NANOPARTICULAR-COATED 3D-PRINTED TISSUE SCAFFOLDING PRODUCTION METHOD AND THE PRODUCT PRODUCED USING THIS METHOD.

Abstract

This invention relates to a method for producing nanoparticle-coated 3D-printed tissue scaffolds. The method involves producing a 3D-printed tissue scaffold using a 3D printer and polylactic acid (PLA) filament, modifying the surface of the 3D-printed tissue scaffold by treating it with a NaOH solution, synthesizing chitosan nanoparticles, and coating the surface of the 3D-printed tissue scaffold with chitosan nanoparticles. The nanoparticle-coated 3D-printed tissue scaffold produced by this method consists of a 3D-printed tissue scaffold made of polylactic acid (PLA) filament and a chitosan nanoparticle coating on the surface of the 3D-printed tissue scaffold. Chitosan nanoparticles can be loaded with at least one bone healing factor.

Description

1 TARIFF PRODUCTION METHOD OF NANOPARTICULAR-COATED 3D-PRINTED TISSUE SCAFFOLDING AND PRODUCT MANUFACTURED USING THIS METHOD Field of Application of the Invention 5 The invention relates generally to the field of tissue engineering and, more specifically, to bone healing. It relates to methods for producing nanoparticle-coated 3D-printed tissue scaffolds for use. This invention has implications for the field of tissue engineering in general, and more specifically, for bone healing. It is related to methods for producing nanoparticle-coated 3D-printed tissue scaffolds for use. 10 The technology described here can display depth information in physical environments such as the body. It is concerned with systems, methods, and devices that produce geometric representations based on this technology. The application emphasizes that accurate representation of the damaged tissue environment is vital for successful treatment. It is particularly relevant to the formation of tissue scaffolds for bone healing. State of the Art Regarding the Invention (Prior Art) Bone problems are generally caused by trauma, tumor formation, congenital disorders, and significant other issues. It can result from infection or surgery. Damage or disease in the bone structure. It significantly affects people's quality of life. By its very nature, it is able to regenerate itself and... Although it is a regenerative tissue, it cannot correct some major defects on its own, this 20 Clinical intervention may be necessary. The most commonly used methods today for treating bone defects... The most common method is autologous bone grafts. In addition, there are allograft and xenograft bone grafts. It is also used. Due to the problems that can be experienced with these methods, tissue engineering is preferred. These approaches are coming to the forefront. Three main elements are fundamental to tissue engineering. It contains; tissue scaffold, growth factors (BF) and cells. 25 Tissue engineering involves the functional transformation of cells, supporting structures, and biological factors in humans. Tissue engineering is a highly disciplined field aimed at improving tissue quality. The main goal of tissue engineering is... Functional structures that repair, protect, or restore damaged tissues or entire organs. 2 The goal is to create tissue engineering, which aims to direct the growth of new functional tissues. It involves the use of scaffolds as templates for tissue regeneration. 3D printing technology is a powerful tool for producing tissue scaffolds with complex structures and functions. It emerged as a tool. This technology, the skeleton, its size, its shape, 5 It provides precise control of porosity and internal structure. Polylactic acid (PLA), Due to its biocompatibility and biodegradability, biodegradable fiber is widely used in 3D printing. However, PLA scaffolds alone are not the desired material for tissue engineering applications. It generally lacks bioactive and cell adhesion properties. Although there are many methods for producing tissue scaffolds, 3D printing has become increasingly popular in recent years. It is frequently preferred. With this technology, the structures of scaffolding can be controlled at the micro level. This has made it possible to develop complex, patient-specific 3D materials. Since BF'n n let mb rçok is important in many respects, various studies have been conducted in this field and some 15 Methods have been developed. These methods include physically confining BF within the scaffold, the scaffold... covalent bonding is achieved using micro or nanoparticles (NP) as carriers. The use of various release methods for BFs with different molecular structures. It has been developed. While 3D-printed tissue scaffolds are popular, they can also be used in extrusion-based devices. Materials are limited. Synthetic polymers are generally preferred. Material