A composition for a wound dressing facilitating skin wound healing and a method of manufacture thereof

Abstract

The invention relates to the technical field of biomedical engineering and pharmacology and relates to, but is not limited to, a method for the composition and production of a wound dressing for the healing of complete and chronic skin wounds.

Description

[0001] A COMPOSITION FOR A WOUND DRESSING FACILITATING SKIN WOUND HEALING AND A METHOD OF MANUFACTURE THEREOF

[0002] TECHNICAL FIELD

[0003] The invention relates to the technical field of biomedical engineering and pharmacology and relates to, but is not limited to, a method for the composition and production of a wound dressing for the healing of complete and chronic skin wounds.

[0004] PRIOR ART

[0005] Skin wounds are classified as superficial / partial-thickness or full-thickness wounds according to the depth of the damaged tissue. Superficial / partial wounds involve skin damage only to the lower epidermis, while full-thickness wounds include subcutaneous tissue. Skin wounds are also categorized based on their development process as either acute or chronic wounds. Acute wounds are caused by temporary factors and tend to heal within a short period of time. These wounds are subject to relatively few inhibitory factors, and healing is typically completed within 8 to 12 weeks, depending on the size and depth of the wound. Chronic wounds are wounds caused by a persistent venous, arterial or neuropathic agent and the wound healing process does not proceed as expected. Chronic wounds therefore heal very slowly (more than 3 months), recur frequently and the healing process is hampered by many systemic (inflammation) and local (lack of growth factors) factors.

[0006] Three-dimensional (3D) tissue scaffolds, such as wound dressings, not only cover the wound and provide a physical barrier against external infections, but also support the formation of skin tissue by serving as a scaffold for both dermal fibroblasts and keratinocytes. The ideal tissue scaffold should resemble natural ECM, support cell adhesion, proliferation and differentiation, exhibit appropriate physical and mechanical properties to protect the wound against external infection, be biocompatible and biodegradable to contribute to new tissue formation, not lose biocompatibility while degrading, not form toxic products, form a porous structure and have a high surface / volume ratio, come together with cells and biologically active molecules to support tissue regeneration and repair.

[0007] As is known, the natural ECM in the dermis layer is composed of nanoscale collagen fibers and provides structural integrity and mechanical strength to skin tissues. Therefore, for wound healing, the biologically inspired scaffold production approach is to create ECM analogs composed of nanoscale fibers that possess structures and functions similar to the natural ECM. In the literature, various tissue scaffolds are produced based on many different natural or synthetic materials and using different methods. In particular, one production technique that allows the design and fabrication of biomimetic structural scaffolds offering potential applications in wound healing is the electrospinning method.

[0008] The electrospinning method is an efficient technique that provides a flexible and relatively simple approach for nanofiber production. Among the materials used for wound healing studies are hydrogels, decellularized porcine dermal scaffolds, and freeze-dried or gas-foamed scaffolds. However, these materials lack the ability to mimic the ECM architecture of the skin. Recently, the electrospinning method has attracted considerable interest in wound healing, and with this technology, biomimetic nanofibrous materials with biologically desirable properties are produced from a wide variety of natural and synthetic polymers. The ECM of the skin is composed of fibrous structural proteins including collagens, elastins, laminins, various polysaccharides, and proteoglycans (such as dermatan sulfate, hyaluronan, etc.). During the wound healing process, the ECM plays an active role by physically providing a scaffold that supports the cells and by creating the necessary conditions for cell adhesion, proliferation, migration, and differentiation. The signaling pathway for healing is rapidly initiated, and fibroblasts are attracted to the dermal layer. In this way, the secretion of collagen in the ECM and various cytokines (growth factors, angiogenic factors) for the repair of the damaged tissue begins. Nanofibers, due to their formation, possess a large surface area and a three-dimensional porous structure. Owing to their nanometer-scale dimensions and randomly oriented structures, they mimic the ECM structure of the skin. Wound dressings are used for wound closure in scientific studies in the literature and in some clinical applications. However, since their combination with cellular elements can provide higher tissue regeneration, their combination with mesenchymal stem cells is preferred. Nevertheless, as the cells must be added to the wound dressing immediately before application, the requirement for additional equipment and personnel for cell thawing procedures at application centers poses a challenge and limits clinical implementation. In addition, in order to preserve MSCs for a long period, they currently need to be frozen at -80°C or -196°C, and during the thawing process at the application stage, there is a cellular loss of approximately 10-20% in number. In addition to cellular loss, it also has disadvantages such as a decrease in cell viability. MSCs need to be transported to hospitals or application centers in a cold chain logistically. These also bring additional costs to the product. Current wound treatments (antibacterial agents) are not sufficient for complete tissue regeneration, and deep wounds generally heal with scar formation.

