A composition that enables the complete healing of wounds in individuals suffering from diabetes

Abstract

The invention pertains to the technical field of biomedical engineering and pharmacology and, without being limited thereto, specifically relates to a composition that enables the healing of full-thickness and chronic wounds (particularly in the foot region) observed in individuals suffering from diabetic disease.

Description

[0001] A COMPOSITION THAT ENABLES THE COMPLETE HEALING OF WOUNDS IN INDIVIDUALS SUFFERING FROM DIABETES

[0002] TECHNICAL FIELD

[0003] The invention pertains to the technical field of biomedical engineering and pharmacology and, without being limited thereto, specifically relates to a composition that enables the healing of full-thickness and chronic wounds (particularly in the foot region) observed in individuals suffering from diabetic disease.

[0004] PRIOR ART

[0005] Diabetes is a chronic disease in which the body's ability to regulate blood sugar (glucose) is impaired. It occurs due to the insufficiency of the insulin hormone produced by the pancreas or the body's development of resistance to insulin. Diabetes is classified into two main types: type 1 , which is characterized by the pancreas's inability to produce sufficient insulin, and type 2, which is characterized by reduced sensitivity to insulin.

[0006] If not controlled, diabetes can lead to serious complications in various parts of the body, such as the eyes, kidneys, and nervous systems. Additionally, one of the most significant complications of diabetes is defined as diabetic foot, which involves the formation of hard-to-heal wounds on the patients' feet. Wounds observed in diabetic diseases typically arise as a result of complications such as impaired blood circulation and nerve damage (neuropathy) due to prolonged high blood sugar levels. These complications reduce the body's ability to heal wounds and increase the risk of infection. Diabetic wounds can lead to serious health issues, including infection, gangrene, and even amputation. Therefore, diabetes management and foot care are of vital importance in preventing such wounds. In the relevant technical field, inadequate treatment methods such as wound dressing and the use of specialized wound care products are applied for the treatment of these wounds in patients.

[0007] In addition to these techniques, various methods are applied to promote wound healing, including moist dressing applications, clinical approaches such as arterial reconstruction, the use of xenograft and graft transfer methods in cases of significant tissue loss, and debridement procedures.

[0008] The disadvantages of these methods include the risk of disease transmission from the donor, the formation of new wounds at the graft harvesting site, and the necessity of surgical intervention. Therefore, the development of new therapeutic strategies is of great importance to shorten the treatment duration and prevent complications such as infections.

[0009] Therefore, the development of new therapeutic strategies aimed at shortening the healing process by regulating inflammatory responses and promoting rapid tissue regeneration in the treatment of full-thickness and chronic wounds, particularly those observed in diabetic diseases, is of great importance. Consequently, research and development activities in the relevant technical field have become a necessity.

[0010] BRIEF DESCRIPTION OF THE INVENTION

[0011] In particular, wounds that occur in diabetic disease are chronic and full-thickness wounds. Full-thickness wounds contain subcutaneous fat or deeper tissues in addition to the epidermal and dermal layers and are more difficult to heal than superficial / partial-thickness wounds. The healing of such wounds usually requires the use of skin autografts or artificial skin substitutes.

[0012] The treatments for similar chronic and full-thickness wounds in the relevant technique present a range of issues, including wound contraction, delayed vascularization, scar formation, poor integration with healthy tissue, and high costs. The present inventors propose a composition that enables wound healing by eliminating these technical drawbacks and challenges.

[0013] To achieve this, the composition of the invention is designed to possess an ideal tissue scaffold. Accordingly, the aim is to provide 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, and features a biodegradable, biocompatible, and porous structure that promotes tissue regeneration.

[0014] Another objective of the present invention is to provide a composition that includes an additive component contributing to the tissue scaffold, which ensures the mentioned technical solution and advantages by providing immunomodulatory and regenerative effects. In this way, a composition with enhanced wound healing properties can be obtained, demonstrating high performance with the support scaffold and enabling a reduction in healing time.

[0015] In another aspect, the present invention provides a method for producing a composition that includes such an additive material and a tissue scaffold. The method disclosed in the invention, with its essential process steps, aims to enable the rapid and cost-effective production of the tissue scaffold while ensuring the efficient incorporation of the additive material into the obtained tissue scaffold.

[0016] BRIEF DESCRIPTION OF THE FIGURES

[0017] Figure 1 shows the SEM image of the tissue scaffold in the composition of the invention.

[0018] Figure 2 shows the PCL / Gelatin diameter distribution graph in the tissue scaffold.

[0019] Figure 3 shows flow cytometry analysis images for positive surface markers CD29, CD73, CD90, CD105. Figure 4 shows flow cytometry analysis images for negative surface markers (BD Biosciences, USA) for CD28 (PerCp), CD14 (PE), CD34 (APC), HLA-DR (FITC) analysis.

