PRF Biohybrids Modified with Gelatin-Based Hydrogel Microspheres

Abstract

The invention relates to a biohybrid and its preparation method, which utilizes gelatin-based hydrogel structures in the form of microspheres for improving the functional, mechanical, and biological properties of blood concentrates such as platelet-rich fibrin (PRF).

Description

PRF MODIFIED WITH GELATIN-BASED HYDROGEL MICROSPHERES BIOHYBRIDS Technical Field to Which the Invention Relates The invention has implications for biomedical engineering, biomaterial technologies, tissue engineering, and It relates to the field of regenerative medicine. More specifically, the invention involves platelet-rich fibrin. Functional, mechanical and biological properties of blood concentrates such as (PRF) The use of gelatin-based microsphere hydrogel structures for improvement purposes, The subject is the preparation of hydrogel structures in a way that will provide controlled release and a biohybrid tissue loaded with antibacterial phages to protect against infections It relates to the creation of a repair system. The invention also involves these biohybrid structures. preparation, combination with PRF and especially open wounds, tissue damage and for use in clinical settings where there is a risk of infection. It describes the application methods. State of the Art Tissue damage and open wounds; trauma, surgical operations, burns, due to many reasons such as infections, chronic diseases and circulatory disorders This can occur especially in areas involving large surface areas or deep tissues. Wounds cannot be fully repaired by only superficial healing processes, and most This situation requires advanced biomedical interventions. These types of wounds... The main goal in treatment is to support the regeneration of damaged tissue. The goal is to reduce the risk of infection and speed up the healing process. Therefore, the last In recent years, in the fields of regenerative medicine, tissue engineering and biomaterial technologies Important studies are being conducted. Traditional wound treatment methods include antiseptics, antibiotics, and surgery. Interventions and various dressing materials are used. However, These methods are often sufficient, especially in wounds involving extensive tissue loss. It is known that this is not the case. Also, in chronic wounds or in people with weakened immune systems... Patients have a very high risk of developing infection. This affects wound healing. slows down and in some cases can progress to serious systemic infections. 1 This can lead to complications. Therefore, in modern medical practice... The use of biologically active materials is becoming increasingly important. In recent years, an important biological agent has been used to promote wound healing. One of the materials is blood concentrates. These materials are derived from the patient's own blood. obtained and containing various growth factors and biological signaling molecules These are structures. Among blood concentrates, "platelet-rich fibrin" (PRF) is particularly prominent. Second-generation blood concentrates, also known as blood concentrates, are widely used in clinical practice. PRF is used by centrifuging blood taken from the patient in a controlled manner. It is a fibrin-based biomaterial obtained. Within this structure, platelets, leukocytes, and Various growth factors are held within a natural fibrin hydrogel network. One of the most important features of PRF is that the growth factors it contains affect the tissue. It has effects that support healing. Released by platelets “Platelet-Derived Growth Factor” (PDGF), “Transforming Growth Factor beta” (TGF- β), “Vascular Endothelial Growth Factor (VEGF), “Insulin-like Growth Factor (IGF) and similar growth factors and other bioactive molecules promote cell proliferation, It plays an important role in angiogenesis and tissue regeneration processes. Therefore, PRF is particularly relevant for dentistry, maxillofacial surgery, orthopedics, and plastic surgery. to support bone and soft tissue healing in areas such as these It is used. The PRF structure basically consists of three main components. The first of these is the fibrin hydrogel structure. Fibrin is a natural biopolymer that helps with wound healing. a suitable microenvironment for cell attachment and migration during healing This provides the fibrin hydrogel structure for the controlled release of growth factors. It contributes to its release in this way. The second important component is platelets, It contains granules that store various growth factors, and after activation these It synthesizes and releases molecules into the environment, triggering healing processes. The third component. These are leukocytes. Leukocytes play a role in immune system functions and wound healing. It plays important roles in infection control and tissue regeneration in the region. It undertakes this. Despite the advantages that PRF provides in clinical applications, this material... There are some limitations. PRF is usually obtained in the form of a solid fibrin mass. This form is suitable for some clinical applications, but it is used in different tissue types. or limitations in terms of use in complex wound geometries It can generate. Furthermore, the release kinetics of the biological components within the PRF are precise. 