[0009] Therefore, in order to overcome the above-mentioned technical problems and disadvantages, the development of a nanofiber wound dressing composition together with mesenchymal stem cells is of great importance. In this regard, conducting research and development activities in the relevant technical field has become a necessity.

[0010] BRIEF DESCRIPTION OF THE INVENTION

[0011] The invention relates to the technical field of biomedical engineering and pharmacology, and is not limited thereto, but is concerned with a wound dressing composition that enables the healing of full-thickness and chronic skin wounds.

[0012] The wound dressing composition subject to the invention is provided with an ideal tissue scaffold. To achieve this, it is aimed to present a tissue scaffold that resembles the natural extracellular matrix (which may be abbreviated as ECM), supports cell adhesion, proliferation, and differentiation, protects the wound against external infections, is biodegradable and biocompatible, has a porous structure, and supports tissue regeneration.

[0013] An object of the invention is to provide a wound dressing that preserves the viability of mesenchymal stem cells.

[0014] Another objective of the present invention is to provide a wound dressing that contributes to the tissue scaffold providing the aforementioned technical solution and advantages by imparting immunomodulatory and regenerative effects. In this way, a wound dressing with enhanced wound healing properties and high performance supported by the scaffold can be achieved, also allowing a reduction in healing time.

[0015] The treatments of similar chronic and full-thickness wounds in the current technique have a series of problems such as wound contraction, delayed vascularization, scar formation, poor integration with healthy tissue, and high cost. The present inventors provide a composition that enables wound healing by eliminating the above-mentioned technical drawbacks and deadlocks.

[0016] DETAILED DESCRIPTION OF THE INVENTION

[0017] In this detailed description, the subject of the invention relates to a wound dressing composition suitable for use in the technical fields of pharmacology and biomedical engineering.

[0018] The wound dressing composition subject to the invention contains components having wound healing functions; in this way, it is provided with a structure that will function particularly in the treatment of challenging wounds considered as chronic and full-thickness wounds.

[0019] The wound dressing subject to the invention comprises at least one tissue scaffold component and at least one active component. The present inventors include at least one from the group consisting of polycaprolactone, polyethylene oxide, and polyvinyl alcohol as the tissue scaffold component in obtaining the composition.

[0020] Polycaprolactone (abbreviated as PCL) is included in order to provide or improve properties such as mechanical strength and structural integrity, enhanced processability, and a cell-friendly surface. PCL is a biocompatible (accepted by the body), biodegradable (can dissolve in the body over time), non-toxic, and FDA- approved semi-synthetic polymer.

[0021] In the invention, polyvinyl alcohol (PVA) and polyethylene oxide (PEO) are used as a modifying agent to prepare new medical wound dressings with improved properties. PVA and PEO nanofiber membranes with different PVA and PEG contents are prepared using electrospraying technology. The biomimetic natural extracellular matrix structure of the nanofiber membranes is obtained by adjusting the PVA and PEO contents. Nanofiber membranes containing PVA and PEO possess improved biocompatibility.

[0022] The composition subject to the invention comprises at least one mesenchymal stem cell as the active component. In this invention, the reinforcing component is included in the composition to accelerate the wound healing process and to improve healing performance.

