A PCL / gelma hybrid scaffold cultured with HUC-MSCS and its use in pelvic reconstruction

Abstract

The present invention relates to PCL / GelMA (polycaprolactone / gelatin methacrylate) tissue scaffolds cultured with HUC-MSCs (human umbilical cord mesenchymal stem cells), a method for their production, and their use in pelvic floor reconstruction. Experimental results demonstrating the efficacy of the tissue scaffolds obtained according to the invention are also presented.

Description

[0001] A PCL / GELMA HYBRID SCAFFOLD CULTURED WITH HUC-MSCS AND ITS USE IN PELVIC RECONSTRUCTION

[0002] Technical Field

[0003] The present invention relates to PCL / GelMA (polycaprolactone / gelatin methacrylate) tissue scaffolds cultured with HUC-MSCs (human umbilical cord mesenchymal stem cells), a method for their production, and their use in pelvic floor reconstruction.

[0004] Prior Art

[0005] Although polypropylene is considered an ideal material for pelvic floor reconstruction in the treatment of Pelvic Organ Prolapse (POP), it has also given rise to various serious complications, such as mesh exposure or displacement into organs like the vagina and bladder, chronic pain, painful sexual intercourse (dyspareunia), and infections. The primary cause of these complications is the inflammatory response induced by synthetic meshes in the tissue, fibrosis, mesh shrinkage, and their permanent nature. Furthermore, properties of polypropylene and other synthetic materials such as porosity, design type, degradability, and primarily stiffness can also alter the tissue healing response and, together with excessive collagen production, may lead to a higher, unhealthy ratio of collagen VIII. This, in turn, can result in the formation of excessive fibrosis, which causes the aforementioned complications.

[0006] Upon reviewing previous studies that combine cellular therapy with synthetic scaffolds, it is evident that an ideal cell-scaffold combination for the treatment of pelvic floor disorders does not yet exist. Hansen et al. used biodegradable, electrospun polycaprolactone (PCL) scaffolds loaded with either fibroblast growth factor (bFGF) or connective tissue growth factor (CTGF) and rat mesenchymal stem cells (MSCs) in a rat model of a full-thickness abdominal wall defect. As a result, they found that the meshes delivering connective tissue growth factor and rat MSCs improved the biochemical and biomechanical properties of the weakened abdominal wall without causing complications. [1]

[0007] In another study, B. Aghaei-Ghareh-Bolagh et al. previously conducted a comparative study in rats, comparing a PP mesh with a newly designed knitted product, and they demonstrated enhanced tissue responses, new collagen deposition, and greater neovascularization in response to the new mesh. [2, 3] In this study as well, the newly designed knitted product was prepared via the electrospinning method, and human dermal fibroblast cells were used as the cell source.

[0008] Although the electrospinning method is a widely used production technique in research for the production of meshes for use in the pelvic floor, the scaffolds produced with this technique may impede cellular infiltration and tissue integration due to their irregular and small pore sizes.

[0009] WO2017064667A2 discloses multilayered cylindrical tissue scaffolds. The scaffold comprises GelMA layers, encapsulated cells, and PCL networks. The GelMA layers are obtained through iterative solution dipping and photopolymerization processes and are coated with a solution blow-spun PCL network. The potential use of mesenchymal stem cells is also mentioned, and an example application concerning human umbilical cord cells is presented.

[0010] US11213614B2 discloses a scaffold produced by 3D printing from materials including PCL, and the seeding of cells onto this scaffold within hydrogels, including GelMA. It is also mentioned that the cells can be human mesenchymal stem cells or umbilical cord vein endothelial cells. The document particularly focuses on bone tissue.

[0011] CN114681679A discloses a printing material suitable for 3D printing techniques and its use in tissue scaffolds. The printing material is a hydrogel that may contain GelMA. It is also stated that a reinforcing agent, such as polycaprolactone, can be added during printing. The seeding of cells onto the printing material is also mentioned. Furthermore, it is specified that the cells can be umbilical cord mesenchymal stem cells.

[0012] Objectives of the Invention

[0013] The object of the present invention is to develop a hybrid tissue scaffold suitable for use during pelvic floor reconstruction for the treatment of Pelvic Organ Prolapse (POP), and a method for producing said scaffold.

