhibition zones (mm) MIC (pg / ml)

[0185] Sample coli

[0186] A 9 8 150 150

[0187] B 9 8 - -

[0188] C 8 7 150 150

[0189] D 9 9 150 150

[0190] E 11 10 75 75

[0191] F 12 14 37.5 37.5 In vitro wound healing studies

[0192] To study the wound healing potential of the scaffolds, their biocompatibility was conducted on the BHK-21 (fibroblasts) cell line by MTS proliferation assay. Fibroblasts are responsible for the synthesis of collagen, which is a crucial protein that plays a role in cell repairing and development and forms part of Extracellular Matrix (ECM) deposition. As a result, collagen maintains the integrity of tissues, muscles, and other structures, which is crucial in wound healing. Therefore, understanding the biocompatibility or cytotoxicity of the scaffolds towards such cells is crucial as much as wound healing is concerned. The biocompatibility studies showed that exposing the scaffolds to the BHK-21 cells does not harm their viability. As shown in Figure 9, the cells treated with the scaffolds were as viable as the control cells (i.e., untreated cells). This was an indication that the scaffolds were not toxic to the cells and are therefore suitable for skin tissue repair.

[0193] To further investigate the biocompatibility of the scaffolds and their ability to be used in skin tissue repair, a cell adhesion study was conducted. In this study, cells were cultured on the surface of the scaffolds. Figure 10 shows the optical images of the cultured cells with (a) being the control culture (untreated) and (b-d scaffolds cultures after (A) 24 h and (B) 48 h. Uniform growth and morphology of fibroblasts were observed from both untreated, and scaffolds-treated cultures after 24 h. Cell adhesion within the matrix of scaffolds was also observed. Cells treated with scaffolds, as shown in Figures 10 (b-d) showed rapid growth compared to untreated cells within 24 h of culture. This is an indication that the scaffolds can influence cell proliferation. After 48 h of culturing, the cells appeared to have grown further compared to 24 h for both untreated and scaffolds-treated. The ability of these scaffolds to influence proliferation while maintaining the fibroblasts morphology makes them an excellent candidate for skin tissue repair.

[0194] In-vivo wound healing studies using 3D scaffolds: Study A

[0195] Figures 11(a) and (b) show the extent of wound closure in diabetic-induced and non-diabetic rats, respectively. The degree of wound closure was quantified by measuring the sizes of wounds on days 0, 3. 7, 11 and 14. The bar graph in Figure 12 represent the quantified degree of wound closure with time (in days). The scaffolds exhibited fast wound healing process as compared to the gauze. These observations were much clear on day 7, wherein over 60% wound closure was achieved in both diabetic and non-diabetic wounds. The gauze treated wounds displayed a delayed healing with just above 50% closure after 11 days. On day 3, the wound closure on diabetic-induced rats appeared to be slow compared to nondiabetic. As time passed by, the rate of wound healing on both scaffold-treated diabetic-induced and non-diabetic rats was comparable, which can be clearly observed on day 11 and 14. The scaffold-treated wounds, on both diabetic and non- diabetic rats were completely closed on day 14 whereas wound closure on the gauze treated rats was approximately 80%. These observations confirm the effectiveness of the scaffolds for the treatment of wounds even in compromised health.

[0196] In-vivo wound healing studies using 3D scaffolds: Study B

[0197] In study A it was observed that the wound healing process predominantly occurred by the skin contraction mechanism. To evaluate the wound healing by re- epithelization mechanism the wound model was modified to the splinted excisional wound model to minimize wound contraction. As shown in Figure 13, silicon rings were placed around the wound and fixed in position using surgical-grade superglue. The silicon rings acted as splints and prevented the skin around the wound from moving. This minimized the occurrence of wound healing by skin contraction and allowed for a more qualitative and quantitative evaluation of the wound healing process.