2 It cannot be controlled in this way. Growth factors and other bioactive molecules are mostly... Time is being released relatively quickly, and this situation affects long-term tissue. It may not provide the desired effect for regeneration. Another important aspect of PRF is... Its limitation is related to preserving the viability of biological components. PRF The functional characteristics of platelets and leukocytes within it change over time. This can decrease. This situation is particularly important in terms of the continuity of biological activity. This constitutes a limitation. Furthermore, there is a risk of infection during PRF applications. It is not completely eradicated. Open wounds harbor pathogenic microorganisms. It can create suitable environments for its development. Therefore, biological wounds the treatment materials will also provide antibacterial protection Design has become an important area of ​​research. Another approach developed to support wound healing is... This involves the use of biomaterial-based carrier systems. These systems are generally They are prepared using biopolymers or synthetic polymers and are bioactive. It aims to ensure the controlled release of molecules. Biopolymers include polypeptides such as collagen and its denatured form gelatin, And naturally occurring polysaccharides such as chitosan, alginate, and hyaluronic acid are common. These materials are used because of their biocompatibility and suitability for use in biological environments. They are preferred in biomedical applications because of their biodegradability. Hydrogel structures, in particular, are important in the fields of wound healing and tissue engineering. This allows them to be included among biomaterials. Hydrogels have high water retention. They are three-dimensional polymer network structures with the capacity to interact with biological tissues. They can exhibit similar mechanical properties and provide a suitable microenvironment for the cells. They are formed. Hydrogels also facilitate the transport of various biological molecules and It also offers suitable platforms for controlled release. In recent years, hydrogel systems have been developed in the form of nano or micro-scale particles. Many studies are being conducted on their design. In nano and microsphere form. Hydrogel structures, thanks to their high surface area, allow for the loading of bioactive molecules and This can provide advantages in terms of release. Such particles also offer different It can show a more homogeneous distribution in biological environments and has various applications. They can adapt to various methods. Gelatin-based hydrogel systems are used in biomedical applications. Gelatin is one of the most researched biopolymer platforms in applications. 3 It is a natural polymer obtained by the hydrolysis of collagen and is biocompatible, biodegradable and They possess properties that promote cell adhesion. Gelatin-based hydrogels offer various... Different mechanical and physical properties are obtained by using cross-linking methods. It can be produced in such a way as to include growth factors within the gelatin matrix. Drugs or other biological agents can be transported. However, hydrogels can deliver them. There are some challenges in using these systems in biomedical applications. The mechanical strength of hydrogels is often limited, and rapid growth in biological environments is problematic. It can be broken down in this way. Additionally, some hydrogel systems are bioactive. The release kinetics of the molecules cannot be controlled sufficiently. Therefore, hydrogel optimizing the structural properties of systems and with different biological materials Studies on combining these methods are ongoing. In wound treatment, infection control is a crucial aspect. Traditionally... Antibiotics are commonly used to treat bacterial infections. However, in recent years, the number of bacteria that have developed resistance to antibiotics has increased. A significant increase has been observed. Antibiotic resistance is a major concern for global health systems. This poses a threat. This situation necessitates alternative antibacterial approaches. This has made its development necessary. One of these alternative approaches is "Bacteriophage". This method is known as "bacteriophage therapy." Bacteriophages (phages) infect bacteria and These are viruses that replicate. These viruses are able to specifically recognize target bacteria and They can destroy them by breaking them down. Phages are nature's antibacterial agents. The reason for their existence is that they use pathogenic bacteria as hosts and multiply – this Through a natural process, lysis eliminates the pathogenic bacteria that they use as hosts. They do this, thus controlling the population of pathogenic bacteria in nature. Phages were a key element of the 20th century. They have been defined from the outset and are particularly used in clinical practice in some countries. It is used. In recent years, with the increase in antibiotic resistance, "Phage Therapy" has become popular. Phages have once again become an important research topic in clinical applications. Suitable carrier systems need to be developed in order for them to be used. Phages It is important that it remains stable in the biological environment and reaches the target area in a controlled manner. This is a topic. Therefore, phages are being transported using different biopolymer-based carrier systems. Studies are being conducted on the stabilization and controlled release of these systems. Among them, hydrogel structures and micro / nano particle platforms stand out. 4 Current techniques involve biomaterial systems, blood concentrates, and antibacterial agents. There are many studies in which the agents are used individually. However, this In order for biological systems to be used effectively in clinical applications many aspects such as compatibility, release kinetics, mechanical properties and infection control The parameters