A magnetic substrate based tissue scaffold and a method for the production of this tissue scaffold with the support of a 3D printer

Abstract

The invention relates to a magnetic substrate based tissue scaffold suitable for use in the growth, proliferation, and organization of cells in the technical field of tissue engineering, provided with intelligent behavior characteristics and thus dynamic structure, and a method for the production of this tissue scaffold with the support of a 3D printer.

Description

[0001] DESCRIPTION

[0002] A MAGNETIC SUBSTRATE BASED TISSUE SCAFFOLD AND A METHOD FOR THE PRODUCTION OF THIS TISSUE SCAFFOLD WITH THE SUPPORT OF A 3D PRINTER

[0003] TECHNICAL FIELD

[0004] The invention relates to a magnetic substrate based tissue scaffold suitable for use in the growth, proliferation, and organization of cells in the technical field of tissue engineering, provided with intelligent behavior characteristics and thus dynamic structure, and a method for the production of this tissue scaffold with the support of a 3D printer.

[0005] PRIOR ART

[0006] Tissue engineering is an interdisciplinary field of study that develops biological systems designed to perform or support the functions of tissues and organs, or to replace them completely, using the principles of engineering, medicine, and life sciences. With the tissue engineering method, researchers aim to eliminate waiting lists for organ transplantation and to produce suitable tissues and organs for patients from the patient's own stem and primary cells in a laboratory environment.

[0007] Tissue engineering combines the principles of cell transplantation, materials science, and engineering to develop biological substitutes that can restore and maintain the normal function of diseased or injured tissues / organs. Despite technical advancement in recent decades, the use of these approaches has been limited to research applications, and few have been used in the clinic. Many clinicians are still using biodegradable polyesters, which were first approved for use in humans 30 years ago. This is a serious concern because morphogenesis is strongly influenced by interactions between cells and the extracellular environment during normal tissue development. While simple synthetic polymers currently in use provide support for neo-tissue development, they fail to successfully mimic the complex interactions between tissuespecific cells and tissue-specific extracellular matrices (ECMs) that promote functional tissue regeneration. In tissue engineering, three strategies are used to modify or stimulate targeted tissues: (1) the use of cells alone, (2) the use of biocompatible biomaterials, (3) the use of a combination of cells and biomaterials both. These cells and biomaterials are combined into scaffolds through various processes, which can often be classified as top-down or bottom-up

[0008] As a technology that has been frequently mentioned in recent years, 3D printing has entered tissue engineering as 3D bioprinting, which allows the co-printing of cells and biologic agents. 3D bioprinting is used in many areas in the literature, especially in tissue repair, cell carrier scaffold production, disease and defect modeling, and drug delivery and monitoring applications.

[0009] Bioprinting is a 3D production technology used to precisely dispense cell-loaded biomaterials for the construction of complex 3D functional living tissues or artificial organs. Although still in its early stages, bioprinting strategies have shown their potential use in regenerative medicine to produce a variety of transplantable tissues, including skin, cartilage, and bone. However, current bioprinting approaches still have technical challenges in terms of innovation in high-resolution cell deposition, controlled cell distributions, vascularization, and complex 3D tissues. While a one-size-fits-all approach to bioprinting has not emerged, it remains an optional, versatile production technique that can address the growing shortage of organs, as well as provide a highly efficient method for micrometer-scale cell modeling for broad biomedical engineering applications.

[0010] Accordingly, the difficulty of printing complex structures using 3D printing is proportional to the printing precision and the complexity of the structures to be printed. The materials used for 3D bioprinting are called bioinks. Bioinks are aqueous solutions of hydrogels that also contain cells for the formation of a 3D cell environment. Bioinks, which have a high water content and low crosslinking density, are materials that are often inversely proportional to their biological performance and printability. In order for the 3D bioprinting process to be successful, it is necessary to achieve a balance between these two parameters.