[0020] Figure 5-a shows after three weeks, the cells were fixed with 10% formaldehyde by removing the medium and staining with Alizarin Red staining solution, and osteogenic colonies and calcium deposits are seen under a light microscope.

[0021] Figure 5-b shows the appearance of the chondrocytes under light microscopy, with the nucleus and proteoglycans stained blue and the chondrocytes / cartilage formation in dark blue.

[0022] Figure 5-c shows light microscope images of adipocytes and fat drops after 2 weeks, with Oil Red O Staining and hematoxylin-eosin staining (Sigma).

[0023] Figure 6 shows the luminescence intensity of fluorescently labeled exosomes as 83.4±5.8 MFI.

[0024] Figure 7 shows the FESEM image of DF-MKH exosomes.

[0025] Figure 8 shows FESEM images after exosome seeding on tissue scaffolds.

[0026] DETAILED DESCRIPTION OF THE INVENTION

[0027] In this detailed description, the subject of the invention relates to a wound-healing composition and is explained with examples that serve solely to enhance understanding of the invention without imposing any limiting effect.

[0028] The composition of the invention is specifically designed to possess wound-healing properties and is structured to function in the treatment of challenging wounds, particularly those classified as chronic and full-thickness wounds.

[0029] In another aspect, the invention relates to providing a composition that enables the treatment of wounds, particularly those caused by diabetic diseases, which lead to full-thickness and chronic wounds. Diabetic foot ulcer disease is specifically cited as an example of this condition. However, it is evident that a composition functioning in the treatment of this challenging disease can also serve as a therapeutic agent for wounds with a simpler healing mechanism. Additionally, it can play a therapeutic role in neuropathic ulcers and ischemic ulcers, similar to diabetic foot ulcers.

[0030] The composition of the invention contains a tissue scaffold. For the tissue scaffold to possess wound-healing properties in the treatment of the aforementioned wounds, it must have a structure similar to the natural extracellular matrix (which may be abbreviated as ECM), supporting cell adhesion, proliferation, and differentiation, protecting the wound against external infections, and being biodegradable, biocompatible, porous, and capable of promoting tissue regeneration.

[0031] The present inventors utilize a gelatin-based structure as the tissue scaffold in the composition. Gelatin is a protein-based substance typically derived from animal sources such as bovine skin and bones. It is obtained from collagen and consists of a water-soluble polypeptide mixture. Collagen can be hydrolyzed into gelatin through a prolonged boiling process. Gelatin possesses properties such as biocompatibility and biodegradability, high film- and gel-forming ability, sensitivity to temperature changes, and the ability to provide a suitable environment for cell growth and tissue regeneration. In addition to these properties, its ease of shaping, low production costs, and high efficiency in supporting cell growth and tissue regeneration make it a highly suitable material for use in the invention.

[0032] The present inventors also incorporate polycaprolactone as an additional component into the gelatin-based tissue scaffold. Polycaprolactone (abbreviated as PCL) is included to provide or enhance properties such as high mechanical strength and structural integrity for the gelatin, improved processability, and a cellfriendly surface. PCL is a biocompatible (accepted by the body), biodegradable (capable of degrading over time in the body), non-toxic, and FDA-approved semisynthetic polymer. The combination of PCL and gelatin creates a synergistic effect within the tissue scaffold. While PCL provides mechanical strength and processability to the tissue scaffold, gelatin contributes biocompatibility, cell repair, and regeneration.

[0033] The tissue scaffold obtained with a structure containing gelatin and PCL can have a more porous structure compared to hydrogel-based tissue scaffolds. This allows the scaffold to accommodate healing components at higher efficiency and concentration.

[0034] In the present invention, the tissue scaffold within the composition is produced using manufacturing methods that allow it 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, especially those with a superficial porous and tightly stacked two-dimensional structure that restricts cell infiltration / proliferation for the treatment of diabetic ulcer disease, whose size and geometry are uncertain, provide inadequate technical solutions and advantages. Therefore, in the present invention, low-density, compressible, and easily shapeable three-dimensional tissue scaffolds are provided for the treatment of full-thickness wounds with irregular geometry, such as diabetic ulcers. The three-dimensional nature of the tissue scaffold ensures that the incorporated components remain more stable throughout the wound bed and promotes their homogeneous distribution, facilitating infiltration as well as dermal repair and regeneration.

[0035] The inventors have conducted characterization studies to determine the suitability of the tissue scaffold containing PCL and gelatin in achieving the targeted technical solutions and advantages.