need to be evaluated together. Therefore, biological New approaches that combine materials with biopolymer systems Research is ongoing. As a result, tissue regeneration and wound healing are possible. Although significant progress has been made in the areas of recovery and infection control Current technologies still have some limitations, especially biologically. stability of active ingredients, controlled release, optimization of mechanical properties and Issues such as ensuring infection control are still subjects of research. Research continues in these areas, including the development of new biomaterial systems. application methods are important in the fields of regenerative medicine and biomedical engineering. It has the potential to make contributions. Brief Description and Objectives of the Invention The invention describes the biological and functional effects of blood concentrates such as platelet-rich fibrin (PRF). hydrogel in the form of gelatin-based microspheres for the improvement of its properties A biohybrid approach using these structures is described. The hydrogel in question... The structures will provide controlled and delayed release of bioactive components. being prepared and treated with bacteriophages to impart antibacterial properties. is loaded. In addition, the preparation of the specified hydrogel structures is included within the scope of the invention. The creation of biohybrid structures by combining them with PRF and these structures especially clinical settings with open wounds, tissue loss, or a risk of infection. The application processes for its use in applications are also explained. The aim of the invention is to analyze the physical, mechanical and rheological properties of platelet-rich fibrin (PRF) structures. improving its properties for more controlled and wider use in clinical applications. The aim is to provide this opportunity. In the current technique, PRF is usually in the form of a solid fibrin mass. It is found and used in some clinical applications for shaping, spreading and application. Limitations may arise in terms of ease of use. Within the scope of the invention, these The technical element that enables the achievement of the goal; gelatin-based microspheres. This involves preparing hydrogel microspheres (GEL) in this form and combining them with PRF. This is obtained thanks to the dispersion of these hydrogel microspheres within the PRF matrix. The biohybrid structure alters the mechanical strength and rheological behavior of PRF, resulting in different... It contributes to creating a structure more suitable for clinical applications. Another aim of the invention is to utilize the growth factors naturally present in PRF and by preventing the rapid release of other bioactive molecules, thus providing a more controlled experience. and to create a long-term release mechanism. In current applications, PRF The bioactive components within are released relatively quickly, and this affects the tissue. This can make it difficult for the regeneration process to proceed optimally. The technical element that enables the achievement of the objective; the liquid phase when mixed with PRF. This involves the use of gelatin-based hydrogel microspheres (GEL) that have absorbent properties. These hydrogel structures absorb the liquid phase within the PRF, thus promoting growth. controlled and delayed release of factors and other bioactive molecules This allows biological activity to continue for a longer period. is provided. Another aim of the invention is to analyze platelets and leukocytes found within PRF. increasing the lifespan and biological activity of cellular components The aim is to provide this. In PRF applications, the activity of these cells may decrease over time. And this situation can limit the effectiveness of the tissue healing process. The invention The technical element that enables the achievement of this goal within this scope is gelatin-based. It involves the use of hydrogel microspheres within a PRF matrix. These hydrogel structures, The physical and microenvironment of the microenvironment containing the cellular components within PRF by improving their biochemical properties, the functional activities of cells are enhanced This contributes to its long-term preservation. Furthermore, the biodegradation of gelatin... The resulting amino acids also support tissue repair processes. It has effects. Another aim of the invention is to prevent bacterial infections that can occur, especially in open wounds. to reduce the risk of infection and, if an infection develops, an antibacterial The goal is to create an effect. Open wounds provide a suitable environment for the multiplication of pathogenic bacteria. This can create a condition that negatively affects the healing process. The technical element that makes this goal possible is the antibacterial phage of GELs. It involves loading them with cocktails and combining these structures with PRF. In this way... The prepared biohybrid structures deliver controlled phage release to the wound area. 6 It creates an antibacterial protection mechanism against pathogenic bacteria and It reduces the risk of infection. One aim of the invention is to adapt PRF-based treatment applications to different clinical needs. The goal is to develop a modular biohybrid system that allows for adaptability. In this context, biohybrids containing only hydrogel additives are available according to different clinical situations. the use of structures or antibacterial phage-loaded hydrogel structures This is the goal. The technical element that enables the achievement of this goal is two different This is the definition of the biohybrid