[0011] Therefore, smart biomaterials that actively participate in functional tissue regeneration should be developed and used for future applications. As a result, it has been determined that a tissue that can show high biological performance in the field of tissue engineering and have a form that can be compatible for patients after transplantation, and innovations for the production thereof should be provided.

[0012] BRIEF DESCRIPTION OF THE INVENTION

[0013] In the technical field of tissue engineering, appropriately produced / patterned tissue scaffolds are needed to enable cells to form target tissues in an organized manner in tissue transplantation for patients.

[0014] In the present art, 3D printer method is used for the production of tissue scaffolds to be transplanted. In particular, said synthetic tissue scaffolds have known disadvantages in the art, especially when it comes to their production in 3D-dimensional printers and their bioinks that can be used as raw materials in them. The most important disadvantage is that synthetic tissue scaffolds obtained from 3D printers obtained with bioinks have a static structure. However, in order for this static structure to be compatible with the environment and the person's body after transplantation to the patient, having a dynamic structure is the characteristic expected by the advancing technology.

[0015] The inventors of the present invention aim to provide additional technical characteristics to the tissue scaffolds obtained from 3D printers, giving them a dynamic characteristic. To achieve this, the use of smart materials with shape memory or selfactivating characteristics as biomaterials in tissue scaffolds is proposed.

[0016] Accordingly, the primary object of the present invention is related to obtaining smart materials with shape memory or self-activating characteristics for use in the technical field of tissue engineering.

[0017] Another object of the invention is to introduce a tissue that exhibits a controlled and functional change in physical or chemical characteristics when exposed to a magnetic stimulus. Another object of the invention is to introduce a tissue production method for eliminating the need for extra device use.

[0018] Another object of the invention is to introduce a tissue production method for maximizing cell viability.

[0019] DETAILED DESCRIPTION OF THE INVENTION

[0020] In this detailed description, the subject of the invention relates to a tissue scaffold comprising smart materials with shape memory or self-activating characteristics as biomaterial for use in the technical field of tissue engineering, and the production thereof, and is explained by way of example only for a better understanding of the subject, which shall not create any limiting effect.

[0021] In the invention, "complex tissue" is expressed as a biological structure that combines different cell types and components to perform a unique function.

[0022] In the invention, "smart material" is expressed as materials that react to environmental conditions or applied stimuli. Said smart materials exhibit changes in their physical or chemical characteristics when exposed to a specific stimulus. These changes can occur in response to various stimuli, such as temperature, pH, light, electric field, magnetic field, or chemical components.

[0023] As is known in the art, the use of 3-dimension (3D) bioprinting production method is suitable as a tissue scaffold production method. However, in said 3D bioprinting methods, the need to use support materials in the production of very complex geometries arises. Accordingly, this situation poses a technical challenge in terms of production process and cell viability.

[0024] In order to eliminate the aforementioned technical disadvantage, the inventors of the present invention have worked to save time in tissue production and maximize cell viability for the relevant technical field.

[0025] Based on what has been mentioned herein, the present invention relates to a method for the production of tissue scaffold systems by 4D printing for the technical field of tissue engineering and is detailed in the following lines below. Said 4D printing is integrated with the 3D printing known in the art and comprises process steps for the products obtained from this method to gain additional characteristics.

[0026] - Obtaining guide molds with a 3D printer

[0027] Said molds are designed by means of design software known in the art and are produced in 3D printers, preferably using PLA filaments. Here, PLA may vary in the future. But today, it is preferred since it is the most well-known and frequently used material. The scope of protection of the invention is not limited to this. Likewise, design and software used for design are not decisive for the subject of the invention. What is desired to be achieved here for the relevant technical field is to obtain tissue scaffolds that are more effective than the products to be provided by 3D printing, that can be compatible with the environment and the patient and, if preferred, have a more complex design.

[0028] Accordingly, there is a mold formation with a 3D printer. And in this invention, the term guide mold for mold is preferred to avoid ambiguity. In this invention, there are existing embodiments belonging to the inventors of the present invention for obtaining the mold, however, since the innovative aspect of the invention is not included here, a detailed explanation is not necessary.

