PRODUCTION METHODS OF CRYOGELS FROM NATURAL BIOMATERIALS AND THE USE OF THESE CRYOGELS AS SOFT TISSUE SCAFFOLDS OR DRUG DELIVERY SYSTEMS

Abstract

This invention relates to the production of cryogels from various natural biomaterials and the use of these cryogels as drug delivery systems and / or cellular or cell-free soft tissue scaffolds. The aim of the invention is to create a porous tissue scaffold from natural and biocompatible materials such as LBG, XG, and MG, on which cells can adhere and grow, providing an environment for the release of molecules such as drugs, growth factors, hormones, etc., if needed, or to use the produced cryogels as a separate drug delivery system that provides extended release of molecules loaded into it.

Description

25418.96 1 TARIFF PRODUCTION OF CRYOGELS FROM NATURAL BIOMATERIALS THE METHOD AND SOFT TISSUE SCAFFOLDING OF THESE CRYOGELS OR USE AS A DRUG DELIVERY SYSTEM Technical Area This invention develops cryogels derived from natural biomaterials as drug delivery systems. or is related to its use as a cellular or acellular soft tissue scaffold. Previous Tech 10 Tissue engineering provides permanent solutions for millions of people who have suffered tissue damage and loss. It is an emerging field with the potential to provide solutions. Tissue The basic components of engineering are cells, natural or artificial polymeric scaffolds. and are stimulating factors. Materials alone or in conjunction with other tissue engineering techniques. It can be used in combination with its elements. There are 15 in use today. orthopedic solutions, invasive surgical methods, tissue or cell transfers, and These are various implants. However, these methods involve long recovery times and immunological complications. It can have disadvantages such as rejection and toxicity. Artificial polymeric scaffolds have less biocompatibility compared to natural ones. 20 They exhibit acidic degradation products of artificial polymeric scaffolds. This causes toxicity. Therefore, in our invention, natural biomaterials are used. These have been used. In addition, they are used during the formation of tissue scaffolds. The inability to achieve sufficient pore size and porosity in most methods, High degradation rate of materials, low mechanical properties, 25 the fact that the scaffolding structures created do not form the same way when repeated Problems are also encountered. Locust bean gum is obtained from the carob fruit. LBG), xanthan gum produced by the bacterium Xanthomonas campestris 30 (Xanthan gum - XG) and is produced by the gum tree called Pistacia lentiscus. 25418.96 2 Mastic gum (MG) is commonly used in the food and pharmaceutical industries. It is used in applications. When the literature is examined, hydrogels prepared with an LBG-XG structure or 5 Cryogels are not used in any tissue engineering applications. It has been observed that in published articles, the LBG-XG mixture is only a drug carrier. in the creation of edible film, tablet or microparticle structures It has been observed to be used [1, 2, 3]. When the relevant patent studies were researched, patent number CA1093466 (A) 10 The document describes various LBG-type devices developed specifically for transporting ophthalmic drugs. XG compositions are mentioned. Patent number EP0818991 (A1) The document describes insufflators that perform controlled release of dust. It is mentioned that LBG and XG are used in their formulations. PT78813 In patent document number (A), pharmaceutical 15 containing LBG and XG. The preparation of the compositions is discussed. When articles and patents in the literature are examined, it is seen that the LBG-XG-MG mixture... in the field of any drug delivery system or tissue engineering It was observed that there was no scaffolding structure available for use. 20 Brief Description of the Invention The aim of the invention is to create a biodegradable ecosystem from natural and biocompatible materials such as LBG, XG, and MG. the creation of a porous tissue scaffold and the attachment of cells to these scaffolds to enable it to grow, medication, growth factors, hormones, etc. if needed. 25 It provides a medium for the release of molecules. Another aim of the invention is to utilize natural and biocompatible materials such as LBG, XG, and MG. The materials prepared with this method are used as cell-free tissue support. 