[0198] Figure 14 shows digital images of the wound healing progression from Day 0 to Day 18. On day 4 it can be observed that the scaffold integrates into the skin. This is a good characteristic, as it shows that the scaffold can be left inside the wound until it is completely healed. Furthermore, the scaffold absorbs the exudate and keeps the moisture regulated in the wound site. The untreated wound appears fresh and moist, indicating an earlier stage of healing compared to the treated wound. On day 8, it is observed that both wounds are progressively healing and closing. The untreated wound appears very pink and moist, which indicates that it is predominantly in the proliferative phase, which is characterized by active granulation tissue formation, high cellular activity, and ongoing re-epithelialization. The treated wound appears slightly dry but still retains a pink colouration. This is an indication of an advanced wound healing stage, transitioning from the late proliferative phase to the remodeling phase, characterized by reduced granulation tissue, advanced re-epithelialization, and less cellular activity. Furthermore, the untreated wound appears deeper, indicating some missing tissue, while the treated wound appears shallower, most likely due to the assistance of the scaffold in regenerating the tissue. On day 14, it can be observed that the treated wound is almost completely healed, it appears crusty and less active, indicating a transition into the remodeling phase. For the untreated wound, the healing process seems incomplete, healing is observed to be on the surface, with a more active wound under the dried exudate. This is an indication of higher cellular activity and ongoing re-epithelization. By day 18 all animals were completely healed and only the scar tissue was observed. However, the untreated wound still had a slight pink colouration, indicating a wound-healing process that is still active (slow healing rate). While the untreated wound appeared white, indicating tissue maturation and strengthening.

[0199] Figure 15 shows a bar graph representation of the wound healing in terms of percentage wound closure, as an average of 3-5 rats. A faster healing rate was observed for the treated wounds with approximately 40 % of wound closure in the initial stages (day 4) of wound healing compared to the untreated wounds which showed about 20% wound closure. By day 8 the wound closure for the untreated wound was approximately 60 %, while it was about 65 % for the treated wound. By day 14, 100% closure was observed for the scaffold-treated wounds and about 90% for the untreated wounds. There are no significant differences in wound closure rates between the wounds on days 8 and 14. This is because the wound healing mechanism in this study was predominantly re-epithelialization instead of contraction, due to the presence of the silicon rings that acted as splints, minimizing skin movement. The scaffold showed very promising wound-healing properties, especially in the early stages of healing.

[0200] The healing process of the treated and untreated wounds was further assessed by H&E staining of the healing wound tissue on days 4, 8, 14 and 18 as shown in Figure 16. On Day 4 both wounds were still in the early stages of healing, however, the treated wound was in a later stage of healing compared to the untreated wound. The treated wound spans the epidermis and the dermis, while the untreated wound extends into the subcutaneous tissue and the underlying skeletal muscle. The treated wound indicates a well-established epithelialization and granulation tissue is formed, suggesting later stages of the proliferative phase. The untreated wound indicates an earlier to mid-stages of the proliferative phase, with initial epithelialization and ongoing formation of granulation tissue. Furthermore, the untreated wound has a moderate number of fibroblasts, while the treated wound has a moderate to large number of fibroblasts, indicating the production of collagen and extracellular matrix components. On Day 8, the treated wound is well covered with granulation tissue, indicating a wound that is in the later stages of the proliferative phase of healing, characterized by significant fibroplasia, moderate epithelialization, and ongoing cellular activities necessary for tissue repair and wound closure. The untreated wound is still in the mid to later stages of the proliferative phase. The wound is mostly filled up by granulation tissue indicating an ongoing cellular activity. By day 14, the wounds have healed and are undergoing tissue maturation. The treated wound indicates more advanced progress and deeper tissue repair. On day 18 the wounds are in the remodelling phase. The treated wound has healed and has minimal remaining activity in the deeper tissues. The epidermis shows signs of previous healing activity with hyperplasia and hyperkeratosis, indicating a mature, remodelled state. The untreated wound has also healed at the surface but with more remodelling and tissue organization in the dermal and subcutaneous tissues, indicated by the presence of moderate fibroblasts, fibrous tissue, and mild immune cell infiltrates.