[0023] In this invention, “mesenchymal stem cells (abbreviated as MSCs)” refer to stromal cells isolated from tissues that, in addition to their tissue regeneration capacity, exhibit anti-inflammatory effects in immune responses. MSCs are involved in the tissue regeneration process either directly (by differentiating into tissue cells) or indirectly (by secreting growth factors), and they show great potential in tissue regeneration including myocardium, blood vessels, bone, cartilage, and skin.

[0024] Stem cell-based therapies have recently become the focus of numerous studies aimed at accelerating wound healing. An important step in this regard is the development of suitable scaffolds for cell delivery that promote the viability and homogeneous distribution of cells throughout the wound bed. These systems must be rigid enough to facilitate handling, yet soft enough to limit damage to the newly formed wound tissue and ensure patient comfort. However, in the treatment of critically sized and geometrically undefined wounds, the clinical use of two- dimensional (2D) densely packed nanofiber membranes is limited due to their superficial porous structures and tightly packed arrangement, which restrict cell infiltration and proliferation. On the other hand, three-dimensional (3D) scaffolds composed of electrospun nanofibers promote cellular infiltration and are developed for dermal repair / regeneration, serving as a synthetic scaffold.

[0025] As the active component, mesenchymal stem cells may be embedded, entrapped, or seeded within the tissue scaffold components.

[0026] What is critical here is that the tissue scaffold component must be capable of releasing the active agent in order to provide therapeutic efficacy against the wound. To enhance this efficacy, the scaffold component is expected to be three- dimensional and porous.

[0027] The invention relates to a wound dressing production method combined with mesenchymal stem cells, developed for use in the healthcare sector together with centers performing cellular therapy.

[0028] In the composition subject to the present invention, production methods are provided that allow the tissue scaffold to be obtained in a three-dimensional nanofiber form in a preferred application. In the relevant technical field, it has been determined that tissue scaffolds in a two-dimensional structure, which are superficially porous and tightly packed, restricting cell infiltration / proliferation, provide insufficient technical solution and advantage, especially for the treatment of wounds with undefined size and geometry. Therefore, in the present invention, tissue scaffolds in a three-dimensional structure that are low-density, compressible, and easily shapeable are provided for the treatment of full-thickness wounds with irregular geometry. Thanks to the three-dimensional structure of the tissue scaffold, the components it contains can remain more stable throughout the wound bed and their homogeneous distribution can be promoted, thereby enabling infiltration and dermal repair / regeneration. The inventors have conducted characterization studies to determine the suitability of the PCL, PEO, and PVA-based tissue scaffold contained therein for providing the intended technical solution and advantages.

[0029] Firstly, in order to analyze whether the obtained MSCs possess stem cell characteristics, analyses were performed using a cytometry device and by applying differentiation-inducing media and staining methods.

[0030] In Figure 1 , flow cytometry analysis was performed after staining the cells with Annexin V antibody and 7AAD antibody. In this analysis, the lower left quadrant (LL = 94.11%) indicates the percentage of live cells. It is observed that the percentage of live cells is 94%.

[0031] In Figure 2, flow cytometry analysis was performed after staining the cells with Annexin V antibody and 7AAD antibody. In this analysis, the lower left quadrant (LL = 87.10%) indicates the percentage of live cells. It is observed that the percentage of live cells is 87%.

[0032] Figure 3 is a microscopic image of the viability analysis and staining performed with trypan blue solution after the production of live MSCs together with the nanofiber material. It is observed that more than 75% of the cells appear bright blue and are viable.

[0033] Figure 4 shows the scanning electron microscope image of the PVA material.

[0034] Figure 5 shows the scanning electron microscope image of the PVA nanofiber produced together with live mesenchymal stem cells. Cellular structures are observed in the areas indicated by the arrow on Figure 5.