25418.96 3 Another aim of the invention is to prepare biomaterials with LBG, XG, and MG, as a drug delivery system that provides an extended release of the molecule loaded into it It is also used. Detailed Description of the Invention The purpose of this invention was achieved through "Natural Biomaterials". Cryogel Production Method and Soft Tissue Scaffolds Made from These Cryogels or its use as a drug delivery system” is shown in the attached figures; this from the shapes: 10 Figure 1 – 2% (grams / milliliter) LBG, XG, LBG-XG, LBG-XG-MG This is a graph showing the percentage weight loss of cryogels after 60 days. Figure 2 – 3% (grams / milliliter) LBG, XG, LBG-XG, LBG-XG-MG This is a graph showing the percentage weight loss of cryogels after 60 days. Figure 3 – 60-day weight percentage of cryogels containing only LBG and XG. It is a representation of the loss graph. Figure 4 – Phosphate of cryogels containing 2% (grams / milliliter) LBG, XG, and MG. Graph of pH change in buffered saline solution over 60 days It is a representation. Figure 5 – Phosphate 20 of cryogels containing 3% (grams / milliliter) LBG, XG and MG. Graph of pH change in buffered saline solution over 60 days It is a representation. Figure 6 – Phosphate-buffered saline of cryogels containing only LBG or XG. This is a graph showing the pH change of the solution over 60 days. Figure 7 – 25% LBG-XG prepared in 1:1 and 2:1 ratios by mass. with examples and by adding 5 mg / mL and 10 mg / mL MG to them This is a visualization of SEM micrographs of the generated samples. Figure 8 – 3% LBG-XG prepared in 1:1 and 2:1 ratios by mass. with examples and by adding 5 mg / mL and 10 mg / mL MG to them This is a visualization of SEM micrographs of the generated samples. 30 25418.96 4 Figure 9 – Pore size distribution of samples 1-12 in Table 1. It is a representation of the graph. Figure 10 – Graphical representation of FTIR results obtained for LBG, XG, and MG. Figure 11 – Samples containing 2% and 1:1 ratio of LBG-XG (MG ratio) (0%, 5% and 10% respectively) FTIR spectra. 5 Figure 12 – Samples containing 2% LBG-XG at a 2:1 ratio (MG ratio) (0%, 5% and 10% respectively) FTIR spectra. Figure 13 – Samples containing 2% LBG-XG at a 2:1 ratio (MG ratio) (0%, 5% and 10% respectively) FTIR spectra. Figure 14 – Samples containing 3% LBG-XG at a 2:1 ratio (MG ratio 10 (0%, 5% and 10% respectively) FTIR spectra. Figure 15 – Graphical representation of the tensile analysis (Young's Modulus) results of cryogels. It is a representation. Figure 16 – Graphical representation of the results of cryogel shear analysis (Young's Modulus). It is a representation. 15 Figure 17 – Cumulative concentration of Cartogenin released from cryogels It is a graphical representation of the change over time. Figure 18 – Percentage cumulative release of Cartogenin from cryogels It is a graphical representation of the change over time. Figure 19 – Graphical representation of the viability test of cells seeded onto tissue scaffolds. 20 It is a representation. Figure 20 – Chemical structure of LBG (Locust bean gum) It is a representation. Figure 21 – Chemical structure of XG (xanthan gum - XG)) It is a representation. 25 The invention includes natural and biocompatible materials such as LBG, XG, and MG. the creation of a porous tissue scaffold and the attachment of cells to this scaffold The aim is to provide an environment for it to grow. Also, tissue scaffold 30 a drug carrier system that provides an extended release of the molecule loaded into it 25418.96 It is also intended to behave as a system. The invention made in this direction is a material. engineering, biomaterials, tissue engineering, bioengineering and It is related to biotechnology fields. The invention is made entirely from natural materials. This 5 When materials are combined, macro-porous hydrogel (cryogel) scaffolds are formed. Hydrogels are three-dimensional in structure and have a high water retention capacity. These are tissue scaffolds. They can be film, microparticle, nanoparticle, coating, or tissue. They can be used in scaffolding forms. The porous structure of the product subject to the invention. It is suitable for loading micro- or macromolecular drug structures. Same 10 It can also provide prolonged drug release depending on the diffusion rate over time. LBG involves grinding carob seeds to form a fine-grained flour. It is produced by subsequent precipitation and purification. Biocompatible, It is a biodegradable, excretable, and