[0029] - Preparation of bioink

[0030] In this invention, bioink is considered as the raw material of the final product. Accordingly, the final product is expected to consist of that material according to the characteristic to be obtained. In a preferred embodiment, bioink may be a polymer material manufactured from one or more biocompatible materials.

[0031] If preferred, bioink can be a polymer material in which one or more synthetic polymers are combined.

[0032] If preferred, the final product can be a product with a configuration in layers. In this case, each layer can be a synthetic or natural polymer material with a different function. According to the physical and chemical characteristics of the final product to be obtained in this invention, the composition of the bioink is very important. In addition, the bioink to be obtained also determines the print quality.

[0033] The present invention preferably utilizes gelatin methacrylate polymer material derived from the natural polymer gelatin. After the crosslinking processes of natural polymer gelatin, gelatin methacrylate polymer material can be obtained.

[0034] A photo-crosslinkable solution is prepared for the production of bioink of the invention. In this solution, gelatin methacrylate polymer material is added as raw material. Then, at least one photoinitiator and at least one catalyst are added to the solution.

[0035] In a preferred embodiment, the gelatin methacrylate (abbreviated as GELMA) suitable for use in obtaining bioink is contained in the solution at a value between 10% to 30% by weight.

[0036] In a preferred embodiment, the solution prepared for bioink production contains alginate solution as a crosslinking component. Said alginate solution is present in the solution at a value in the range of 0.5% to 1 .5% by weight.

[0037] In a preferred embodiment, said solution contains at least one photoinitiator component at a value between 0.5% to 2% by weight. It comprises at least one of the compounds EY (known as Eosin Y in the art) and / or TEA (known as triethanolamine) as a photoinitiator component. In a preferred embodiment, it comprises both EY and TEA compounds as photoinitiators.

[0038] When preferred, the bioink solution contains V2P compound (1 -vinyl-2-pyrrolidone) to accelerate the polymerization processes. The V2P compound is at a value in the range of 0.5% to 1 .5% by weight in the solution.

[0039] - Obtaining of magnetic substrates

[0040] Magnetic substrates are present in the tissue scaffold for shaping the bioink to be printed thereon into a tubular shape with the help of a magnetic field after printing. For this reason, each substrate produced must have the same thickness and dimensions. It has been determined that there must be a folding neck on the substrate so that each product to be obtained can be folded in the same way every time.

[0041] In order to achieve these objectives, the magnetic substrate is manufactured from silicone rubber materials, which are non-cytotoxic, easily curable, and flexible materials.

[0042] Said silicone rubber material contains corrosion-resistant carbonyl iron powders for magnetic effect.

[0043] The silicone rubber used in the invention has a fluid form at room temperature. In this way, in order to obtain magnetic substrate in the desired dimensions and designs, carbonyl iron-containing silicone rubber is cured in the guide mold obtained by means of a 3D printer in the previous process step. To achieve this, the present invention comprises at least one silicone rubber curing agent. Said magnetic substrate is in the form of a 1 :1 mixture of silicone rubber:curing agent by weight at room temperature prior to curing of the substrate. Carbonyl iron powder at a value of 30% by weight is added into this mixture.

[0044] Then, this mixture is poured into guide molds and curing process is carried out for at least 4 hours. This process step is preferably carried out at a temperature in the range of 20 to 24 °C.

[0045] - Surface modifications of magnetic substrates

[0046] Due to the hydrophobic nature of silicone rubber surfaces used as raw materials in obtaining magnetic substrate, it is very difficult to print polymers with low viscosity. In order to overcome this obstacle, the surfaces of the magnetic substrates obtained in the previous process step are preferably made hydrophilic by UV-Ozone application and APTES ((3-Aminopropyl) triethoxysilane) binding.