compatibility, capacity to attract cells, and bioactivity are natural. It is not as high as the materials. For this reason, the natural surface of the scaffolding has been examined through various studies. It has been observed that it is covered with materials. K tosan stands out in this field with its compatible natural br 25 pol merd r. K direct integration of tosa into the surface, sten len biologic k compatibility increase While it can provide this, it does not allow for the acquisition of a different advantage. One of the BF release methods is via groups of reactants located between the scaffold surface and the BF. The goal is to establish a bond. Accordingly, BF mod f ye ed lmel ve reakt fbr grup eklenmel d r. This 30 3 This method allows the BF to remain on the scaffold for a longer period of time while simultaneously releasing the material slowly. The side effects of an extrabreathing reaction are also prevented. The release usually occurs through hydrolysis and reduction reactions. or it occurs with enzyme mat k factors. Although the benefits of this method are interesting, the method is lengthy. Time constraints, increasing workload, and high costs limit its use. Furthermore, The region where the modified BF covalent bond binds may limit protein activation. 5 and therefore the effect can be reduced. Another release method is non-covalent bonding. BFs are physically encapsulated within the scaffold structure. For this, physical encapsulation, protein n Adsorption or ion interactions can be used. With these methods, BF, 3D matrix structure can be obtained. It is immobilized inside. However, in this method, the release rate is controlled because it depends on diffusion. It is very difficult to control and consequently rapid release can occur. 10 The Chinese patent certificate numbered CN110484225A, which is included in the technical status, concerns k tosan. The method describes a nanoparticles loaded onto a 3D-printed scaffold, but the method... It involves complex procedures and the resulting scaffold contains irregularly arranged nanoparticles. The distribution is as follows: 15 In the patent application document numbered WO2018031491 A1, which is included in the technical status, To enable tissue regeneration; three-dimensional printed tissue scaffolds and production methods. It has been explained. In the patent application document numbered WO2022271255 A1, which is included in the technical status Tissue scaffolds, scaffold construction and usage methods are explained. In the method in question, A textile layer consisting of numerous plies is obtained. For this purpose, many a number of plates, consisting of one polymer or a mixture of two polymers It consists of interlocking fiber bundles. In addition, it has a predefined thickness of 25 a layer of textile containing one or more of two polymers or a mixture of two polymers. An additional substrate layer is being created. Specifically, PLA filament is mentioned in the document. It is not mentioned. 4 Purposes and Brief Description of the Invention The aim of the invention is to further apply chitason nanoparticles to the surface of chitason-coated 3D-printed tissue scaffolds. to enable the formation of a uniform and stable LBR coating and therefore, bone matrix from nanoparticles It increases the controlled release of y- ... For this purpose, the invention involves 3D-printed tissue scaffolding surfaces coated with NaOH solution. Processing and modification of chitosan nanoparticles, synthesis of chitosan nanoparticles and 3D printed tissue a study involving the steps of coating the surface of the skeleton with synthesized k tosan nanoparticles The method is explained. Definitions of the Figures Illustrating the Invention Results and related graphs regarding tissue scaffolds obtained using the method discussed in the invention. This is explained below. Figure 1: Structural and morphological characteristics of a 3D-printed PLA scaffold at 60% infill. Characterization: (A) Digital image, (B) Light microscope image; scale: 200 μm, (C) SEM 15 (D) Top view with SEM analysis; scale: 200 μm, (D) Cross-sectional view with SEM analysis; scale: 100 μm. Figure 2: TEM micrographs of empty chitosan nanoparticles at (A) 200 nm and (B) 100 nm scale. Figure 3: (A) untreated, (B) alkali-treated, (C) nanoparticle-coated tissue. SEM images of the piers. 