edible natural polymer. American Food Codex 15 and GRAS, meaning generally accepted as safe by the Food and Drug Administration (FDA). It is classified under the category of LBG, β-mannanase, β-mannisodase and α- It can be broken down by galactosidase enzymes, and its degradation products are eliminated from the body. It can be easily discarded. Türkiye has a 6% share in carob fruit production. It ranks 6th in the world. XG is Xanthomonas 20, a gram-negative pathogenic bacterium. It is a polyanionic exopolysaccharide produced by the plant campestris, with a wide range of uses. It is a stable material within the temperature range and is resistant to shear force. Biocompatible, repeatable, non-toxic, highly water-soluble. It is a substance. The U.S. Food and Drug Administration quantifies it as a food additive. It has been approved without limit. In addition, from the petroleum industry to pharmaceuticals 25 It is used in many areas, from industry to the production of cosmetic products. In our country, it grows in the western Aegean region (between Çeşme and Karaburun) and is called Pistacia lentiscus. Mastic gum (MG), produced by the mastic tree, which is called mastic, has healing properties. It is known for its properties and is especially used in gastrointestinal disorders. It is a material. Recent studies have shown that mastic gum is 30 its resin has antimicrobial, antifungal, antioxidant, anticancer properties and 25418.96 6 It is known to have anti-inflammatory properties. Considering all these features... Macroporous hydrogels composed of LBG, XG, and MG are biodegradable, bio- Soft tissues are preferred because they are compatible, inexpensive, renewable, and repeatable. It is highly suitable for use in engineering and as a drug delivery system. The invention involves the preparation of cryogels from LBG-XG natural biomaterials. It is a production method developed for this purpose, - LBG and XG biomaterials should have a ratio of 1:1-3:1 (preferably 1:1, 2:1) to each other. and weighing in a ratio of 3:1 (grams / grams) and in stages. Mixed in pure water at a concentration of 1-4% (grams / milliliter) 10 Dissolving in such a way as to form a solution within the concentration range, - The solution temperature is maintained between 90-100°C using a magnetic stirrer. stirring continuously for 30 minutes with the help of... - After the solutions are transferred to glass petri dishes, they are stored at -20°C. It should be placed in the refrigerator and left for 24 hours, 15 - In the final stage, it is completely dried by applying the lyophilization process. - Cryogels, which are the final product after drying by lyophilization The steps involved in obtaining it are being carried out. Within the scope of the invention, cryogels made from LBG-XG-MG natural biomaterials are 20 It is a production method developed for the purpose of preparation, - Firstly, magnetic resonance imaging (MRI) in the concentration range of 0.1-50 mg / mL. mixing with a mixer,  Following this step, LBG and XG biomaterials are mixed in a 1:1 ratio. 25 in a ratio of 3:1 (preferably 1:1, 2:1 and 3:1) (grams / gram) weighed and 1-4% (gram / milliliter) (Preferably 2% and 3% (gram / milliliter)) to form a solution within the concentration range Adding to MG solution at these concentrations, - Pure water by maintaining the solution temperature between 90-100 °C. 30 to be resolved gradually 25418.96 7 - After the solutions were continuously stirred for 30 minutes, they were transferred to glass petri dishes. pouring into containers - The mixture should be quickly placed in a refrigerator at -20°C for 24 hours. waiting, - Lyophilization of tissue scaffolds for solvent removal 5 the process is applied - Cryogels, which are the final product after drying by lyophilization The steps involved in obtaining it are being carried out. These cryogels, obtained within the scope of this invention, can be used on cellular or acellular soft tissue. They can be used as scaffolds for tissue engineering. In addition, these cryogels, inside are substances such as drug molecules, small molecules, growth factors, etc. By being confined, it can be used as a drug delivery system for transporting drugs. Experimental Studies The experimental studies conducted as part of the development of the invention are described as follows: can be done:  Topographic and mechanical analysis of cryogels formed from LBG-XG and LBG-XG-MG 20 and their chemical characterizations have been carried out.  