[0201] PART 2: SYNTHESIS OF 2D SCAFFOLDS, EXPERIMENTS AND DISCUSSION Synthesis of Ag-PDA / CS 2D film / scaffold

[0202] To synthesise the PDA-coated chitosan 2D films decorated with Ag NPs, 1 .5 g of CS was dissolved into 2% (v / v) glacial acetic acid and then blended with pre-synthesised PDA. To this, a solution of AgNOs was added. The mixture was stirred for 30 min at room temperature. Catechol and amine groups from PDA were able to completely reduce Ag ions to form Ag-PDA / CS hydrogel. The gel was poured into a petri dish and stored in an oven for 18 hours for drying and formation of the films.

[0203] Synthesis of Ag-PDA / CS / Gel 2D film / scaffold

[0204] To synthesise of PDA-coated chitosan / gelatine 2D films decorated with Ag NPs, 1.5 g of CS was dissolved into 2% (v / v) glacial acetic acid. To the CS solution, 1% (w / v) of genipin was added while stirring for 2 h. This was then followed by the addition of 1 g of gelatin which was pre-dissolved in water. The mixture was stirred at room temperature to crosslink the two polymers. The cross-linked CS / Gel hydrogel was then blended with pre-synthesized PDA, and to this, a solution of AgNO3was added. The mixture was stirred for 30 min at room temperature. Catechol and amine groups from PDA were able to completely reduce Ag ions to form Ag@PDA / CS / Gel hydrogel. The gel was poured into a petri dish and stored in an oven for 18 hours for drying and formation of the films.

[0205] In vivo studies using 2D scaffolds

[0206] Ten to eleven weeks-old male Sprague Dawley rats, weighing between 400 and 450 g, were individually placed in cages. The rodent food pallets and water were readily available for the rats to access as required. The room temperature was kept at ±25 °C and maintained under 12h light:dark cycle. The food and water intake were monitored, and weight was recorded accordingly. Diabetic induction was performed as mentioned above in the section describing 3D scaffolds.

[0207] Suroery / wound creation

[0208] A biopsy punch was used to create wounds in the dorsal area of the rats. One hour before surgery, 0.1 mL of morphine was administered through intraperitoneal injection. One hour after the morphine injection the rats were anaesthetized with isoflurane gas to maintain anesthesia. Hair over the dorsal area was shaved off the skin and the area was cleaned for incision before the biopsy. Two full-depth wounds (2 mm) with a diameter of ±6 mm were created on the left and right sides using the biopsy punch. Forceps were used to lift the incised skin and scissors to cut the skin tissue from the body. 10 mm silicon rings were fixed onto the skin around the wound using surgical-grade superglue. The animals were treated for pain for three days post-wound creation.

[0209] Wounds treatment

[0210] The wounds on the left were not treated, and the wounds on the right were filled with the 2D scaffold sheets as shown in Figure 17. The wounds on the left were enclosed with Tegaderm tape and fixed with an adhesive bandage. The Tegaderm tape and bandages were changed on days 4, 7, and 14. The wound-healing process was monitored by measuring the wound contraction over 14 days and imaging the wounds on days 7 and 14. To measure the wound contraction, the bandages from at least three animals were removed, followed by the careful removal of the Tegaderm tape and the scaffold. The wounds were then imaged, and the three animals were terminated. This was done on days, 7 and 14. The images were analyzed for wound contraction measurements by using Image J software to measure the area of the wound. At the end of the experiments, the rats were euthanized using sodium phenobarbital. The degree of wound healing was determined using the equation below: 100

[0211] Discussion on 2D scaffold wound treatment

[0212] Figure 18 shows the wound healing progression of the 2D scaffold treated wounds. The wounds on the left were left untreated while the wounds on the right were treated with the 2D scaffolds. To measure the wound size, the scaffold was removed from the wounds. On day 7 it is observed that the wounds have progressed to later stages of healing, although the untreated wounds still appear big with bright red colouration. The treated wounds appear smaller, with the Ag-PDA / Gel / CS treated wound showing signs of a more advanced stage of healing as indicated by a wound surface that appears dry with less pink colouration. From the images, it can be deduced that the scaffold-treated wounds are showing faster healing rate, especially the Ag-PDA / Gel / CS-treated wound. On day 14 all wounds have healed. The wounds have a white crusty surface, which indicates muscle maturation and strengthening.