[0014] Another object of the present invention is to develop a tissue scaffold that provides mechanical support in the region where it is implanted. To this end, the object is to develop a tissue scaffold that provides both strength and creates pores for the delivery of cells into the tissue.

[0015] A further object of the present invention is to develop a tissue scaffold that disappears from the tissue over time, concurrently with the repair of damaged or lost connective tissue.

[0016] A further object of the present invention is to develop a tissue scaffold that promotes the regeneration of damaged or lost connective tissue and accelerates healing.

[0017] Detailed Description of the Invention

[0018] The tissue scaffold, developed to achieve the objects of the present invention, is described with reference to the accompanying drawings.

[0019] Figure 1 Fluorescence microscopy images after cell seeding of: (a) PCL with a small pore size, (b) GelMA-coated PCL with a small pore size, (c) PCL embedded in GelMA with a small pore size, (d) PCL with a large pore size, (e) GelMA-coated PCL with a large pore size, and (f) PCL embedded in GelMA with a large pore size.

[0020] Figure 2 Representative images of the HUC-MSCs used (a) explant and (b) passage 1.

[0021] Figure 3 Fluorescence and confocal microscopy images of PCL / GelMA scaffolds and TCP on (a) day 2 and (b) day 5 after cell seeding.

[0022] Figure 4 XTT colorimetric assay results for the HUC-MSC samples at day 2 and day 5.

[0023] Figure 5 Microscopy images of the scaffolds after the in vivo study, obtained with (a) hematoxylin and eosin staining for group 1, (b) hematoxylin and eosin staining for group 3, (d) Mallory's Azan staining for group 1, and (e) Mallory's Azan staining for group 3.

[0024] Figure 6 Microscopy images of samples from a non-incised rat, obtained with (a) Mallory's Azan staining and (b) hematoxylin and eosin staining.

[0025] Figure 7 Scanning Electron Microscopy (SEM) images of (a) the PCL-only scaffold and (b) the PCL / GelMA biomaterial according to the invention. Figure 8 Stress-strain curves for (a) the GelMA-coated-1 scaffold and (b) the non-GelMA- coated-1 scaffold, and (c) a comparison of the Young's modulus for the samples.

[0026] The method for producing the tissue scaffold of the invention essentially comprises; forming a polycaprolactone (PCL) fibrous material in the form of a mesh via MEW (melt electrowriting), incubating the fibrous material in a 5-10% (w / v) gelatin methacrylate (GelMA) solution containing a photoinitiator, and subsequently removing the excess GelMA solution, applying a photopolymerization process to the PCL / GelMA hybrid material, sterilizing the resulting scaffold, incubating human umbilical cord mesenchymal stem cells (HUC-MSCs) by seeding them in a mesenchymal stem cell expansion medium, seeding the expanded HUC-MSCs onto the scaffold.

[0027] According to a preferred embodiment of the invention, said process steps are implemented as follows; forming the PCL fibrous material in the form of a mesh via MEW, with a nozzle diameter of 0.25±0.05 mm, an infill rate between 20-50%, a pressure of 23-25 PSI, and a voltage of 2 to 10 kV, incubating the fibrous materials for 1 hour in a 5-10% (w / v) GelMA solution containing 0.5% Irgacure as a photoinitiator, and removing the excess GelMA solution, applying a photopolymerization process to the PCL / GelMA hybrid material, incubating the scaffold for 1 hour in a 2% antibiotic solution (preferably penicillin and / or streptomycin), seeding the HUC-MSCs in an incubator in a mesenchymal stem cell expansion medium under conditions of 37°C, 5% CO_2, 95% air, and 80% relative humidity, seeding approximately 500,000 to 700,000 cells for each 20*20* 1 mm dimension of the scaffold.

[0028] To demonstrate the efficacy of the invention, several experiments were also conducted, and the results of the studies performed with scaffolds produced according to an example embodiment of the invention are described below. Preparation of the PCL Fibrous Material: A fibrous material was printed from bead PCL using the MEW (Melt Electro Writing) method with the following parameters: a nozzle diameter of 0.25 mm, an infill rate between 20-50%, a pressure of 23-25 PSI, and an applied voltage of 2 to 10 kV. The fibrous material can be printed as a single piece or in multiple parts in dimensions suitable for the application area. To create the samples for the experiments, PCL microfibrous structures with dimensions of 20x20x 1 mm, various pore sizes in the millimeter range, and an average fiber diameter of 103 pm were printed using MEW. The printing was performed using an Axolotl 3D bioprinter with an integrated MEW apparatus.