Degradation of cryogels formed from LBG-XG and LBG-XG-MG Analyses have been conducted.  Cryogels formed from LBG-XG and LBG-XG-MG are small molecule... A release study was conducted. 25  Cells placed on cryogels formed from LBG-XG and LBG-XG-MG Viability studies were conducted by planting the seeds. 25418.96 8 1. Preparation of Cryogels from LBG-XG When cryogels are prepared separately from LBG and XG biomaterials, they are weaker. It has been observed that tissue scaffolds are obtained, therefore LBG and XG are used in 1:1, 2:1 and 3:1 ratios. (grams / gram) ratios in the concentration range of 1-4% (grams / milliliter). It was mixed and gradually dissolved in pure water. The temperature of the solution was 90-5°C. Maintaining the temperature around 100°C and stirring with a magnetic stirrer for 30 minutes. The solutions were continuously stirred. After being transferred to glass petri dishes, they were stored at -20°C. It was refrigerated and left for 24 hours, and in the final stage, the lyophilization process was carried out. It was applied and completely dried. The dried tissue scaffolds were placed in 24-well trays. cut into plates to fit and for the analyses to be done 10 It has been prepared. Initially, LBG and XG are mixed in a ratio of 1:1-2:1-3:1 (grams / gram), or 1-4% (grams / milliliter). They were combined within a concentration range of two percent and three percent. Polymers with these concentrations form tissue scaffolds in terms of density. It is more suitable for production compared to polymers with other concentrations. It has been found that solutions with a four percent concentration have higher densities. It could not be distributed homogeneously in glass petri dishes, therefore the tissue scaffold Their use was deemed unsuitable. As a result of these observations, the total 20 on samples with polymer concentrations of two percent and three percent Experiments have continued with increased intensity. LBG polymer is more effective compared to XG. LBG was used in the experiments because its durability was observed in previous cryogel trials. The ratio was continued by increasing it by mass. At this stage, LBG-XG composites Tissue scaffold experiments were conducted by combining the materials in mass ratios of 2:1 and 3:1. 2. Preparation of Cryogels from LBG-XG-MG Cryogel experiments were conducted by combining LBG, XG, and MG. First, MG 0.1- To be mixed with a magnetic stirrer within a concentration range of 50 mg / mL and It was dissolved in pure water while maintaining the temperature between 90-100°C. This 30 Following this step, LBG and XG are mixed in a 1:1-2:1 (gram / gram) ratio by mass, at 2% and 3%. 25418.96 9 The solutions were gradually dissolved in (grams / milliliter) concentrations. After being stirred continuously for a few minutes, it was poured into glass petri dishes and It was quickly placed in a refrigerator at -20°C and left there for 24 hours. Lyophilization process on tissue scaffolds for the purpose of solvent removal. This process was applied. LBG and XG were prepared in the same way at a 2:1 mass ratio. 5 were obtained. 2% and 3% (grams / milliliter) solutions containing 1:1 and 2:1 LBG and XG by mass. 5, 10, 25 and 50 mg / mL MG were added to the solutions at the specified concentrations. Two percent. Composites containing LBG-XG (1:1) (gram / gram) with 25 mg / mL and 50 mg / mL MG They were observed to be very concentrated. For this reason, studies were conducted at 5 mg / mL and 10 The process continued with polymers containing mg / mL MG. It included 10 components in a 2:1 ratio by mass. LBG-XG and 2% and 3% solutions containing 5 mg / mL or 10 mg / mL MG. Tissue scaffolds have been successfully created from polymers (grams / milliliter). 