[0213] Figure 19 shows a bar graph representation of the wound healing in terms of percentage wound closure, as an average of at least 3 rats. On day 7 a faster healing rate was observed for the Ag-PDA / Gel / CS treated wounds, with approximately 70 %. The Ag-PDA / CS treated wounds showed approximately 60% wound closure, while the untreated wounds showed about 50% wound closure. On day 14, a 100% healing rate was observed for all the wounds.

[0214] CONCLUSION

[0215] The inventors of the present invention were able to showcase for the first time the coating of crosslinked chitosan / gelatine scaffolds with PDA as well as the properties and therapeutic effects obtainable by the use of these scaffolds. The inventors also showed that the therapeutic effects of the scaffolds were obtainable even in scaffolds that did not have Ag NPs, although such scaffolds have a reduced antimicrobial activity. The inventors found that the presence of a PDA coating layer containing functional groups had the ability to reduce AgNOs metal ions into Ag, facilitated good polymer / metal interaction, and resulted in the dispersion of Ag NPs on the polymer surface. The use of the 3D scaffold significantly accelerates the wound healing process compared to untreated wounds. The scaffold not only integrates well into the wound, regulating moisture and absorbing exudate but also facilitates faster closure and more advanced healing. By day 14, scaffold-treated wounds achieve full closure and enter the remodelling phase, while untreated wounds show slower healing rates and ongoing activity. Histological analysis confirms that scaffold-treated wounds exhibit more mature and organized tissue repair, highlighting the scaffold's efficacy in enhancing early and overall wound healing processes.

[0216] In addition, the use of the 2D scaffolds, particularly the Ag-PDA / Gel / CS composite, significantly accelerates the wound healing process compared to untreated wounds. By day 7, 2D scaffold-treated wounds, especially those treated with Ag-PDA / Gel / CS, exhibit a more advanced healing stage. This demonstrates the effectiveness of the 2D scaffold treatments in enhancing the wound healing rate.

[0217] The 2D and 3D scaffolds of the present invention, especially those incorporated with Ag NPs, endowed biodegradable properties with excellent antimicrobial activity against gram-negative and gram-positive bacterial strains. Moreover, the 2D and 3D scaffolds demonstrated appropriate biocompatibility with good cell adhesion on their surfaces. Therefore, it is expected as evidenced by the results reported above, that the novel 2D and 3D scaffolds of the present invention should accelerate the woundhealing process since they possess relevant wound healing properties.

[0218] While the invention has been described in detail with respect to a specific embodiment and / or example thereof, it will be appreciated that those skilled in the art, upon attaining an understanding of the foregoing may readily conceive of alterations to, variations of and equivalents to these embodiments.

Claims

CLAIMS1 . A tissue scaffold comprising at least one polymer material and a coating agent coating the at least one polymer material, wherein the tissue scaffold is optionally incorporated with an effective amount of an antimicrobial agent.

2. The tissue scaffold according to claim 1 , wherein the at least one polymer material comprises a first polymer material and a second polymer material.

3. The tissue scaffold according to claim 2, wherein the first polymer material is bonded with the second polymer material.

4. The tissue scaffold according to claim 2 or 3, wherein the at least one polymer material comprising the first and second polymer materials is selected from synthetic polymers, natural polymers such as organic polymers, and combinations thereof.

5. The tissue scaffold according to claim 4, wherein the first polymer material is a polysaccharide such as chitosan.

6. The tissue scaffold according to claim 4 or 5, wherein the second polymer material is a protein-based polymer material such as gelatin.

7. The tissue scaffold according to any one of claims 2 to 6, wherein the first and second polymer materials are bonded by cross-linking.

8. The tissue scaffold according to claim 1 , wherein the at least one polymer material comprises or consists of a first polymer material.