20 Figure 4: Size distribution graphs of nanoparticles synthesized with magnetic stirrer: (A) empty (A) DIPQUO-charged nanoparticle (NP), (B) DIPQUO-charged nanoparticle (D-NP), (C) GS4012-charged nanoparticle (G-NP) and (D) DIPQUO and GS4012 co-charged nanoparticles (DG-NP). Figure 5: Zeta potential distribution of nanoparticles synthesized with magnetic stirrer. Graphics: (A) empty nanoparticle (NP), (B) DIPQUO charged nanoparticle (D-NP), (C) GS4012 25 (D) charged nanoparticle (G-NP) and (D) DIPQUO and GS4012 co-charged nanoparticle (DG-NP). Detailed Description of the Invention With the method described in the invention, the surface of a 3D-printed tissue scaffold is treated with an NaOH solution. It is being modified. The invention concerns the method of synthesizing chitosan nanoparticles and 3D the steps of coating the printed tissue scaffold surface with nanoparticles It contains. 5 The invention relates to a method for producing 3D-printed tissue scaffolds coated with nanoparticles. Furthermore... Specifically, the method involves 3D printing using a 3D printer and polylactitic acid (PLA) filament. The process involves producing a tissue scaffold. Then, the surface of the 3D-printed tissue scaffold is coated with an NaOH solution. It is modified by processing. Following this, chitosan nanoparticles are synthesized and 3D printed 10 It is used to coat the surface of the tissue scaffold. A preferred application of the invention is the production of tissue scaffolds in .stl format, 10 mm. A three-dimensional cylindrical model with a diameter of 2.5 mm and a height of 2.5 mm was used. Zaxe X1 3D Tissue scaffolds were created by attaching PLA filament to the printer. The scaffold consists of 0.05 mm sheets. Its thickness is 42 layers. The internal design is based on a 0° / 90° cross-hatching pattern. It has been determined that the occupancy rate of the selected scaffolds in the device's Xdesktop software v2.1.6 is 60%. It has been determined that while molten PLA is extruded through a 0.4 mm diameter nozzle, the printing temperature is... The temperature was set to 210°C and the printing plate was heated to 60°C. In a preferred application of the invention, chitosan nanoparticles are used in Calvo's ionic gelling 20 It was synthesized using this method. Empty nanoparticles were synthesized by adding them drop by drop to the chitosan solution. Sodium tripolyphosphate (TPP) was added. The chitosan:TPP mass ratio was increased to 3.33, and the pH of the medium was increased to 4.8. It is adjusted and continues to operate at 400 rpm for 30 minutes in the magnetic stirrer. It has been done. For the synthesis of GS4012-loaded nanoparticles, GS4012 was directly added to the chitosan solution and 25 Mixed for 30 minutes to ensure homogeneous distribution. 0.1% for DIPQUO-loaded nanoparticle synthesis. A complex was formed by adding DIPQUO to a chitosan solution containing Tween 20, and It was mixed for 30 minutes to ensure homogeneous distribution. Finally, this medium was added for nanoparticle synthesis. TPP was added dropwise. Chitosan containing 0.1% Tween 20 was used for the dual encapsulation study. DIPQUO is added to the solution, and then 30 drops of this mixture are added using a magnetic stirrer. Nanoparticle synthesis is carried out by adding droplets of GS4012 and TPP complex. 6 In one application of the invention, in the step of coating the tissue scaffold with nanoparticles, the tissue To open active –OH groups on the scaffolds, they were treated at a concentration of 0.5 M for 4 hours. It is treated with NaOH. Washing is done with phosphate buffered salt solution (PBS). Non-binding groups are removed. Nanoparticles 5 are applied to the modified tissue scaffold surface. It is added directly and dried in a vacuum desiccator. In one application of the invention, a 3D-printed tissue scaffold is produced using a 3D printer with a nozzle diameter of 0.4 mm. It is produced using [method name]. In one application of the invention, PLA filament is used in the production of 3D printed tissue scaffolds. It is extruded at a temperature of 210°C. The extrusion temperature is the temperature at which the PLA filament is extruded by the 3D printer. to ensure that the nozzle is sufficiently molten for smooth extrusion and at the same time It was chosen to solidify rapidly in order to preserve the skeletal structure over time. The use of a PLA filament extruded at 210°C results in a skeleton that is 15 inches thick. The technology provides a technological advantage in terms of mechanical properties. In particular, the skeletal healing process. the advanced strength required to support the load-bearing function of the bone throughout and rj tl ğ serg lemekted r. In one application of the invention, 20 are used to modify the surface of 3D-printed tissue scaffolds. The concentration of the NaOH solution is 0.5 M. Using a 0.5 M NaOH solution on the surface... This provides a technical advantage in terms of the efficiency of the modification process. In particular, this NaOH concentration to trigger the formation of hydroxyl groups on the PLA filament surface This is sufficient, and in the next steps, it will demonstrate the ability to bind to ktosan nanoparticles. This increases the uniformity and stability of nanoparticles on the scaffold surface. 