[0036] The SEM image of the resulting tissue scaffold is shared in Figure 1 . SEM analyses were performed with the ZEIS GEMINI-500 FESEM device. The analysis was performed on gold-coated samples under low pressure. The diameters of the scaffold fibers obtained as tissue matrix were analyzed using SEM images using Imaged (NIH, Bethesda, MD) software. The diameters of 50 nanofibers were measured from the images, and the measurements were analyzed using the software. Average fiber diameters, along with fiber diameter distribution graphs and porosity images, were obtained. The PCL / Gelatin fiber diameter distribution graph is shared as Figure 2.

[0037] The porosity and pore size distribution of the nanofiber tissue scaffolds were analyzed using a Micromeritics Autopore IV Mercury Porosimeter. The analysis of the produced three-dimensional tissue scaffolds was conducted using low-pressure analysis (0-50 psi), which allows measurements for pores ranging in size from 3 nm to 360 pm. In the test, mercury intrusion data were used with a mercury surface tension of 485 dyne / cm and a contact angle of 130° between the mercury and the pore walls. The tissue scaffold pores were filled with mercury by gradually increasing the pressure from 0 psi (for larger pores) to 50 psi (for relatively smaller pores). As a result of the measurement, after determining the amount of mercury intruded into the sample under pressure, the material's porosity, pore volume distribution, pore size distribution, bulk density, and apparent density were obtained. For the three-dimensional PCL / GEL tissue scaffold disclosed in the invention, the average pore size was found to be 11.8 pm, and the porosity ratio was determined to be 70%.

[0038] As another study, the mechanical strength values of the tissue matrix in question were tested.

[0039] A tensile test was applied to the obtained three-dimensional PCL / Gelatin tissue scaffold. The tensile tests were conducted using a TA Instruments Q800 DMA (Dynamic Mechanical Analysis) device. The tests were performed at a body temperature of 37°C with a force application rate of 0.1 N / min. The tested tissue scaffold was found to have a tensile strength of 1 .151 MPa, an elongation at break of 18.93%, and a Young’s modulus of 0.03499 MPa.

[0040] Compression tests were conducted using a TA Instruments Q800 DMA (Dynamic Mechanical Analysis) device. The tests were performed at a body temperature of 37°C with a force application rate of 0.1 N / min. The tested tissue scaffold was found to have a compression strength of 0.2544 MPa, a compression strain at break of 26.91%, and a compression modulus of 0.073 MPa. As another study, an in vitro biodegradability test was conducted for the three- dimensional PCL / Gelatin tissue scaffold. To determine the biodegradability properties of the tissue scaffolds, their initial dry weights were recorded, and each sample was incubated in two sets of 10 mL phosphate-buffered saline (abbreviated as PBS) solution (pH: 7.4) at 37±0.5°C in an incubator. On the 3rd, 7th, 14th, 17th, and 21st days of incubation, the tissue scaffolds were removed from the buffer solution, and excess liquid was removed using an air pump. The tissue scaffolds were then dried at 37±0.5°C for one day and reweighed. The time-dependent percentage mass loss of the tissue scaffolds was determined using Formula 1 .

[0041] %Degradation=[(lnitial Weight -Final Weight)-? Initial Weight ]x100

[0042] Formula 1 .

[0043] At the end of 21 days, the three-dimensional PCL / Gelatin tissue scaffold was found to have undergone approximately 90% biodegradation.

[0044] As another study, an in vitro biocompatibility test was conducted for the three- dimensional PCL / Gelatin tissue scaffold. The keratinocyte cell line from cell isolates was suspended in keratinocyte growth medium and seeded at a density of 1 x105cells per well in 24-well culture plates. The cells were cultured for 24 hours until they reached confluence. After 24 hours, a 1 x1 cm2nanofiber tissue scaffold was placed on the confluent cells and incubated with the culture medium for 24 hours, 7 days, and 14 days. At the end of the culture period, the cells were stained with Calcein AM (green) for live cells and Ethidium Homodimer-1 (EthD-1 ) (red) for apoptotic cells. The images were captured using a fluorescence microscope. In the fluorescence microscopy analysis, the fluorescence intensity of the negative cell control (a well without nanofiber) stained with Calcein AM at 24 hours was considered as 100% cell viability. The fluorescence intensity of wells containing the biomaterial, as well as equivalent wells cultured for 7 and 14 days, was compared accordingly. The percentage of cell viability was recorded, and all culture wells were tested in triplicate. Cell Viability Rate:

[0045] 1 . Control Culture Well (Keratinocyte Cells) - 24 hours: 100% ± 0%

[0046] 2. PCL / GEL Culture Well (Keratinocyte Cells + PCL / GEL) - 24 hours: 97.33% ± 0.66%

[0047] 3. Control Culture Well (Keratinocyte Cells) - 7 days: 98.12% ± 0.48%

[0048] 4. PCL / GEL Culture Well (Keratinocyte Cells + PCL / GEL) - 7 days: 94.82% ± 0.64%