3. Degradation Analysis of Cryogels 15 In the first stage, degradation analysis was performed on 16 samples. These samples were 2% and Having a total polymer concentration of 3% (grams / milliliter), with 1:1 and 2:1 ratios by mass. Polymers containing LBG-XG and having 5 mg / mL and 10 mg / mL MG in their structure. These are cryogels containing 2% and 3% (grams / milliliter) LBG or XG alone. (Table 1). Cryogels prepared for degradation analysis were weighed and mixed with 0.09% solution (20). It was placed in an isotonic salt solution containing sodium azide and shaken at 37°C. The samples were kept in a water bath. The samples were removed from the water bath on the 7th, 15th, and 30th days. They were collected, the pH of the solutions they were in was measured, and they were stored at -80°C. They were cryopreserved and lyophilized. Percentage degradation graphs show the samples completely. The percentage weight loss between the initial and final weights after drying is 25. It was created by finding it. 25418.96 Table 1. Samples selected for degradation analyses. Sample Number Sample Content 1 2% (gram / milliliter) - 1:1 (gram / gram) LBG-XG 2 2% (gram / milliliter) - 1:1 (gram / gram) LBG-XG + 5 mg / mL MG 3 2% (gram / milliliter) - 1:1 (gram / gram) LBG-XG + 10 mg / mL MG 4 2% (gram / milliliter) - 2:1 (gram / gram) LBG-XG 2% (gram / milliliter) - 2:1 (gram / gram) LBG-XG + 5 mg / mL MG 6 2% (gram / milliliter) - 2:1 (gram / gram) LBG-XG + 10 mg / mL MG 7 3% (gram / milliliter) - 1:1 (gram / gram) LBG-XG 8 3% (gram / milliliter) - 1:1 (gram / gram) LBG-XG + 5 mg / mL MG 9 3% (gram / milliliter) - 1:1 (gram / gram) LBG-XG + 10 mg / mL MG 3% (gram / milliliter) - 2:1 (gram / gram) LBG-XG 11 3% (gram / milliliter) - 2:1 (gram / gram) LBG-XG + 5 mg / mL MG 12 3% (gram / milliliter) - 2:1 (gram / gram) LBG-XG + 10 mg / mL MG 13% 2 (grams / milliliter) LBG 14 3% (grams / milliliter) LBG 2% (grams / milliliter) XG 16% 3 (grams / milliliter) XG 25418.96 11 Looking at the percentage weight loss graphs of cryogels, it is 2% (grams / milliliter). Weight loss in samples containing LBG-XG at the end of 60 days was 24%-37%. Weight loss of samples containing 3% (grams / milliliter) LBG-XG while within the range It ranges from 23% to 32% (Figures 1 and 2). The highest percentage weight loss is 2%. (grams / milliliter) - 1:1 (grams / grams) and 2% (grams / milliliter) - 2:1 (grams / grams) LBG-5 While seen in XG samples (37%), the lowest weight loss was 3% (grams / milliliter) - 1:1. (gram / gram) LBG-XG + 10 mg / mL MG sample showed a 20% increase. Figure 1 and When compared in Figure 2, samples containing 2% (grams / milliliter) LBG-XG contained 3% According to samples containing (grams / milliliter) LBG-XG, more was observed at the end of day 60. It was determined that they experienced weight loss. 10 containing the same concentration of LBG-XG In cryogel samples containing MG, the percentage weight loss was higher compared to those without MG. It has been observed that it is less. Samples containing different concentrations of MG. No significant difference is observed in the percentage weight loss values ​​between them. Degradation analyses of cryogels containing only XG or only LBG 15 Upon examination, it was found that these cryogels completely degraded on the 15th and 30th days, respectively. It has been observed (Figure 3). According to this result, the decay rate of XG is higher than that of LBG. There are too many. During degradation analysis, the percentage weight loss is determined, and at the same time, 20 The pH values ​​of the solutions containing the cryogels have also been determined. A significant decrease in pH at the site where the tissue scaffold is implanted. This is undesirable because it causes necrosis in cells and surrounding tissues. This is a situation. Therefore, the products that arise during the degradation of cryogels The changes it causes in the pH of the environment should be examined. The first 12 25 in Table 1 When the samples were compared, the sample with the lowest pH after 60 days was 3%. (grams / milliliter) - 1:1 (grams / grams) LBG-XG + 10 mg / mL MG (pH: 7.06), maximum The sample with pH is 2% (grams / milliliter) - 2:1 (grams / gram) LBG-XG (pH: 7.27) (Figures 4 and 5). 12 samples compared by percentage weight loss and pH analysis. The pH of the samples that experienced the greatest percentage weight loss was 30 from the initial pH. It was determined that it was closest to 7.4. It ranked lowest in percentage weight loss. 25418.96 12 One example showing a loss is 3% (grams / milliliter) - 1:1 (grams / gram) LBG-XG. + It was determined that 10 mg / mL MG had the lowest pH (7.06). The reason is the acidic chemicals in the structure that are released when MG decomposes. and that XG contains glucuronic acid and pyruvic acid in its structure. It is generally believed that the pH of samples containing MG is 5 times lower than that of other samples. It was determined to be lower compared, but the pH determined at the end of the 60th day... None of the pH values ​​dropped