[0029] Coating of the Fibrous Material with GelMA: Following the printing process, the biomaterials were coated with GelMA in order to enhance their biocompatibility.

[0030] Accordingly, the fibrous materials prepared by MEW were placed in 10% (w / v), 7% (w / v), and 5% (w / v) GelMA solutions containing 0.5% Irgacure; after a 1-hour incubation, the excess GelMA solution was removed, and the resulting MEW PCL / GelMA hybrid materials were photopolymerized. For this purpose, UV light at a power of 23mW / cm2and a wavelength of 285 nm was applied for 10 minutes. Subsequently, the materials were washed with PBS to remove any remaining unpolymerized GelMA. Additionally, to demonstrate the effect of GelMA on cell attachment, uncoated PCL fibrous material samples and fibrous material samples completely embedded in GelMA were also prepared as controls. For sterilization, the materials were incubated for 1 hour in a 2% antibiotic (penicillin / streptomycin) solution, after which they were ready for cell culture applications. The GelMA used in this study was synthesized by I§ik et al. [4],

[0031] Characterization of the Scaffold: SEM (Scanning Electron Microscopy): The physical and morphological structure of the PCL / GelMA scaffold of the invention was also examined using SEM (scanning electron microscopy). For this purpose, the prepared biomaterial scaffolds were dehydrated using techniques such as CPD (critical point drying) or freeze- drying and were thus made suitable for examination. The prepared samples were examined with a QUANTA 400F Field Emission SEM instrument. Tensile testing was performed using a Cell Scale micromechanical testing device.

[0032] Isolation of HUC-MSCs: During a Cesarean delivery, a 10 cm segment of the umbilical cord was taken from the end proximal to the placenta, and stem cells were isolated from the Wharton's jelly of this tissue. In this application, the Can and Balci technique was used for the isolation of stem cells from the tissue. [5]

[0033] Seeding of HUC-MSCs onto the Scaffold: The HUC-MSCs were cultured in an incubator in MSC expansion medium (Millipore) under conditions of 37°C, 5% CO2, 95% air, and 80% relative humidity. The medium was refreshed every 2-3 days, and when the cells reached 80-90% confluency, they were washed with DPBS (Dulbecco's Phosphate-Buffered Saline) and detached from the plastic using 0.05% trypsin / 0.02% EDTA to be passaged. Cells between passages 3 and 4 were used, and unused cells were stored at -86 °C. The cells were seeded onto each 20^20x 1 mm scaffold at a density of 500,000 cells per scaffold.

[0034] Cell Proliferation and Viability Assay: Cellular proliferation was evaluated after HUC- MSCs were cultured and then seeded onto the scaffold (the MEW PCL / GelMA hybrid material). Following an MTT assay on days 2 and 5, the proliferation of HUC-MSCs was measured by taking absorbance readings at 590 nm. Cell growth was then analyzed by plotting a growth curve based on the absorbance readings. To visualize cell viability, a Live / Dead assay was performed. The cells were stained for 10 minutes with the fluorescent dyes calcein (stains live cells, green wavelength) and ethidium homodimer (stains dead cells, red wavelength) (Thermo Fisher). The samples were then examined under a confocal microscope at the green and red wavelengths. Cell counts were calculated by counting cells per unit area in 10 different regions.

[0035] In Vivo Application of the Biomaterial in Rats: Forty 12-month-old female Wistar Albino rats were divided into five groups, with 8 rats in each group.

[0036] Group 1 : A PCL / GelMA scaffold cultured with stem cells are implanted.

[0037] Group 2: A Type 1 polypropylene mesh is implanted.

[0038] Group 3: A PCL / GelMA scaffold is implanted.

[0039] Group 4: The animals underwent a surgical incision which was then closed with polyglactin sutures, but no implant was placed.

[0040] Group 5: No incision was made in the animals. For this purpose, in two of the three rats, the procedures for group 1 and group 3 were performed in the left scapular region, while in the same rats, the procedures for group 2 and group 4 were performed in the right scapular region. The third rat was kept without an incision, serving as group 5. As of the filing date of this application, experiments for the same five groups, being conducted with 32 rats, are also ongoing.