9. The tissue scaffold according to claim 8, wherein the first polymer material is selected from synthetic polymers, and natural polymers such as organic polymers.

10. The tissue scaffold according to any one of the preceding claims, wherein the coating agent is not optional, and wherein the coating agent is a biopolymer material, such as polydopamine (PDA).

11. The tissue scaffold according to any one of the preceding claims, wherein the antimicrobial agent is silver or copper nanoparticles, preferably silver nanoparticles.

12. The tissue scaffold according to any one of the preceding claims, incorporating pores arranged to absorb wound exudate, wherein the pores are on a surface of the tissue scaffold and at least partly traverse into the tissue scaffold.

13. The tissue scaffold according to any one of claims 8 to 12, wherein the tissue scaffold is a 2D scaffold comprising the at least one polymer material comprising or consisting of the first polymer material.

14. The tissue scaffold according to any one of claims 2 to 7 and 10 to 12, wherein the tissue scaffold is a 2D scaffold comprising the at least one polymer material comprising the first polymer material and second polymer material.

15. The tissue scaffold according to any one of claims 2 to 7 and 10 to 12, wherein the tissue scaffold is a 3D scaffold comprising the first polymer material and second polymer material.

16. A method of making a tissue scaffold, the method including: contacting at least one polymer with a coating agent and an effective amount of an antimicrobial agent, thus obtaining a mixture containing the at least one polymer, coating agent and antimicrobial agent; anddrying the mixture containing the at least one polymer, coating agent and antimicrobial agent, thus obtaining the tissue scaffold.

17. The method according to claim 16, wherein the at least one polymer is comprised in a solution comprising a first polymer and a second polymer.

18. The method according to claim 17, wherein prior to forming the solution comprising the first and second polymers, the method includes: contacting a first solution comprising the first polymer with a second solution comprising the second polymer, and contacting a bonding agent with one of the first and second solutions, preferably the first solution, prior to contacting the first solution with the second solution.

19. The method according to claim 18, wherein the bonding agent is a crosslinking agent that is arranged to facilitate the crosslinking of the first polymer with the second polymer.

20. The method according to claim 18 or claim 19, wherein the bonding agent is a natural crosslinking agent such as genipin.

21. The method according to any one of claims 17 to 20, wherein the first polymer is a biomaterial, in particular a polysaccharide such as alginate or chitosan, preferably chitosan.

22. The method according to any one of claims 17 to 21 , wherein the second polymer is a protein-based polymer such as gelatine.

23. The method according to any one of claims 16 to 22, wherein the coating agent is in the form of a precursor of the coating agent.

24. The method according to claim 23, comprising facilitating the precursor of the coating agent to polymerize, thus forming the coating agent.

25. The method according to claim 24, wherein in facilitating the precursor of the coating agent to polymerize, the method comprising exposing the solution comprising the first and second polymers to polymerizing conditions, such as adding a base solution to the solution comprising the first and second polymers, thus causing the precursor of the coating agent to self-polymerize.

26. The method according to claim 25, wherein the base solution is an aqueous solution of ammonia.

27. The method according to any one of claims 23 to 26, wherein the precursor of the coating agent is dopamine and the coating agent is polydopamine.

28. The method according to any one of claims 16 to 22, wherein the coating agent is pre-synthesized polydopamine.

29. The method according to any one of claims 16 to 28, wherein the drying step comprises freeze-drying, in particular lyophilization.

30. The method according to any one of claims 16 to 28, where in the drying step comprises thermal drying.

31. The method according to claim 16, wherein the at least one polymer comprises or consists of a first polymer.

32. The method according to claim 16 or 31 , wherein the coating agent is presynthesized polydopamine.

33. The method according to any one of claim 16 and 31 to 32, wherein the drying step comprises thermal drying.

34. A tissue scaffold produced in accordance with the method of any one of claims 16 to 30, or any one of claims 16 and 31 to 33.

35. A tissue scaffold of claim 34 for use in the treatment of a wound.

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