[0049] 5. Control Culture Well (Keratinocyte Cells) - 14 days: 92.28% ± 0.84%

[0050] 6. PCL / GEL Culture Well (Keratinocyte Cells + PCL / GEL) - 14 days: 90.26% ± 0.48%

[0051] As another study, a cytotoxicity test was conducted for the three-dimensional PCL / Gelatin tissue scaffold. The 1 .6 cm diameter tissue scaffold was washed three times with phosphate-buffered solution containing 1% penicillin / streptomycin. The tissue scaffold sample was exposed to ultraviolet light for 2 hours. Fibroblasts were used for the in vitro culture. Fibroblast cells were seeded at a density of 10x103cells per well in 24-well culture plates and cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% FBS and 1% penicillin-streptomycin under 37°C and 5% CO2conditions in a CO2incubator. When fibroblast cells reached 80% confluency, they were detached using 0.25% trypsin-EDTA solution. For cytotoxicity testing, 1.6 cm nanofiber materials were placed in 24-well plates, and 10x103human dermal fibroblast (HDF) cells were seeded onto them with DMEM medium, followed by 3 days of incubation. Additionally, for control purposes, HDF cells were cultured alone in a separate well. At the end of the culture period, the DMEM medium was discarded and replaced with fresh DMEM medium. MTT solution (5 pg / L) was added to each well along with the fresh DMEM, and the plates were incubated at 37°C for 4 hours. After incubation, the medium was removed, and 1 pL of DMSO was added to dissolve the formed formazan. After the formazan crystals were dissolved, 100 pL aliquots were transferred to a 96-well culture plate, with five replicates per sample. Absorbance was measured at a 570 nm wavelength using a microplate spectrophotometer. The absorbance values obtained were compared to the control well, which was considered 100% cell viability. The percentage of cell viability was determined as follows: Cell Viability Rate:

[0052] 1 . Control Culture Well (Fibroblast Cells): 100% ± 0.00

[0053] 2. PCL / GEL Culture Well (Fibroblast Cells + PCL / GEL): 92.78% ± 1 .22

[0054] As another study, a water retention test was conducted for the three-dimensional PCL / Gelatin tissue scaffold. For the tests, each tissue scaffold sample was cut into 1 cm2pieces in two sets. First, the dry weights of the scaffolds were measured. Then, the samples were incubated in 10 mL phosphate-buffered saline (PBS) solution (pH 7.4) at 37±0.5°C in an incubator. On the 3rd, 7th, 14th, 17th, and 21st days of incubation, the tissue scaffolds were removed from the buffer solution, and excess liquid was removed using an air pump. The wet weights of the samples were then measured to determine their water retention percentages. The timedependent water retention ratios (%) of the tissue scaffolds were calculated using Formula 2, based on their dry and wet weights.

[0055] %Water Retention=[( Wet Weight-Dry Weight) / Dry Weighty 100 Formula 2.

[0056] According to the obtained test results, the approximate water retention capacity of the three-dimensional PCL / Gelatin tissue scaffold is 1060%.

[0057] As another study, a contact angle measurement was conducted for the three- dimensional PCL / Gelatin tissue scaffold. Contact angle measurement is a widely used and appropriate method for investigating the hydrophilic properties of scaffolds. The contact angle value is directly related to the hydrophilicity of the tissue scaffold; the lower the contact angle value, the higher the hydrophilicity of the scaffold. The surface contact angle measurements of the tissue scaffolds were tested using a Biolin Scientific Attension Theta Lite device. The measurements were performed by dropping approximately 10 pL of distilled water onto the tissue scaffolds. The three-dimensional PCL / Gelatin tissue scaffolds exhibited highly hydrophilic properties, with contact angles approaching zero within a maximum of 5 seconds. As another study, a water vapor permeability test was conducted for the three- dimensional PCL / Gelatin tissue scaffold. The water vapor permeability tests were performed using the cup method for the three-dimensional PCL / Gelatin scaffolds. For the tests, glass containers were filled with approximately 70 mL of purified water, and their initial weights (Winitiai) were recorded. Next, PCL / Gelatin scaffolds were placed and secured over the openings of the glass containers. The containers were then incubated at 37±0.5°C for 24 hours. After incubation, the final weights (Wfinai) of the glass containers were recorded after removing the scaffolds. The measurements were performed in triplicate. Using the obtained data, the water vapor permeability rate (g / m2h) of the scaffolds was calculated using Formula 3.

[0058] Water Vapor Permeability Rate= (WinitiarWfinai) / (Scaffold Surface AreaxTime) Formula 3.