[0041] Surgical Procedure: Following an intramuscular (IM) injection of ketamine / xylazine (90 / 10 mg / kg), the dorsal skin of the experimental animal was shaved and a vertical incision was made. Each 20^20 mm biomaterial / PP mesh was placed on the muscle between the two scapulae of the rat through a 3 cm skin incision and a 1 cm vertical incision in the muscle tissue. The materials were fixed to the muscle tissue with a single polyglactin suture, and the skin was closed with simple interrupted sutures. In Group 4 (sham), only the skin and muscle tissue incisions were made, followed by closure with polyglactin sutures. Post-operatively, IM antibiotic prophylaxis was administered for 3 days.

[0042] Follow-up of Experimental Animals: Following a 60-day follow-up period, the experimental animals were sacrificed, and the changes that occurred in the dorsal subcutaneous and muscle tissues were analyzed histologically. The implants were excised in blocks with a 1 cm margin of adjacent tissue, including the underlying adherent muscle. Each tissue sample from the first and second groups was divided into three strips; one strip was stored in a 2% antibiotic PBS solution for biomechanical testing, while the other strips were fixed in 10% buffered formalin and 4% paraformaldehyde solution, respectively, to prepare paraffin and cryo-blocks. Tissues from the other two groups were divided into two strips and were similarly fixed in 10% buffered formalin and 4% paraformaldehyde solution, respectively, for the preparation of paraffin and cryo-blocks.

[0043] Tissue Processing. Sectioning, and Staining: Following routine histological processing procedures, tissue sections obtained from paraffin blocks and cryosections were subjected to histochemical and immunofluorescent staining. They were then evaluated for tissue morphology, the relationship of the scaffold with surrounding tissues and cells, inflammation, angiogenesis, fibrosis, and biodegradation. For the assessment of inflammation, paraffin block sections were stained with Hematoxylin-Eozin and analyzed semi -quantitatively. For angiogenesis, microvascularization rates were compared between groups by counting endothelial cells labeled with a vWF antibody in the cryosections. The level of fibrosis was evaluated by determining the collagen I / III ratio in cryoblocks labeled with collagen I and III antibodies. Additionally, antibodies against the inflammation marker MMP2-9, the anti-inflammatory macrophage marker CD206, and the inflammatory macrophage marker CD80 were examined.

[0044] After the tissues were processed into paraffin blocks and cryoblocks, paraffin sections were cut at a 5pm thickness on a microtome, and cryosections were cut at a 10pm thickness on a cryomicrotome. For histochemical staining, Hematoxylin-Eosin and Mallory's Azan stains were performed. For immunofluorescent staining, the tissue sections were circled with a PAP -Pen and washed with PBS. Primary and secondary antibodies, diluted at the recommended concentrations, were incubated for 2 hours at 37°C within the wells created by the PAP -Pen and were subsequently washed off with PBS. Following the incubations, the sections were coverslipped with a mounting medium containing a nuclear stain (Hoechst) and were then examined, photographed, and evaluated under an Airyscan Super-Resolution Confocal Microscope (Zeiss LSM880).

[0045] As a result of the experiments, it was observed that on the PCL-only scaffold, cells did not adhere sufficiently to the material, with most passing through the pores. The effect of pore size was not significant, and it was noted that 650,000 cells (small pore size) and 500,000 cells (large pore size) had adhered to the underlying culture plastic. On the GelMA-coated PCL scaffold, however, the effect of pore size was significant; it was observed that 240,000 stem cells (small pore size) and 700,000 stem cells (large pore size) passed through to the culture plastic and were viable. Consequently, it was found that approximately 400,000- 500,000 viable stem cells remained on the small-pored, GelMA-coated material. For the PCL scaffold embedded in GelMA, it was seen that cells did not adhere sufficiently, with 120,000 cells (small pore size) and 100,000 cells (large pore size) remaining on the material. In conclusion, stem cell attachment and viability were successfully established with the small-pored, GelMA-coated PCL. (Figure 1)

[0046] For the viability assay, the created tissue scaffolds were grouped as PCL-only, GelMA- coated PCL, and PCL embedded in GelMA. For each group, the scaffold material was fabricated with either small (density < 80%) or large (density < 20%) pores. To each culture well containing a scaffold, 100,000 adipose tissue-derived mesenchymal stem cells at passage six, cultured in DMEM / F12 medium and provided by the laboratory, were added. The scaffolds were then incubated for 10 minutes at 25 °C, and 6 random fields were selected for imaging under an epifluorescence microscope at *400 magnification. The cells were provided with a high-glucose and amino acid-rich medium. After three days, cell attachment and viability were examined under a fluorescence microscope after staining with Calcein AM (CaAM). The cells that had passed through to the culture plastic were detached via trypsinization, centrifuged, and counted using a hemocytometer.