[0047] As the first process step, the magnetic substrates obtained by the curing process are kept in the UV-Ozone cleaner for at least 30 minutes. Then, it is preferably treated with APTES solution. Preferably, ethanol is included as a solvent in the APTES solution. The amount of APTES in solution is at a value between 1 % to 4% by volume. T reatment with APTES solution continues for at least 16 hours.

[0048] The obtained hydrophilic magnetic substrate is subjected to washing processes. Ethanol and distilled water are used in washing processes.

[0049] - Addition of bioink to prepared magnetic substrates

[0050] The bioink obtained in the previous process step is placed on the robotic head where 3D printing processes will be performed. This involves setting the bioink to a temperature between 20 to 25 °C. This temperature value is the set temperature as it is the minimum temperature allowed by the robotic head. The temperature of the tray where the printing processes will be carried out is at a value between 17 °C to 23 °C. Printing processes are carried out with bioink polymer composition on magnetic substrates obtained from reference molds. For these printing processes, printing processes are carried out with the values given in Table 1 below. The shared parameter values for the printing processes were obtained as a result of research and development activities to ensure that the final product has the desired mechanical and chemical characteristics.

[0051] Bioink, as preferred in this invention, is in the form of a cross-linked composition of gelatin methacrylate, a natural polymer, with an alginate solution.

[0052] Table 1. Printing parameters for 3D printing processes used in the method of the invention

[0053] The bioink to be printed is prepared in a sterile manner in the composition mentioned in the previous process steps; GELMA and alginate powders are sterilized by being kept under UV light for at least 30 minutes, and other materials in liquid form are sterilized after dissolving in distilled water, preferably by passing them through a 0.22 micron filter. All tools such as plunger, needle tip, syringe to be used in the 3D printer are sterilized by drying under UV light after a 1 -hour 70% ethanol bath. All joining and filling processes are carried out in the laminar flow cabinet. The needle tip insertion and filling sections of the bioink-filled printer syringe are carefully wrapped with parafilm. It is then covered with a plexiglass cabinet and placed on the robotic head of the 3D printer, which is UV-irradiated for 1 hour, and made ready for printing. On the other hand, magnetic substrates to be used for printing substrates are sterilized by drying under UV light after being left in 70% ethanol for 1 hour. The substrates transferred to the 3D printer cabinet in sterile petri dishes are placed on the 3D printer printing tray sterilized with UV light and made ready for printing. After all placements are completed, the robotic head temperature is set to 20 to 25eC and the print tray temperature is set to a value between 17eC to 23eC. After the syringes and substrates reach the determined temperatures, the printing process is started. After the printing process is completed, GELMA is crosslinked using 50 W, 470 nm wavelength blue LED light and alginate crosslinking is performed using 0.25 M sterile CaCh solution.

[0054] In the study where blue light was used first; after printing, GELMA was crosslinked with blue LED light for 1 minute at a distance of 5 cm on the tray at 17 °C, followed immediately by alginate cross-linking with 4 °C, 0.25M CaCh spraying. In the study where CaCb was used first, alginate crosslinking was performed by spraying 0.25M CaCh at 4 °C after printing, and after 5 minutes of waiting, GELMA crosslinking was performed with blue LED light for 1 minute at a distance of 5 cm on a cold tray. The GELMAIg101 and GELMAIg201 samples were gelled in this way by double crosslinking.

[0055] - Application of Alginate Lyase

[0056] Alginate, which is added to the structure in order to increase 3D printability and to provide the mechanical strength required in the folding stages, is removed from the system with the application of alginate lyase (AL) enzyme in order to increase cell spreading in the hydrogel matrix after crosslinking and folding process. 24 hours after printing, 0.05 U / ml alginate lyase enzyme was added to the culture medium of the printed and folded hydrogel. As a result of this process, alginate was removed from the samples. TESTS

[0057] Swelling Ratio Measurement

[0058] Swelling ratio calculation was made to measure the liquid absorption capacity of the samples obtained after the printing processes. The obtained hydrogels were washed with PBS buffer for 5 minutes and then lyophilized for 24 hours. After lyophilization, the dry weights of the samples were measured. The samples were then incubated at 37eC in PBS for 24 hours and then their wet weight was measured. The swelling ratio was defined as the ratio of the mass value after swelling to the mass value of the dried samples after lyophilization.