[0059] The water vapor permeability rate of the three-dimensional PCL / Gelatin tissue scaffold was found to be 219.2622 g / m2h.

[0060] The air permeability of the three-dimensional PCL / Gelatin tissue scaffolds was measured using the Proser K008 air permeability test device. The air permeability tests were conducted according to the EN ISO 9237 SPECIAL standard, measuring airflow (mm / s) through a 20 cm2area at 100 Pa pressure. For the air permeability measurements, three-dimensional PCL / Gelatin scaffolds with an average thickness of 1.56 mm were used. The air permeability value was determined to be 483.5 mm / s.

[0061] As observed in the conducted studies, the use of gelatin and PCL materials as sources in the production of the tissue scaffold described within the composition is suitable for the treatment and healing of chronic and full-thickness wounds.

[0062] The composition of the invention contains at least one active agent with woundhealing properties. In this invention, the active agent is included in the composition to accelerate the wound healing process and enhance healing performance. In this invention, at least one mesenchymal stem cell is used as the active agent. In a preferred embodiment, the mesenchymal stem cell is a dental follicle mesenchymal stem cell.

[0063] In this invention, dental follicle mesenchymal stem cells (DF-MSCs), which are a specialized type of stem cell derived from the dental follicle, a component of tooth development, are utilized. These cells exhibit the characteristic properties of mesenchymal stem cells, including self-renewal, differentiation into various cell types, and significant regenerative potential.

[0064] DF-MSCs contain various bioactive components, including microRNA, mRNA, transforming growth factor-beta (TGF-[3), indoleamine 2,3-dioxygenase (IDO), hepatocyte growth factor (HGF), prostaglandin E2 (PGE2), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), and insulin-like growth factor (IGF). The presence of these components together enables DF-MSCs to be used as an active agent for wound healing and tissue regeneration.

[0065] The composition of the invention primarily consists of a three-dimensional PCL and Gelatin-based tissue scaffold enriched (supplemented) with DF-MSC exosomes.

[0066] In a preferred embodiment, the DF-MSC component can be medically sourced, which may further reduce the overall cost of the composition.

[0067] For this invention, surface antigen analysis and in vitro multipotent differentiation analysis were conducted on the obtained DF-MSCs.

[0068] To determine whether the obtained MSCs exhibit stem cell characteristics, their positive surface markers were analyzed using a flow cytometry device, and their differentiation potential was assessed through stimulating media and staining techniques.

[0069] For DF-MSCs, surface marker analysis was performed on third-passage cells using flow cytometry-compatible antibodies. A total of 2x105cells were trypsinized, washed twice with PBS, and then incubated at 4°C in the dark for 30 minutes in 200 pL PBS containing a fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, or allophycocyanin (APC)-conjugated specific monoclonal antibody. Cell surface markers were analyzed using flow cytometry (BD Accuri C6 Plus, USA). For positive surface markers, CD29, CD73, CD90, and CD105 were analyzed, and the flow cytometry analysis images are shared in Figure 3. For negative surface markers, CD28 (PerCP), CD14 (PE), CD34 (APC), and HLA-DR (FITC) were analyzed using BD Biosciences (USA) antibodies, and the results are shared in Figure 4.

[0070] The osteogenic differentiation potential of DF-MSCs was analyzed through specific testing. DF-MSCs were seeded in 6-well plates at a density of 1 x105cells per well and cultured in 2 mL DMEM / F12 medium until they reached confluence. Once DF- MSCs reached 60% confluency, the culture medium was replaced with a commercially available osteogenic induction medium (StemPro™, ThermoFisher). The medium was refreshed every 3 days. After three weeks, the cells were fixed with 10% formaldehyde, stained with Alizarin Red staining solution, and analyzed under a light microscope to observe osteogenic colonies and calcium deposits. The results are shared in Figure 5-a.

[0071] The chondrogenic differentiation potential of DF-MSCs was analyzed through specific testing. DF-MSCs seeded in 6-well plates were cultured in chondrogenic induction medium (StemPro™, ThermoFisher) for three weeks. The chondrogenic medium was refreshed every 3 days. Cells attached to the well surface were fixed with 10% formaldehyde and stained with Alcian Blue. The nucleus of chondrocytes and proteoglycans appeared blue, while differentiated chondrocytes / cartilage formations appeared dark blue. The results were observed under a light microscope and are shared in Figure 5-b.

[0072] The adipogenic differentiation potential of DF-MSCs was analyzed through specific testing. DF-MSCs were cultured in a commercially available adipogenic induction medium (StemPro™, ThermoFisher). After two weeks, the cells were stained with Oil Red O and hematoxylin-eosin staining (Sigma), and adipocytes and lipid droplets were observed under a light microscope. The results are shared in Figure 5-c.