[0047] In the viability test, it was observed that scaffolds embedded in GelMA and scaffolds coated with 10% GelMA did not support cellular viability. Subsequently, 200,000 HUC-MSCs were seeded onto each of the scaffolds, which were prepared separately and coated via a fiber coating method or a dipping method, with GelMA concentrations of 10%, 7%, and 5%. Representative images of the explant and passage 1 HUC-MSCs that were used are shown in Figure 2a and Figure 2b.

[0048] Seven days after cell seeding, HUC-MSC proliferation was measured using a Cell Counting Kit-8 (CCK-8) via absorbance measurements at 450 nm, as specified by the manufacturer. Cell growth was analyzed by plotting a cell growth curve based on the absorbance readings. Cell viability on the four scaffolds was evaluated using a LIVE / DEAD viability / cytotoxicity kit and compared with Tissue Culture Plastic (TCP), which was used as a control. Representative Z-stack confocal images (300pm) of encapsulated HUC-MSCs, stained with the Calcein AM (live) and ethidium homodimer (dead) viability staining kit, were obtained on days 2 and 5. Live cells were stained green, while dead cells were stained red. It was found that the scaffold with the maximum adhesion and cell viability consisted of PCL fibers coated with 5% GelMA. (Figure 3)

[0049] Maximum cell viability rates were observed only at the 5% GelMA concentration. Very few viable cells were seen at the 10% GelMA concentration. Although more viable cells were observed in the field of view at the 7% GelMA concentration compared to 10%, this was not considered significant when compared to 5% GelMA. The proliferation capacity of HUC- MSCs on the GelMA / PCL membranes was observed via the XTT assay. In the XTT study, the experiments on day 2 and day 5 were conducted using the combinations with 5% and 10% GelMA concentrations. In light of this data, the XTT assay was performed according to the kit protocol to observe the difference in proliferation on day 2 and day 5. For this purpose, a colorimetric method for detecting cell viability and proliferation was used. This method is based on the principle that, at the designated time points of day 2 and day 5, the XTT tetrazolium compound is reduced to soluble, orange-colored formazan crystals in the cell culture medium. This reduction is mediated by nicotinamide adenine dinucleotide phosphate (NADPH)-dependent dehydrogenase enzymes found only in metabolically active cells. HUC-MSC cells were seeded onto the GelMA / PCL membranes in a 24-well plate at a density of 100,000 cells per membrane. The cells were incubated at 37°C and 5% CO2 until the time of analysis. At the designated time points, the culture medium in the wells containing the samples was removed, and after washing with PBS, 200pl of fresh medium was added. The reaction solution was prepared by adding 0.1 mL of activation solution to 5 mL of XTT reagent. 100 pl of the prepared solution was then added to each well containing a sample, followed by a 2-hour incubation in an incubator at 37°C with 5% CO2. Afterward, once the color change was observed to be complete, the supernatant containing the medium- XTT reaction solution was collected, transferred to a new 96-well plate, and the absorbance was recorded at a wavelength of 490 nm using a spectrophotometer. (Figure 4)

[0050] Only the scaffolds coated with 5% GelMA demonstrated a level of cell proliferation comparable to that of the positive control (TCP) (Figure 3). Based on these results, the samples coated with 5% GelMA were used for the in vivo studies.

[0051] In the in vivo study, in the experimental groups (groups 1 and 3), the scaffold was observed to be integrated with the surrounding connective and striated muscle tissues. Inflammatory cells and a single layer of squamous epithelium-like cells were observed lining the scaffold pores. Compared to the control group (in which the striated muscle and connective tissue appeared normal) and to groups 2 and 4, an increase in the amount of connective tissue and vascularization was also observed in groups 1 and 3 (Figure 5 a,b hematoxylin-eosin; and 5 c,d Mallory's Azan).