[0035] In this invention, a method for obtaining the composition is also provided. In this invention, the electrospinning method is first used for obtaining the three- dimensional tissue scaffold. Accordingly, a composition comprising the raw materials PCL, PEO, and PVA, which constitute the tissue scaffold, is obtained. The wound dressing composition subject to the invention has two configurations: single-layer and double-layer. The first layer enables the proliferation of the cellular content, while the second layer is provided to ensure the adhesion of the cellular content to the wound area.

[0036] A single-layer wound dressing composition comprising at least one tissue scaffold and at least one mesenchymal stem cell, and comprising the following process steps:

[0037] - preparing a polymer solution comprising one of the polymers from the group of poly(£-caprolactone), polyethylene oxide), or poly(vinyl alcohol) in at least one solvent,

[0038] - adding at least one mesenchymal cell into the obtained polymer solution and obtaining a homogeneous mixture by means of a mixing process,

[0039] - feeding the obtained homogeneous mixture into the electrospinning injector and obtaining the fibrous structure by means of the electrospinning device,

[0040] - performing a drying process to remove the solvent from the obtained fibrous structure.

[0041] The above-mentioned solvent is at least one selected from the group consisting of trifluoroethanol, water, or ethanol.

[0042] In the single-layer wound dressing composition, the amount of the said polymer is in the range of 5% to 15% by weight in the solution.

[0043] In the single-layer wound dressing composition, the mixing process for obtaining the said polymer solution is carried out at a temperature in the range of 25 to 65 °C.

[0044] In the single-layer wound dressing composition, 2 million mesenchymal stem cells are added to 5 ml of polymer solution, and a mixing process is applied for at least 1 hour. In the single-layer wound dressing composition, the said electrospinning process step is performed by depositing the homogeneous mixture onto the collector at a flow rate in the range of 2 to 4 ml / h.

[0045] In the single-layer wound dressing composition, the said drying process is carried out in a vacuum drying oven for at least 10 hours.

[0046] The wound dressing composition subject to the invention, in its double-layered structure, comprises a double-layer wound dressing composition containing at least one tissue scaffold and at least one mesenchymal stem cell.

[0047] The wound dressing composition subject to the invention consists of a doublelayered structure comprising a tissue scaffold containing PCL as the lower layer, and a tissue scaffold containing at least one of PVA and / or PEO and at least one mesenchymal stem cell as the upper layer, and comprises the following process steps:

[0048] - preparing a polymer solution containing PCL in an amount of 5% to 15% by weight in TFE,

[0049] - in parallel, preparing a polymer solution containing PEO in an amount of 5% to 15% by weight in water,

[0050] - in parallel, preparing a polymer solution containing polyvinyl alcohol in an amount of 5% to 15% by weight in ethanol and / or water,

[0051] - combining the polymer solution containing PEO and the solution containing PVA, and obtaining the second solution by adding at least one mesenchymal stem cell,

[0052] - feeding the obtained second solution into the electrospinning injector and obtaining the first fibrous structure by means of the electrospinning device,

[0053] - subjecting the obtained first fibrous structure to drying processes,

[0054] - feeding the polymer solution containing PCL into the electrospinning injector and obtaining the second fibrous structure by means of the electrospinning device,

[0055] - subjecting the obtained second fibrous structure to drying processes, forming the fibrous structure such that the second fibrous structure remains at the bottom and the first fibrous structure remains on top.

[0056] In the double-layer wound dressing composition, the said second solution contains the PEO-containing solution and the PVA-containing solution in a weight ratio in the range of 1 :2 to 1 :4.

[0057] In the double-layer wound dressing composition, 2 million mesenchymal stem cells are added to 5 ml of the said second solution, and a mixing process is applied for at least 1 hour.

[0058] In the double-layer wound dressing composition, the said electrospinning process for the first fibrous structure is performed by depositing it onto the collector at a flow rate in the range of 1 to 3 ml / h.

[0059] In the double-layer wound dressing composition, the said drying processes are carried out in a vacuum drying oven for at least 10 hours.