25 This leads to its formation. Therefore, the bone formation factors from nanoparticles are controlled. It is beneficial in terms of release. In one application of the invention, k tosan nanoparticles, k tosan solution, and tripolyphosphate. The addition of n is synthesized using the ionic gelation method. Ionic gelation 30 7 Synthesis of k-tosan nanoparticles using this method, considering the form-size distribution and This enables the production of nanoparticles with high stability, which in turn allows for the production of nanoparticles. It increases the controlled release of bone marrow binding factors. In one application of the invention, the surface of a 3D-printed tissue scaffold is coated with tonic nanoparticles. The coating process is carried out under vacuum conditions. The use of vacuum conditions makes the scaffolding... The uniformity and stability of the nanoparticle coating on the surface offer a technical advantage. This is especially true under vacuum conditions, which enable the adhesion of ktosan nanoparticles to the scaffold surface. This facilitates a smoother and more stable LBR coating. The invention describes 3D-printed tissue scaffolds coated with nanoparticles, produced using a specific method, and made from PLA filament. a 3D printed tissue scaffold and a 3D printed tissue scaffold surface made of brick. It contains nanoparticles coated with a powder coating. K tosan nanoparticles contain osteogenic factors and from a group of angiogenic factors, at least one bone formation factor can be selected. It can be loaded. 15 Nanoparticles coated with 3D-printed tissue scaffolds offer a technological advantage in bone regeneration. Chemical factors loaded with ktosan nanoparticles, controlled release of these factors By providing this, it increases the effectiveness of promoting skeletal bone formation. The use of 3D printers, By enabling the production of patient-specific scaffolds, it further enhances the effectiveness of the treatment. PLA 20 Filament surface modification enables the immobilization of molecules on the surface. Skeletal NB increases the active function of the bone. The combination of all these features reduces bone defects. It offers a single-step solution in treatment and reduces the number of additional steps during and after surgery. can reduce it. Using a PBR 3D printer with a 0.4 mm nozzle diameter ensures accuracy and precision in scaffold production. In this respect, the technology of CBR offers an advantage. This feature, in particular, allows the skeleton to connect to specific parts of the patient's bone. consistent and appropriate applications that can be useful in a defect area This results in the production of scaffolds of repeatable sizes. 8 The invention describes a method involving NBR applications with 3D-printed tissue scaffolds and a layer thickness of 0.05 mm. It is produced in this way. This layer thickness is related to the resolution of the scaffold structure and the production speed. It is chosen to provide balance. A layer thickness of 0.05 mm is the thickness of natural bone tissue. While allowing the production of complex scaffolding structures that closely imitate those of the same architect, This ensures that scaffold production is carried out rapidly in time. This is cell 5 by providing a suitable micro-environment for attachment, multiplication and differentiation in bone technical advantages in the skeleton's ability to promote regeneration It helps. In one application of the invention, 3D-printed tissue scaffolds are produced with a 60% PLA filler. This filler is 10 The ratio is to strike a balance between the skeletal mechanical strength and porosity. It is selected that a 60% filler ratio ensures the bone's load-bearing function throughout the healing process. In addition to a skeleton strong enough to support it, it also supports cell infiltration and nutrition. High porosity that will facilitate fusion and waste removal. This provides a suitable microenvironment for cell attachment, proliferation, and differentiation. By providing this, the skeleton's ability to stimulate bone regeneration is technically enhanced. It provides an advantage. The PLA has a 60% occupancy rate. (Images taken using a light microscope and SEM.) This is shown in Figure 1. The pore diameter is 20° as determined by ImageJ analysis of the top-down images. The cross-sectional area is 464.30 ± 10.60 μm, and the size of the single pores is 346.46 ± 26.46 μm. It has been observed that the porosity was calculated as 43.8%. Only PLA, surface modified... Carbon matrix degradation study for PLA and nanoparticle-coated PLA scaffolds. The study was conducted. Samples were analyzed at the end of days 5, 10, and 15. PLA pier, It deteriorated by losing 14% of its weight. Surface modified scaffolding 5th day 25 While initially experiencing a 7% loss, this loss reached 16% by the end of the 15th day. The study... Finally, a 12% degradation was observed in the nanoparticle-coated scaffold. Specifically... On the nanoparticles coated scaffold, the swelling rate increased to 5% on the 5th day, due to weight loss. The swelling rates on the last day of the study could not be calculated. The rate was 8.4 in all sample groups. It was observed that the pH value dropped sharply from the first few days, reaching as low as 4. 