[0052] In the non-incised rat, the striated muscles and connective tissue appeared normal (Figure 7a Mallory's Azan staining; 7b Hematoxylin-Eosin staining).

[0053] In conclusion, the PCL and GelMA hybrid tissue scaffold has been shown to promote cell loading and accelerate tissue repair. In the XTT findings, a comparison of viability rates between day 2 and day 5 showed that cell viability increased from 40% to 70%. It was observed that the scaffolds implanted with HUC-MSCs rapidly triggered angiogenesis in rats over a 5-week follow-up period; integration with striated muscle tissue, epithelialization within the scaffold pores, and an increase in connective tissue were also noted. Furthermore, leukocyte infiltration was observed in the tissues. As a result of the SEM analyses, it was observed that the PCL scaffold produced by the MEW method had a fiber diameter of approximately 103 pm and that the targeted fiber alignment was achieved. The pore sizes are on the level of approximately 1 mm. (Figure 7a - PCL-only scaffold - SEM image of the PCL scaffold produced via the MEW method. The scaffold fiber diameter is approximately 103 pm. The alignment of the fibers is as targeted. The pore sizes are in the millimeter range.)

[0054] (Figure 7b - PCL / GelMA biomaterial - SEM images of the PCL scaffold coated with 5% GelMA to provide for integration and bioactivity. It is observed that the GelMA partially fills the pores of the PCL scaffold and creates a suitable micro-porous environment for the planned seeding of HUC-MSCs.)

[0055] Biomechanical Testing: Samples were prepared from the scaffolds for mechanical and tensile testing, and tensile tests were performed using a Cell Scale micromechanical testing device. Samples from four separate groups were used for the test:

[0056] 1) GelMA-coated-1 : PCL scaffolds fiber-coated with 5% GelMA;

[0057] 2) GelMA-coated-2: PCL scaffolds fiber-coated with % 10 GelMA;

[0058] 3) Non-GelMA-coated-1 : PCL scaffolds embedded in 5% GelMA;

[0059] 4) Non-GelMA-coated-2: PCL scaffolds embedded in 10% GelMA.

[0060] In conclusion, according to the biomechanical test results, the GelMA-coated-1 material (the PCL scaffold coated with 5% GelMA) was found to be the strongest material among the four samples studied, due to its higher Young's modulus, yield strength, and ultimate tensile strength values. The Non-GelMA-coated-1 scaffold, in contrast, appears to be the weakest material. (Figure 8a - GelMA-coated-1; Figure 8b - Non-GelMA-coated-1; Figure 8c - Comparison of mechanical properties (modulus of elasticity / Young's modulus) based on the stress-strain curves.)

[0061] Comparison of mechanical properties based on the Stress-Strain Curve: The Modulus of Elasticity (Young's modulus) was measured as follows: 13.947 kPa for GelMA-coated-1; 12.8 kPa for GelMA-coated-2; 7.32 kPa for Non-GelMA-coated-2; and 6.03 kPa for Non- GelMA-coated-1. As a result, the Young's modulus of the GelMA-coated-1 material was found to be higher than the other materials, which means it has a greater resistance to elastic deformation under load and is stiffer. The Non-GelMA-coated-1 material, on the other hand, was observed to have the lowest Young's modulus value and to be more flexible. These results are also shown in Figure 8c.

[0062] The advantages obtained with the tissue scaffold of the invention, which are supported by these results, are described below.

[0063] According to the invention, the tissue scaffold enhances the biocompatibility of PCL and improves cell adhesion, thanks to its GelMA content.

[0064] It was observed that cells proliferated very rapidly on the PCL scaffolds coated with 5% GelMA and UV-crosslinked. In the XTT findings, a comparison of viability rates between day 2 and day 5 showed that cell viability increased from 40% to 70%. The MEW method allows for the production of scaffolds / meshes with a high degree of control and precision, and no toxic solvents are used. They possess higher mechanical strength compared to products manufactured via electrospinning. Furthermore, it has the advantage of being customizable to produce scaffolds with properties suitable for the pelvic region, such as the ability to better control fiber thickness, pore size, and scaffold height.