[0060] In another arrangement of the invention, a double-layer wound dressing composition comprising at least one tissue scaffold and at least one mesenchymal stem cell comprises the following process steps:

[0061] - preparing a polymer solution containing PCL in an amount of 5% to 15% by weight in TFE,

[0062] - in parallel, preparing a polymer solution containing PVA in an amount of 5% to 15% by weight in water and / or ethanol,

[0063] - obtaining the second solution by adding at least one mesenchymal stem cell to the PVA-containing solution,

[0064] - feeding the obtained second solution into the electrospinning injector and obtaining the first fibrous structure by means of the electrospinning device,

[0065] - subjecting the obtained first fibrous structure to drying processes,

[0066] - feeding the polymer solution containing PCL into the electrospinning injector and obtaining the second fibrous structure by means of the electrospinning device, - subjecting the obtained second fibrous structure to drying processes,

[0067] - forming the fibrous structure such that the second fibrous structure remains at the bottom and the first fibrous structure remains on top.

[0068] In the double-layer wound dressing composition, 2 million mesenchymal stem cells are added to 5 ml of the said second solution, and a mixing process is applied for at least 1 hour.

[0069] In the double-layer wound dressing composition, the said electrospinning process for the first fibrous structure is performed by depositing it onto the collector at a flow rate in the range of 1 to 3 ml / h.

[0070] In the double-layer wound dressing composition, the said drying processes are carried out in a vacuum drying oven for at least 10 hours.

[0071] In the invention, a needle (nail) collector is used to produce 3D electrospun PCL nanofiber scaffolds with larger pore sizes compared to conventional 2D nanofiber membranes. The produced scaffolds demonstrate potential for dermal tissue engineering by enhancing human dermal fibroblast infiltration, proliferation throughout the scaffolds, and the secretion of ECM proteins from the cells.

[0072] The tissue scaffold, with its three-dimensional and porous structure, is capable of properly conforming to the wound bed to promote healing and may also possess a structure that enables increased release efficiency of active agents.

[0073] The composition subject to the invention not only covers the wound and provides a physical barrier against external infection, as in wound dressings, but also supports both dermal fibroblasts and keratinocytes, thereby enabling the formation of skin tissue.

[0074] As stated in the invention, the tissue scaffold can be produced in a single step and at reasonable costs by means of the electrospinning method, while also having a porous and three-dimensional structure. Additionally, MSCs can be obtained from medical waste. By using low-cost raw materials, a high value-added technological product can be developed, offering a low-cost treatment option. The scope of protection of the invention is specified in the appended claims and cannot be limited to what is described for illustrative purposes in this detailed description. It is clear that a person skilled in the art can produce similar embodiments in the light of what is explained above, without deviating from the main theme of the invention.

Claims

CLAIMS1. A single-layer wound dressing composition comprising at least one tissue scaffold and at least one mesenchymal stem cell, characterized in that;• the tissue scaffold is a three-dimensional and porous structure that supports cell adhesion, proliferation, and differentiation, and provides protection against infections,• the mesenchymal stem cell is incorporated into the scaffold as an active agent to accelerate wound healing and enhance healing performance,• the tissue scaffold comprises at least one polymer selected from the group consisting of polycaprolactone (PCL), polyethylene oxide (PEO), and polyvinyl alcohol (PVA).

2. The wound dressing composition according to claim 1 , characterized in that; it comprises PCL as the tissue scaffold and at least one mesenchymal stem cell as the active agent.

3. The wound dressing composition according to claim 1 , characterized in that it comprises PEO as the tissue scaffold and at least one mesenchymal stem cell as the active agent.

4. The wound dressing composition according to claim 1 , characterized in that it comprises PVA as the tissue scaffold and at least one mesenchymal stem cell as the active agent.

5. The wound dressing composition according to any of the preceding claims, characterized in that it comprises a double-layered structure consisting of a tissue scaffold containing PCL as the lower layer and a tissue scaffold containing at least one of PVA and / or PEO and at least one mesenchymal stem cell as the upper layer.