below 7. Therefore, these pH values... The decline was not considered a significant drop. The pH change of cryogels containing only LBG or XG at the end of 60 days is 10. When examined (Figure 6), the pH of cryogels composed of XG is lower than that of cryogels composed of LBG. It was found that the pH of cryogels was lower than that of XG. glucuronic acid and pyruvic acid, which are present in its structure and are released as a result of its degradation. It is caused by acid. 4. Scanning Electron Microscopy (SEM) Analysis Scanning electron microscopy to observe the surface structure of cryogels. The analysis was carried out. Fully dried samples were thinned to a thickness of 10 nm for analysis. After being coated with gold, Carl 20 was subjected to high vacuum and 10 kV high voltage. The image was magnified 100-500 times using a Zeiss EVO-40 instrument. SEM analysis. Observation of the surface structures of LBG, XG and LBG-XG-MG samples. It has made it possible. SEM micrographs show cryogels composed of LBG-XG and LBG-XG-MG joined together at 25° They show their bonded porous structures. Containing 2% and 3% LBG-XG by mass. 5 mg / mL and 10 mg / mL MG were added to cryogels and SEM analyses were performed. The topographic features and porous structures of the samples were observed in micrographs. It was determined that the amount of MG did not affect the porous structure (Figures 7 and 8). 25418.96 13 Degradation analysis revealed that samples containing only LBG and only XG were different from the others. Because they decompose faster compared to the samples, the sample size is shown in Table 1. limited to the first twelve examples and characterization from this section onwards It was decided to continue the studies with these examples. The materials Pore ​​sizes were analyzed using SEM micrographs, and pore size 5 Size distributions were determined using a pore analysis program written in the C-Sharp programming language. It has been determined that 50 randomly selected cells are used for each sample in the program. The dimensions were measured. Measurements were taken at 0-100 µm, 100-200 µm, 200-400 µm, and ≥400 µm. The results were categorized according to the number of pores corresponding to their intervals. The percentage distribution is given in Figure 9. 10 According to Figure 9, the pore sizes of the cryogels are 400 µm, except for sample 3. It is below. Another observation is that the majority are in the 100-200 µm range (58-66%). 3% of samples with pores mostly in the 100-400 µm range (82-96%). The samples should contain 2:1 LBG:XG (samples 10-12). These samples have a pore size of 15. Its dimensions are more homogeneous compared to the others. At the other extreme is 2% 1:1 LBG:XG. There is a sample set (Samples 1-3) containing samples in the range of 100-400 µm. The porosity ratio decreases to 50-62%. SEM analysis results show a 2:1 ratio. all samples in the ratio of pore size to samples with a 1:1 ratio It has been observed that the concentration of LBG has increased compared to pore 20. It has been determined that it increases its size. As a result of this analysis, the produced Cryogels generally have pore sizes suitable for soft tissue formation. It has been observed that he possesses it. 5. FT-IR Analysis FTIR analyses were performed on the selected samples (the first 12 samples given in Table 1). Figure 10 shows the individual FTIR results for the three materials used (LBG, XG, and MG). It is shown. 25418.96 14 The following four examples are given in order (Figures 11-14). The results of the study are presented. Each graph shows the percentage of polymer and the LBG-XG ratio. They are the same, but the three different curves show samples containing increasing amounts (0%, 5%, and 10%) of MG. It shows. Some bands that changed significantly with MG doping are shown with vertical lines in Figure 11. It is marked with the symbol shown. These bands are similarly represented in the other 3 sample sets as well. It shows a tendency to change. CH2 asymmetric voltage in the 2900-2940 cm-1 band. and the CH3 symmetric voltage of around 2870 cm-1 is present in all samples. The addition of MG makes it more pronounced in each sample set and 2900-2940 10 The cm-1 band is becoming significantly more dominant. Other marked section This indicates carbonyl (C=O) stresses of 1731 cm-1 and 1614 cm-1, and each of the four In the sample group, the 1731 cm-1 peak also increased proportionally with the addition of MG. This is observed. In contrast, it is more dominant in pure LBG and XG samples. The apparent peak of 1614 cm-1 became relatively 15 with the start of MG doping. It is decreasing. The CC and CO voltage of the pyranose ring is between 1200 cm⁻¹ and 970 cm⁻¹. and the COC tension of glycosidic bonds is involved. These bands are present in all samples. It is located there. 