[0059] Alginate-containing GelMA samples were subjected to swelling ratio measurement as described above. Again as described above, the samples were divided into two groups: those with alginate lyase (AL) applied and those without it applied. The samples with AL applied showed liquid absorption performance of approximately 20 times their dry mass due to the opened pores. On the other hand, the samples without alginate lyase applied showed a swelling behavior approximately 10 times compared to their dry weight. These results show that thanks to the AL application, a more porous structure is created in which the cells can spread.

[0060] Mechanical Test

[0061] Mechanical characterization was used for the obtained hydrogels. A tensile test was performed to evaluate the modulus of elasticity of the hydrogel and its structural compatibility with the natural muscle niche. Samples (L0: 14 mm, Cross-sectional area: 3 mm2) placed between the jaws of a mechanical testing device (Shimazdu Autograph AGS-X) were pulled to the breaking point at a speed of 5mm / min using a load cell with a capacity of 50N. A stress-strain curve was created from the data obtained and the modulus of elasticity (Young's modulus) was calculated

[0062] The effect of alginate lyase (AL) application creating pores in the structure on the mechanical strength was examined. As described above, the tensile test was applied and the modulus of elasticity was calculated. The modulus of elasticity of the sample without AL applied was calculated as approximately 115 kPa, whereas the modulus of elasticity of the sample with AL applied was calculated as approximately 22 kPa. Considering that the modulus of elasticity of the natural muscle niche is approximately 12 kPa, it has been ensured that the carrier scaffold produced by AL application has a modulus of elasticity closer to the natural muscle niche, (stiffness)

[0063] Scanning electron microscopy (SEM) Analysis

[0064] The SEM study was conducted to examine the morphology and porosity of the hydrogel before and after alginate lyase application. Samples were washed using PIPES buffer and left overnight at -80eC, then lyophilized and dried. Then, the surfaces were coated with gold and SEM images were taken at Hacettepe University Hunitek Center.

[0065] The microstructure of the carrier scaffold was visualized before and after the AL application. While sufficient porosity was not observed in the pre-AL sample, pores of various sizes between 50-100 microns were observed in the post-AL sample.

[0066] Cytotoxicity Test

[0067] The magnetic substrates produced were subjected to cytotoxicity experiment, taking into account the iron powder used and the chemicals used for surface treatments. Latex material, which is known to have a cytotoxic effect, was used as the negative control group and was treated in the same way as the samples. Standard propagation medium was used as the positive control group. APTES coated and non-coated samples and latex material were sterilized in 70% ethanol solution by continuous agitation for 2 hours and washed 2 times with PBS. Then, the samples were placed in sterile falcon tubes with a capacity of 50 ml and 15 ml of extraction liquid (cell culture medium) was added on top. The tubes were kept under continuous agitation at 60 rpm for 24 hours at 37 °C. At the end of 24 hours, the extraction liquid was passed through a 0.22 micron filter and collected in sterile 50 ml falcon tubes. Passage L929 cell line cells frozen in a liquid nitrogen tank were thawed and transferred to T175 flasks in cell culture medium (10% FBS in high glucose DMEM, 1% penicillin / streptomycin). The cells cultured in the incubator under standard culture conditions (37 °C, 5% CO2) for 24 hours were then separated from the flask by trypsin application and counted. The cells were planted in 12-well cell culture dishes at a concentration of 10,000 cells / cm2. The surface area of the 12-well dishes suitable for cell attachment is 3.8 cm2. When the cells cultured under standard culture conditions in the CO2 incubator for 24 hours reached approximately 70% fullness, the extraction liquids were added onto the cell layers at a rate of 1 ml / well. 24, 48, and 72 hours after the addition of extraction liquids, the spread of the cell layer, the amount of dead cells, and the changes in cell morphology were evaluated by microscopic examination. At the end of 72 hours, the number of viable cells in the wells was determined by the Alamar Blue method.