[0073] For the isolation and characterization of DF-MSC exosomes, DF-MSCs were cultured until they reached 80-85% confluency.Once confluency was achieved, the culture medium was replaced with DMEM / F12 supplemented with 10% exosome- free FBS, and the cells were incubated for an additional 48 hours. The collected culture supernatants were centrifuged at 3000g for 15 minutes to separate cell debris. The obtained supernatant was then processed using the ExoQuick-TC exosome isolation kit (EXOTC50A, SBI System Biosciences, Canada) following the manufacturer’s protocol. The final exosomes were analyzed using a protein quantification kit. According to the exosome isolation protocol, the culture supernatant, which had been centrifuged at 3000g for 15 minutes, was mixed with ExoQuick solution and centrifuged at 10,000 rpm for 30 minutes at +4°C. The supernatant was discarded, and the exosome pellet at the bottom was analyzed using a protein quantification kit. Each 200 pg of exosomes was resuspended in 200 pL PBS and stored at -80°C until use. For the analysis of surface markers, which are considered specific exosome markers, flow cytometric methods were used. The analyzed markers included tetraspanins (CD9, CD63) and chaperones commonly found in exosomes (Hsp90), which were examined using a flow cytometry device. The size and morphology of the vesicles obtained after exosome isolation were imaged using FESEM.

[0074] The protein concentration of the isolated exosomes was determined using the Bicinchoninic Acid (BCA) Protein Assay Kit (Thermo Scientific, Catalog No: 23225). Each 200 pg of exosomes in 200 pL PBS was stored in an Eppendorf tube at -80°C. Before protein quantification, the exosomes were resuspended in PBS, then mixed with an equal volume of RIPA buffer and protease inhibitor (without EDTA) to induce exosome lysis. The mixture was then incubated at room temperature for 5 minutes. The measured exosomes were diluted 1 :10 in ultrapure water, and their absorbance was read at 562 nm. The obtained data were recorded in pg / mL. On average, 220-280 pg of exosomes were obtained from every 2 million MSC cultures. For the fluorescent marking of exosomes for the tracking of exosomes, membrane fluorescent marking was performed for the tracking (migration and localization) of exosomes to be placed in three-dimensional PCL / gelatin-containing tissue scaffolds subject to the invention in the tissue area to be applied in order to evaluate the wound healing process. Exosomes were labeled using Exoglow Membrane Labeling Red Kit (465 nm excitation / 635 nm emission). For labeling, the manufacturer's protocol was followed as follows; for each 50-100 pg of exosomes, 12 pL of reaction buffer solution and 2 pL of labeling solution were mixed and incubated with the exosomes for 30 minutes at room temperature and in the dark, and after centrifugation at 10,000 rpm for 10 minutes, the supernatant was discarded and the bottom exosome pellet was diluted with phosphate buffer solution. Fluorescently labeled exosomes were analyzed by flow cytometry. In flow cytometry, the MFI (mean fluorescence index) or percentage (%) values of the fluorescent population in the FL3 channel (488-620 nm) were recorded. Fluorescently labeled exosomes were observed to have a fluorescence intensity of 83.4±5.8 MFI and are shown in Figure 6.

[0075] The present invention also provides a method for obtaining the composition. In the present invention, an electrospinning method is first used for obtaining a three- dimensional tissue scaffold. Accordingly, a composition consisting gelatin and PCL raw materials forming the tissue scaffold is obtained.

[0076] Preparation of PCL / gelatin polymer solution

[0077] In a preferred embodiment, a previously determined solution of poly(s- caprolactone) and gelatin was prepared in trifluoroethanol (abbreviated as TFE). In a preferred embodiment, PCL:Gelatin is present in this solution in a 1 :1 ratio by weight.

[0078] This solution is obtained by stirring for a period of between 10 and 36 hours. In a preferred embodiment, this process is carried out at room temperature.

[0079] Addition of the Obtained PCL / gelatin Polymer Solution to the Electrospinning System and Obtaining the Tissue Scaffold The solution obtained in the previous process step is first transferred to the injector with a blunt-tipped needle and placed in the injector pump integrated with the electrospinning device.

[0080] The polymer solution is deposited on the collector in the electrospinning device by applying a voltage of 10 to 20 kV and at a rate of 0.4 to 0.5 ml / h.

[0081] Three-dimensional tissue scaffolds can be obtained in nano-sized nanoscale collected with a spherical dish collector.

[0082] In order to obtain the composition subject to the invention, DF-MKH exosomes are added to the tissue scaffold obtained in the previous process steps. These process steps are as follows.

[0083] Application of Preliminary Preparation Procedures Before Transplantation to Tissue Scaffolds Obtained by Application of Electrospinning Method

[0084] The tissue scaffolds obtained in the previous process step are reduced to predetermined sizes. In a preferred embodiment, these scaffolds are cut into plates.