6. Mechanical Tests The results of the cryogel tensile test analysis are presented in Figure 15. As can be seen... Increasing the MG ratio reduces the tensile modulus values. Total polymer concentrations of 2% (grams / milliliter) 1-1 and 3% (grams / milliliter) 1-1 LBG-XG In the samples, it was observed that as the amount of MG increased, the Young's Modulus values ​​decreased, 25 In 2% (grams / milliliter) 2:1 mixed LBG-XG samples, MG... It was found that there was no significant effect on Young's Modulus values. The highest The Young's modulus value was observed at 3% (grams / milliliter) in a 2:1 sample. All cryogels have a Young's modulus (for tensile testing) between 2 and 10 MPa. 25418.96 For the shear modulus analysis of cryogels, the pulling speed of the mechanical device was 0.08. It is set to N / min. The shear modulus determines the hardness of the materials. used for, applied parallel to the surface of materials, uniaxial transverse It is a value that indicates the strength to withstand force. The shear modulus of cryogels. When the values ​​are compared, the highest value is 3% (grams / milliliter) - 2:1 (grams / gram) 5 LBG-XG was observed in cryogels consisting of +5 mg / mL MG (Figure 16). When the shear modulus values ​​of all cryogels were examined, they were 2%-1:1 and 3%-1:1. When the amount of MG was added to the samples as 5 mg, the value increased, but not more. The cutoff modulus value decreased when MG was added. Examples: 2-2:1 and 3-2:1. When evaluated, it was found that increasing the amount of MG reduced the shear modulus values. It was determined that cryogel samples containing 2% and 3% polymer by mass were used. When compared, the shear modulus values ​​of the 3% samples were higher than those of the 2% samples. It has been determined that it is generally higher than expected. 7. Encapsulation and release of the small molecule (cartogenin) within a cryogel. characterization Cryogels consisting of LBG-XG and LBG-XG-MG at 2%-3% (grams / milliliter) combined at their concentrations and in ratios of 1:1-2:1 (gram / gram), for 30 minutes. The temperature is maintained at 90-100 ℃ throughout, and 20 grams of pure water is mixed using a magnetic stirrer. The solutions were dissolved. After transferring the solutions to glass petri dishes, they were kept at -20 ℃ for 24 hours. The samples were kept in the refrigerator. In the final stage, the samples were dried using a lyophilizer. The cryogels formed were then mixed with 1 mL of release medium (PBS) at 150 μM while dry. Cartogenin small molecule that induces cartilage formation. They were added and left to cryogelize overnight. The next day, they were mixed with PBS for 25 minutes. The expanded cryogels were placed in 1 mL of release medium (PBS). 1, 3, 7, 14 and 21. Absorbance measurements were taken from samples collected from the release environment over several days. The amount of released cartogenin is cumulative using the value and calibration curve. The percentages were calculated cumulatively (Figures 17 and 18). Inside the cryogels The PBS solution in which it was placed was changed after each measurement. 30 25418.96 16 Figures 17 and 18 show that all samples exhibited extended release of cartogenin. It has been observed that it is able to do so. In all samples, during the first 3 days, and in other time intervals... A faster release was observed compared to the previous one. This is due to absorption onto the cryogel surface. This is due to the easy passage of the cartogen into the release medium. After the third day, Kartogenin, which was trapped inside the cryogel, began to be released. 5 The release rate has decreased. The release rate remained low until day 21. This shows that cryogels maintain their physical integrity over time. After 21 days of release, the cryogels showed a concentration of 2% containing 5 mg / mL MG. (grams / milliliter) 2:1 (grams / grams), 3% (grams / milliliter) 1:1 (grams / grams) and 3% (grams / milliliter) 2:1 (grams / grams) samples contain more than 10 mg compared to those that do not contain MG. It released cartogenin (6%, 6% and 20% respectively). 