[0068] At 24, 48, and 72 hours, it was observed that the cells examined under the inverted light microscope did not undergo any morphological change. For cell proliferation analysis, the number of cells in the wells was determined by the Alamar Blue test. It was observed that both the APTES-coated samples and the silicone-only rubber material did not cause a cell inhibition that was statistically significant compared to the positive control (non-cytotoxic) group.

[0069] The scope of protection of the invention is specified in the appended claims and cannot be limited to what is described for illustrative purposes in this detailed description. It is clear that a person skilled in the art can produce similar embodiments in the light of what is explained above, without deviating from the main theme of the invention.

Claims

CLAIMS1 . A method for the production of a tissue scaffold comprising smart materials with shape memory or self-activating characteristics as biomaterial for use in the technical field of tissue engineering, characterized in that it comprises the following process steps;- obtaining guide molds by means of a three-dimensional printer- obtaining magnetic substrate in guide molds obtained from three-dimensional printers wherein a silicone-based rubber material is used as a raw material for obtaining said magnetic substrate, and carbonyl iron powders are added to provide magnetic characteristics, subjecting said carbonyl iron powders and silicone-based rubber material to curing processes in a guide mold- obtaining bioink for printing processes in three-dimensional printers wherein said bioink contains the polymer material obtained by the photopolymerization processes of gelatin methacrylate as a raw material, wherein gelatin methacrylate interacts with alginate during photopolymerization processes,- transferring the obtained bioink with a three-dimensional printer on the silicone- based rubber material with carbonyl iron powder content obtained by applying curing processes in the guide mold, and performing printing processes- performing the application of alginate lyase enzyme to the hydrogel in order to remove the alginate from the obtained hydrogel structure.

2. A method according to claim 1 , characterized in that polylactic acid filaments are used as raw material for obtaining the guide mold.

3. A method according to one of the preceding claims, characterized in that at least one curing agent is added to the silicone-based rubber material-carbonyl iron powder mixture in a ratio of 1 :1 by weight to carry out curing processes for obtaining the magnetic substrate.

4. A method according to one of the preceding claims, characterized in that the curing process is performed for a duration of at least 4 hours.

5. A method according to one of the preceding claims, characterized in that the curing process is carried out at a temperature value in the range of 20 to 24 °C.

6. A method according to one of the preceding claims, characterized in that the magnetic substrate is subjected to surface modification processes by UV-ozone application and APTES binding processes.

7. A method according to claim 6, characterized in that said UV-ozone process is carried out for at least 30 minutes.

8. A method according to claim 6 or claim 7, characterized in that the binding processes are performed with a solution comprising APTES at a value in the range of 1% to 4% by volume.

9. A method according to one of the preceding claims, characterized in that the temperature of the bioink for printing processes in the three-dimensional printer is at a value in the range of 20 to 25 °C.

10. A method according to one of the preceding claims, characterized in that the temperature of the tray for printing processes in the three-dimensional printers is at a value in the range of 17 to 23 °C.11 . A method according to one of the preceding claims, characterized in that the pressure value set for printing processes in the three-dimensional printer is at a value in the range of 0.25 to 0.75 bar.

12. A method according to one of the preceding claims, characterized in that the layer thickness value set for printing processes in the three-dimensional printer is at a value in the range of 0.15 to 0.2 mm.

13. A method according to one of the preceding claims, characterized in that the printing speed value set for printing processes in the three-dimensional printer is at a value in the range of 20 to 40 mm / sec.

14. A method according to one of the preceding claims, characterized in that the needle inner diameter value set for printing processes in the three-dimensional printer is at a value in the range of 0.2 to 0.25 mm.

15. A magnetically responsive tissue scaffold obtained by a method according to one of claims 1-14.

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