[0085] The tissue scaffolds, which are reduced to the dimensions as determined, are subjected to sterilization procedures. For this purpose, the tissue scaffolds are treated with ethanol or similar cleaner and then washed with a buffer solution.

[0086] In a preferred embodiment, the tissue scaffolds are incubated by UV irradiation after the aforementioned washing process. Preferably, the duration of these treatments is at least 30 minutes on each side of the scaffolds.

[0087] The tissue scaffolds are then kept in a culture medium. This process is carried out for at least 20 hours. For every 10 mm2of PCL / gelatin tissue scaffold, 200 micrograms of DF-MKH exosomes were added in 200 microliters of phosphate buffer solution (PBS). The tissue scaffolds obtained by applying these process steps are incubated overnight in phosphate-based buffer solution to ensure exosome retention. The incubation temperature is 37°C and preferably carried out with a 5% CO2 incubator.

[0088] Seeding of DF-MKH Exosomes onto Tissue Scaffolds

[0089] Isolated DF-MKH exosomes were inoculated into previously obtained tissue scaffolds in 48-well plates at the rate of 200 pg exosomes / 200 pL PBS and kept in a 5% CO2 incubator at 37°C for 24 hours.

[0090] Subsequently, in a preferred embodiment, DF-MKH exosomes seeded on the tissue scaffold are incubated at 37°C after being seeded in triplicate in 3 sets of 3 replicates for release assay.

[0091] At the end of the 7th, 14th, and 21 st days (based on the degradation time of the tissue scaffolds), the solution inside the wells is aspirated, followed by protein isolation using RIPA solution and protease inhibitors, and then the protein amount is measured using the BCA protein concentration assay kit. Ultimately, at the end of the 21 -day period, as well as on the 7th and 14th days, the percentage of release of the seeded exosomes is determined. For the calculations, a tissue scaffold without DF-MKH exosomes and a 200 pg DF-MKH-only scaffold are used separately as control materials. At the end of the 21 st day experimental period, the positive markers of the DF-MKH exosomes are analyzed using a flow cytometry device. The characterization results of the DF-MKH exosomes released from the tissue scaffolds mentioned in the invention are shared in Table 1 .

[0092] The obtained results (protein amount (pg) in 200 pL of PBS) are given below:

[0093] 7th day

[0094] 1 . MKH Exosomes: 198.00 pg ± 4.00 pg

[0095] 2. Tissue Scaffold (PCL / GEL): 0.00 pg ± 0.00 pg

[0096] 3. Tissue Scaffold (PCL / GEL) + MKH Exosomes: 53.00 pg ± 3.00 pg 14th day

[0097] 1 . MKH Exosomes: 195.00 ng ± 2.00 pg

[0098] 2. Tissue Scaffold (PCL / GEL): 0.00 pg ± 0.00 pg

[0099] 3. Tissue Scaffold (PCL / GEL) + MKH Exosomes: 132.00 pg ± 6.00 pg

[0100] 21 st day

[0101] 1. MKH Exosomes: 197.00 pg ± 5.00 pg

[0102] 2. Tissue Scaffold (PCL / GEL): 0.00 pg ± 0.00 pg

[0103] 3. Tissue Scaffold (PCL / GEL) + MKH Exosomes: 193.00 pg ± 2.00 pg

[0104] Table 1. DF-MKH exosome characterization results

[0105] Characterization of Exosomes Seeded onto Tissue Scaffolds

[0106] The culture medium was removed from the wells containing the tissue scaffolds, and the tissue scaffolds were washed twice with PBS. Then, the DF-MKH exosomes were fixed with 2.5% glutaraldehyde in 0.1 M Dulbecco PBS. After passing the tissue scaffolds through various alcohol series (30%, 50%, 70%, 90%, and 100% v / v), they were treated with hexamethyldisilazane (HMDS) and air-dried.

[0107] The surface morphology of the exosome-seeded tissue scaffolds was analyzed on the 7th day of culture using a ZEISS GEMINI-500 FESEM device. The analysis was conducted on gold-coated samples under low pressure. The FESEM image of the tissue scaffolds taken before the exosome seeding process is shown in Figure 1. The FESEM image of DF-MKH exosomes is shown in Figure 7, and the FESEM images of the tissue scaffolds after the exosome seeding process are shown in Figure 8. The composition discussed in the invention is a structure comprising a three- dimensional PCL and gelatin-based tissue scaffold enriched with dental follicle mesenchymal stem cell exosomes. Each component included in this composition is made from biodegradable and biocompatible materials.

[0108] In the composition subject to the invention, PCL and gelatin components, suitable for use as tissue scaffold materials, ensure high moldability and that the mechanical properties meet the desired values.