2% containing 10 mg / mL MG. (grams / milliliter)-2:1(grams / grams) Cartogenin release in LBG-XG sample The amount did not change compared to the MG-free version; 3% (grams / milliliter) The amount of release in a 1:1 (gram / gram) LBG-XG sample compared to one without MG. more (11%) and 3% (grams / milliliter)-2:1 (grams / grams) in the 15 LBG-XG sample. the amount of release is less (16%) compared to the MG-free version. It was observed that when comparing samples containing 5 mg / mL MG and 10 mg / mL MG, 2% (grams / milliliter)-2:1 (grams / gram) LBG-XG and 3% (grams / milliliter)- In 2:1 (gram / gram) LBG-XG samples, samples containing 5 mg / mL MG are more abundant. emitted (5% and 36% respectively), 3% (grams / milliliter)-1:1 (grams / grams) LBG-XG 20 In the example, it was found that samples containing 10 mg / mL MG released 5% more. It has been done. After 21 days of incubation, 3% (grams / milliliter)-2:1 (grams / grams) 5 mg / mL MG Except for this sample, all other samples had approximately 23-46% cartogenin release. It has been observed that it does the following: 3% (grams / milliliter)-2:1 (grams / gram) 5 mg / mL MG sample. By the end of day 7, 40% of the Cartogenin had been used, and by the end of day 21, approximately 60%. According to these results, all samples showed no release of Cartogenin for 21 days. It has been able to continue. Cryogels are carriers that can release the virus for the desired duration. It has been determined that these are systems. The cryogels created hold small molecules, 30 It can release growth factors and various drugs. 25418.96 17 8. MTS Cell Viability Analysis The interaction of cryogels with cells was investigated using MTS analysis. Each cryogel... 60,000 cells were inoculated onto the sample, and cell viability tests were performed on days 1, 14, and 21. According to the analysis results, the cells adhered to all cryogels and had 5 cells on them. It was able to grow (Figure 19). Cryogels did not show a toxic effect on cells. It has been determined that cryogels can be used as tissue scaffolds for soft tissue. It has been determined that it has the potential to be used in its formation. 25418.96 18 REFERENCES [1]. Kurt, Abdullah, Omer Said Toker, and Fatih Tornuk. 2017. “Effect of Xanthan and Locust Bean Gum Synergistic Interaction on Characteristics of Biodegradable Edible Film.” International Journal of Biological 5 Macromolecules 102: 1035–44. http: / / dx.doi.org / 10.1016 / j.ijbiomac.2017.04.081. [2]. Sharma, Navneet, Rohan D. Deshpande, Deepak Sharma, and Rakesh Kumar Sharma. 2016. “Development of Locust Bean Gum and Xanthan 10 Gum Based Biodegradable Microparticles of Celecoxib Using a Central Composite Design and Its Evaluation.” Industrial Crops and Products 82: 161–70. http: / / dx.doi.org / 10.1016 / j.indcrop.2015.11.046. [3]. Venkataraju, M., D. Gowda, K Rajesh, and H. Shivakumar. 2008. “Xanthan 15 and Locust Bean Gum (from Ceratonia Siliqua) Matrix Tablets for Oral Controlled Delivery of Metoprolol Tartrate.” Current Drug Therapy 3(1): 70–77.

Claims

19 REQUESTS 1. LBG (Locust bean gum) - XG (Xanthan gum) -MG (Mast c Gum) natural A production method developed for the preparation of cryogels from biomaterials. 5 method, -First, mix the MG at a concentration of 5 mg / mL using a magnetic stirrer. -Following this step, I will mix LBG and XG in a 2:1 (gram / gram) ratio by mass, 2%. Adding MG solution at a concentration of 3 (grams / ml) -By maintaining the solution temperature between 90-100 °C, 10 gradual resolution -The solutions were continuously stirred for 30 minutes before being poured into glass petri dishes. shedding, -The mixture should be quickly placed in a refrigerator at -20°C and left for 24 hours. -Lyofl zasyon process on tissue scaffolds for solvent removal 15 implementation, -Obtaining final tissue scaffolds after drying with lyophilization. Producing soft tissue scaffolds with natural biomaterials, including processing steps. method .

2. Cellular or cell-free soft tissue obtained by the GBBR method in Claim 1. Cryogels used for scaffold production.

3. Obtained by the GBBR method in Claim 1, and containing drug molecules, small molecules, 25 Used as a drug delivery system by trapping growth factors and similar substances. kr yojeller.

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