[0065] On the other hand, the tissue scaffold was cultured with MSCs derived from the Wharton's jelly of the human umbilical cord. In addition to their other known properties, MSCs derived from the human umbilical cord express the adult stem cell markers CD73, CD90, and CD 105, and are considered to be adult-type stem cells. Furthermore, another of their advantages is that they also express embryonic stem cell (ESC) markers (Tra-1-60, Tra-1- 81, stage-specific embryonic antigen-1 (SSEA-1), SSEA-4, and alkaline phosphatase), which contributes to a reduced tissue fibrotic response. Another of their significant properties is that they do not produce teratogenic or carcinogenic effects following transplantation into tissues.

[0066] Any synthetic material placed in tissue leads to the release of platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and TGF-P in the tissues, and causes the migration of fibroblasts to the implantation site. These fibroblasts, in turn, synthesize collagen I and III to repair the damaged tissue.

[0067] The tissue scaffolds cultured with stem cells provide adequate mechanical support at the repair site, support cell proliferation, ensure an increase in connective tissue during the tissue repair process, do not trigger excessive and detrimental collagen synthesis, initiate proper healing without causing a fibrotic reaction, and simultaneously trigger angiogenesis. This developed material provides a more suitable and effective repair process compared to polypropylene material.

[0068] References

[0069] [1] Hansen S.G. et.al. 2020. Electrospun nanofiber mesh with fibroblast growth factor and stem cells for pelvic floor repair. J Biomed Mater Res Part B 2020: 108B:48-55.

[0070] [2] B. Aghaei-Ghareh-Bolagh a,b,f , S. Mukherjee c,d, f , K.M. Lockley a,b, S.M. Mithieux a, b, Z. Wang a, b,S. Emmerson c, d, S. Darzi c , C.E. Gargett c,d , A.S. Weiss. A novel tropoelastin-based resorbable surgical mesh for pelvic organ prolapse repair. https: / / doi.Org / 10.1016 / j.mtbio.2020.100081 2590-0064 / © 2020 The Author(s). Published by Elsevier

[0071] [3] Ulrich D. 2012. “A preclinical evaluation of alternative synthetic biomaterials for fascial defect repair using a rat abdominal hernia model”. Pios One7(l l)(2012) e50044.

[0072] [4] Isik, M., Eylem, C. C., Haciefendioglu, T., Yildirim, E., Sari, B., Nemutlu, E., ... & Derkus, B. (2021). Mechanically robust hybrid hydrogels of photo-crosslinkable gelatin and laminin-mimetic peptide amphiphiles for neural induction. Biomaterials Science, 9(24), 8270-8284

[0073] [5] A Can, D Balci. “Isolation, culture, and characterization of human umbilical cord stroma-derived mesenchymal stem cells”. Jour. Mesenchymal stem cell assays and applications 51-62, 2011

Claims

CLAIMS1. A method for producing a tissue scaffold, characterized in that it comprises; forming a polycaprolactone (PCL) fibrous material in the form of a mesh via MEW (melt electrowriting), incubating the fibrous material in a 5-10% (w / v) gelatin methacrylate (GelMA) solution containing a photoinitiator, and subsequently removing the excess GelMA solution, applying a photopolymerization process to the PCL / GelMA hybrid material, sterilizing the resulting scaffold, incubating human umbilical cord mesenchymal stem cells (HUC-MSCs) by seeding them in a mesenchymal stem cell expansion medium, seeding the expanded HUC-MSCs onto the scaffold.

2. The method according to claim 1, characterized in that the PCL fibrous material in mesh form is created via MEW with a nozzle diameter of 0.25±0.05 mm, an infill rate between 20-50%, a pressure of 23-25 PSI, and a voltage of 2 to 10 kV.

3. A method according to claim 1, characterized in that approximately 500,000 to 700,000 cells are seeded for each 20^20x 1 mm dimension of the scaffold.

4. A method according to claim 1, characterized in that 0.5% Irgacure is used as the photoinitiator.

5. A method according to claim 1 , characterized in that the fibrous material is incubated in the GelMA solution for 1 hour.

6. A method according to claim 1, characterized in that the step of sterilizing the scaffold comprises incubating it in a 2% antibiotic solution.

7. A method according to claim 1, characterized in that the HUC-MSCs are seeded in an incubator in a mesenchymal stem cell expansion medium under conditions of 37°C, 5% CO2, 95% air, and 80% relative humidity.

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