30 9 In one application of the invention, chitosan nanoparticles are used to coat the skeleton surface. It is loaded with at least one bone loss factor. Bone loss factor, osteogenic factors and from a group of angiogenic factors or a combination of both The use of calcinant nanoparticles loaded with bone formation factors, skeletal n Technological advancements offer a significant advantage in terms of their ability to promote bone formation. Specifically, bone formation... Controlled release of y- leşme factors from nanoparticles, skeletal nb olojij k akt v tes n It increases and potentially accelerates the bone formation process. Osteogenic factors cause new bone formation. While promoting bone tissue formation, angiogenic factors provide nutrients to the newly formed bone tissue. It stimulates the formation of new blood vessels that will provide oxygen. Thanks to this invention, controlled release of BFs can be achieved by integrating them into nanoparticles. And depending on the desired application area, long-term release profiles can be provided. Materials By coating the scaffolding surface, BF emissions are brought under control. As a BF carrier Among the materials used, k tosan stands out as a natural material. There are many advantages to using nanoparticles. Bone and tissue engineering 15 It is known that mechanical strength is very important in applications. Mechanical properties It can be developed using nanotechnology approaches. The use of nanoparticles creates an environment that enhances cell adhesion and proliferation. By facilitating the imitation of natural bone, it promotes osteointegration, osteoconduction, and 20 Osteoinduction is improved. Nanoparticles are small in size. Therefore, they cover a large surface area. They possess a specific area and maintain the effectiveness of the loaded material. BFs in nanoparticles In vivo, its degradation by enzymes is prevented, and long-term preservation is ensured. It is generally possible to control the release rate, which depends on the particle size. It is possible to release more than one molecule using nanoparticles. 25 Thanks to this discovery, osteogenic and angiogenic factors, which are the most important factors in bone formation, have also been identified. The factors were simultaneously encapsulated within nanoparticles. Thanks to nanoparticles, a carrier mechanism for small molecules has been provided, with this 30 With controlled release, the specified amount of product was released into the environment within the specified time. Multiple molecules are combined in a single carrier, shortening the processing time. 3D printer Thanks to this, personalized treatment has become possible. PLA, an inert material, has been modified. By processing, the surface has been made suitable for molecular mmoblization. In total, all When the materials are combined, they are placed into the damaged tissue in a single step during surgery and This has prevented the need for additional steps afterwards. Synthesis of nanoparticles (NPs) takes 5 months. The next step is determining the dimensions. These are taken directly from the environment after synthesis. Nanoparticle size graphs are shown in Figure 4, and zeta potential graphs are shown in Figure 5. It has been shown. The dimension obtained from the Zeta device (Zavg(nm)), PDI and zeta potential (Zeta (+, mV)) results and encapsulation efficiency (EE), loading capacity (YK) and reaction efficiency (RV) is shown in Table 1. TEM micrographs for empty nanoparticles are shown in Figure 2. This is shown. The results indicate that empty nanoparticles have an average kbr size distribution of 150 nm. He has shown that he owns it. Table 1. Nanoparticle size and zeta potential results. Example Zavg(nm) PDI Zeta (+, mV) RV (%) EE (%) YK (%) Empty-NP 110.9±2.50 0.31±0.03 14.8±2.18 52.46±3.10 - - D-NP 144.2±3.06 0.28±0.00 17.8±0.36 69.54±8.23 87.67±13.39 32.32±17.71 G-NP 148.1±2.90 0.36±0.01 21.1±0.36 56.03±2.02 89.97±08.44 59.50±19.07 DG-NP 128.0±1.08 0.27±0.00 18.0±0.74 60.58±7.47 79.37±13.841 75.15±10.952 41.52±4.221 17.36±0.862 1 This data belongs to DIPQUO. 2 This data belongs to GS4012. 15