[0109] The DF-MKH exosomes in the composition contain not only microRNA, mRNA, and protein content, but also a high amount of paracrine factors such as transforming growth factor (TGF-[3), indoleamine 2,3-dioxygenase (IDO), hepatocyte growth factor (HGF), prostaglandin E2 (PGE2), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), and insulin-like growth factor (IGF), which accelerate the wound healing process and promote regeneration.

[0110] The tissue scaffold, with its three-dimensional and porous structure, is able to effectively support the healing of the wound bed and also provides a structure that enhances the release efficiency of active substances. A low-density, compressible, and easily moldable cotton-like three-dimensional tissue scaffold can be provided for the treatment of full-thickness and chronic wounds, such as diabetic ulcers, which do not have a regular geometry. Three-dimensional tissue scaffolds promote the more stable retention and homogeneous distribution of DF-MKH exosomes along the wound bed, serving as an enhanced synthetic dermal matrix for DF-MKH exosome infiltration and dermal repair / regeneration.

[0111] The composition discussed in the invention not only covers the wound and provides a physical barrier against external infection, like wound dressings, but also supports the formation of skin tissue by providing support for both dermal fibroblasts and keratinocytes.

[0112] As mentioned in the invention, the tissue scaffold obtained through the electrospinning method can be produced in a single step and at a low cost, while being porous and three-dimensional. Additionally, MSCs can be obtained from medical waste. By using low-cost raw materials, a high-value technological product can be developed, offering a cost-effective treatment solution.

[0113] The scope of protection of the invention is specified in the attached claims and cannot be limited to those explained for sampling purposes in this detailed description. It is evident that a person skilled in the art may exhibit similar embodiments in light of the above-mentioned facts without drifting apart from the main theme of the invention.

Claims

CLAIMS1. A composition for use in the treatment of full-thickness and chronic wounds, particularly in individuals suffering from diabetic disease, characterized by comprising:- a three-dimensional and porous tissue scaffold that promotes cell adhesion, proliferation and differentiation and protects against infections,- at least one mesenchymal stem cell exosome as an active ingredient doped into the tissue scaffold to accelerate the wound healing process and improve healing performance,- gelatin and polycaprolactone within said tissue scaffold.

2. The composition according to claim 1 , wherein the said tissue scaffold comprises PCL:Gelatin in a ratio of 1 :1 by weight.

3. The composition according to any one of the preceding claims, wherein the said mesenchymal stem cell comprises dental follicle mesenchymal stem cells.

4. The composition according to claim 3, wherein it comprises mesenchymal stem cells obtained from medical waste teeth.

5. A method for obtaining a composition comprising a tissue scaffold and at least one mesenchymal stem cell exosome, characterized in that it comprises the following main process steps:- preparation of polymer solution containing PCL and gelatin,- the production of a tissue scaffold in three-dimensional and porous form through the processes performed in an electrospinning device, using the obtained PCL and gelatin-containing polymer solution,- the seeding of mesenchymal stem cell exosomes isolated from teeth onto the obtained tissue scaffold, and the production of a tissue scaffold enriched with DF-MSC exosomes.

6. The method according to claim 5, wherein the said polymer solution comprises PCL:Gelatin in a ratio of 1 :1 by weight.

7. The method according to claim 5 or claim 6, wherein the following steps are taken to prepare the polymer solution:- adding poly(£-caprolactone) and gelatin in a 1 :1 ratio by weight in TFE and stirring for a period of time in the range of 10 to 36 hours to obtain a homogeneous solution.

8. The method according to claim 7, wherein the said mixing is carried out at room temperature.

9. The method according to any one of claims 5-8, wherein the said electrospinning process step is carried out by treating the obtained polymer solution at a value in the range of 10 to 20 kV.

10. The method according to any one of claims 5-9, wherein the electrospinning process step is carried out by depositing the obtained polymer solution on the collector at a rate in the range of 0.4 to 0.5 ml / h.

11. The method according to any one of claims 5-10, wherein the tissue scaffolds obtained after the electrospinning process are subjected to preliminary preparatory procedures before inoculation:- reducing the tissue scaffolds to predetermined sizes- carrying out incubation processes by subjecting to UV treatments- completing the sterilization process by keeping them in a culture medium- incubating the sterilized tissue scaffolds in a 5% CO2 incubator.

12. The method according to any one of claims 5-11 , wherein the process of seeding mesenchymal stem cell exosomes into tissue scaffolds comprises the following steps:- DF-MKH exosomes were added 1 :1 by volume to the buffer solution to obtain a mixture,- inoculating the obtained mixture onto the tissue scaffolds.- incubating the tissue scaffolds, onto which the inoculation process has been performed, in a 5% CO2 incubator.

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