6. A method for producing a single-layer wound dressing composition comprising at least one tissue scaffold and at least one mesenchymal stem cell, characterized in that; it comprises the steps of:• preparing a polymer solution comprising one of the polymers from the group consisting of PCL, PEO, or PVA in at least one solvent,• adding at least one mesenchymal stem cell into the obtained polymer solution and obtaining a homogeneous mixture by mixing,• feeding the homogeneous mixture into an electrospinning injector and forming a fibrous structure via electrospinning,• performing a drying process to remove the solvent from the obtained fibrous structure.

7. The method according to claim 6, characterized in that the said solvent is at least one selected from the group consisting of trifluoroethanol, water, or ethanol.

8. The method according to any one of claims 6-7, characterized in that the said polymer amount is in the range of 5% to 15% by weight in the solution.

9. The method according to any one of claims 6-8, characterized in that the said polymer solution is prepared by performing a mixing process at a temperature in the range of 25 to 65 °C.

10. The method according to any one of claims 6-9, characterized in that 2 million mesenchymal stem cells are added to 5 ml of the polymer solution and a mixing process is applied for at least 1 hour.

11. The method according to any one of claims 6-10, characterized in that the electrospinning process step is performed by depositing the homogeneous mixture onto the collector at a flow rate in the range of 2 to 4 ml / h.

12. The method according to any one of claims 6-11 characterized in that the said drying process is carried out in a vacuum drying oven for at least 10 hours.

13. A method for producing a double-layer wound dressing composition comprising at least one tissue scaffold and at least one mesenchymal stem cell, characterized in that; it comprises the steps of:• preparing a polymer solution containing 5% to 15% by weight of PCL in trifluoroethanol (TFE),• preparing in parallel a polymer solution containing 5% to 15% by weight of PEO in water,• preparing in parallel a polymer solution containing 5% to 15% by weight of PVA in ethanol and / or water,• combining the PEO and PVA solutions and adding at least one mesenchymal stem cell to form a second solution,• feeding the second solution into the electrospinning injector and forming the first fibrous layer,• drying the first fibrous layer,• feeding the PCL solution into the electrospinning injector and forming the second fibrous layer,• drying the second fibrous layer,• stacking the fibrous layers such that the PCL layer is at the bottom and the cell-containing layer is on top.

14. The method according to claim 13, characterized in that the second solution contains the PEO-containing solution and the PVA-containing solution in a weight ratio of 1 :2 to 1 :4.

15. The method according to any one of claims 13-14, characterized in that 2 million mesenchymal stem cells are added to 5 ml of the second solution and a mixing process is applied for at least 1 hour.

16. The method according to any one of claims 13-15, characterized in that the electrospinning process for the first fibrous structure is performed by depositing it onto the collector at a flow rate in the range of 1 to 3 ml / h.

17. The method according to any one of claims 13-16, characterized in that the said drying processes are carried out in a vacuum drying oven for at least 10 hours.

18. A method for producing a double-layer wound dressing composition comprising at least one tissue scaffold and at least one mesenchymal stem cell, characterized in that; it comprises the steps of:• preparing a polymer solution containing 5% to 15% by weight of PCL in trifluoroethanol (TFE),• preparing in parallel a polymer solution containing 5% to 15% by weight of PVA in water and / or ethanol,• adding at least one mesenchymal stem cell to the PVA solution to form the second solution,• feeding the second solution into the electrospinning injector and forming the first fibrous structure,• drying the first fibrous structure,• feeding the PCL solution into the electrospinning injector and forming the second fibrous structure,• drying the second fibrous structure,• stacking the fibrous layers such that the PCL layer is at the bottom and the cell-containing layer is on top.

19. The method according to claim 18, characterized in that 2 million mesenchymal stem cells are added to 5 ml of the second solution and a mixing process is applied for at least 1 hour.

20. The method according to any one of claims 18-19, characterized in that the electrospinning process for the first fibrous structure is performed by depositing it onto the collector at a flow rate in the range of 1 to 3 ml / h.21 .The method according to any one of claims 18-20, characterized in that; the drying processes are carried out in a vacuum drying oven for at least 10 hours.

Images