Claims

11 REQUESTS 1. - 3D-printed tissue scaffolds using a 3D printer and polylactic acid (PLA) filament. to produce, - Modifying the surface of 3D-printed tissue scaffolds by treating them with NaOH solution, 5 - To synthesize K-tosan nanoparticles and - Steps for coating the surface of 3D-printed tissue scaffolds with nanoparticles To produce nanoparticles coated with 3D printed tissue scaffolds. method.

2. 3D printed tissue scaffolding with a 0.4 mm nozzle diameter using a PBR 3D printer. The production method is characterized by the production method in system 1.

3. Polylactic acid filament used in the production of 3D printed tissue scaffolds at 210°C The GBBr method in system 1 or 2, characterized by extrusion at a certain temperature. 15 4. NaOH solution used to modify the surface of 3D printed tissue scaffolds. The stem, characterized by having a concentration of 0.5 M, is any of the 1 to 3. br ndek gbbr method.

5. Synthesis of K-tosan nanoparticles using ion gelation method character ze ed len stem 1 la 4'ten herhang br ndek gbbr sistem.

6. Characterization of the ionic gelation method by adding tripolyphosphate to the cyclosan solution. ed len stem 5'tek gbbr method. 25 7. Vacuum coating of the surface of 3D printed tissue scaffold with nanoparticles any of the following systems characterized by being carried out under certain conditions: br ndek gbbr method.

8. Polylactate composite laminate 3D printed tissue scaffold and 3D printed tissue skeles nn surface coated brk tosan nanopart kül le karakter ze ed len stem 1 12 a nanoparticle coated 3D printed using any method from la 7 tissue skeleton.

9. Characterized by chitosan nanoparticles loaded with at least one bone binding factor. STEM 8 GBBR nanopart ash-coated 3D printed tissue scaffold. 5 10. At least one of a group consisting of osteogenic factors and angiogenic factors selected. kem ky leşmeoksiti le Karakter ze ed len stem 9'dak gbbr nanopart kül Kaplama 3D printed tissue scaffolds.

11. For use in bone formation, any of the following from system 8 to 10. Nanopart ash-coated 3D-printed tissue scaffolds. 20

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