structure approach. In the first approach, PRFs and GELs are used. By combining these elements, a biohybrid structure is created that supports tissue regeneration. In the second approach, antibacterial phage-loaded GELs are combined with PRF to achieve both A biohybrid structure that supports tissue repair and provides antibacterial protection. This makes it flexible and adaptable for clinical applications. An adaptable treatment platform is provided. Detailed Description of the Invention The invention relates to gelatin-based lyophilized solid / powder hydrogel microspheres (GEL). preparation of these hydrogel microspheres loaded with antibacterial phage cocktail obtaining the forms and combining these GELs with PRF to create biohybrids methods and compositions related to the preparation and implementation of structures The invention encompasses various fields, particularly tissue engineering, wound healing, and biomaterials. biohybrids that can be used in the fields of development and controlled biomolecule release. It is related to systems. The invention includes a biomaterial containing a gelatin-based hydrogel carrier system. its composition is in lyophilized solid / powder form. the gelatin-based hydrogel microspheres found, the aforementioned hydrogel microspheres It contains antibacterial bacteriophage cocktails and platelet-rich fibrin. It contains the (PRF) phase; these gelatin-based hydrogel microspheres are essentially It may contain gelatin A and / or gelatin B, as well as low molecular weight. Gelatin and / or cationized gelatin may also be added; cationized gelatin amine It has an enriched structure in terms of its groups; furthermore, the aforementioned Hydrogel microspheres contain physical and / or chemical cross-links and have a size of 1-100 µm. They have sizes within this range; bacteriophage cocktails have multiple different types. 7 It contains phages (phage mixtures) specific to pathogenic bacteria and has a concentration of 10⁴-10⁸ PFU / mL. They are located within the concentration range, and the hydrogel microspheres in question are liquid phages. It exhibits a structure that has absorbed the dispersion phase; lyophilized hydrogel microspheres. sucrose, soluble low molecular weight gelatin and / or various pH buffers It may contain protective agents, platelet-rich fibrin (PRF) phase fibrin It is a structure that includes a matrix and contains cells such as platelets, leukocytes, and growth factors. These bacteriophage cocktails contain biological components. Staphylococcus species, especially Staphylococcus aureus, Streptococcus species, especially Group A streptococci, Escherichia coli, particularly Extraintestinal Pathogenic E. coli (ExPEC), Proteus species, especially Proteus mirabilis, and Pseudomonas species, It contains phages that are particularly effective against Pseudomonas aeruginosa bacteria. Within the scope of this invention, gelatin-based hydrogel microspheres (GEL) are prepared and these Microspheres are obtained in dried or lyophilized solid / powder form. The subject is GEL microspheres made from biocompatible and biodegradable gelatin polymers. These are hydrogels containing a high amount of aqueous phase that are being formed. This structure is biological. loading and controlled release of agents within a microsphere structure This provides the possibility. Hydrogel microspheres developed within the scope of the invention. It is loaded with antibacterial bacteriophage cocktails. The phages are embedded in a hydrogel matrix. It is contained within and kept stable within its microspherical structure. This Microspheres are formed into solid or powder form through drying or lyophilization processes. It is prepared and can be stored under sterile conditions. The prepared phage-loaded hydrogel... The microspheres are mixed with platelet-rich fibrin (PRF) during application. It forms a biohybrid structure. Dry GEL microspheres are dispersed within the PRF. It absorbs a portion of the liquid phase and forms microreservoirs within the fibrin matrix. It forms hydrogel microspheres thanks to this microreservoir structure. The growth factors and / or phages contained within are controlled within the PRF matrix. It is released over time. Thus, it has a long-term effect in the application area. Growth factors and antibacterial effects are provided. Hydrogel microspheres. Its gelatin-based structure is biodegradable and undergoes biodegradation. The amino acids produced during this process contribute to tissue regeneration processes. It can provide. 8 The GEL hydrogel microspheres developed within the scope of this invention can be dried or lyophilized. It can be prepared in a sterile form and stored for clinical use. Application during which time the blood taken from the patient is centrifuged by healthcare personnel PRF is being prepared, and the prepared PRF is loaded with a specific amount of phage-loaded GEL microspheres. Biohybrid structures are obtained by mixing them. The resulting biohybrid structure The phages within the gels are released in a controlled manner in the application area. It produces an antibacterial effect against the pathogenic bacteria found. In this way, both both to prevent infections that may occur in the wound area and to treat existing bacteria. It is possible to control / eliminate infections. The system developed within the scope of the invention involves the production of phages from gelatin-based hydrogel microspheres. A biohybrid that provides controlled phage release within a PRF matrix with a high loading capacity. By forming a structure, it promotes both wound healing and infection control. It presents a new biomaterial approach that provides The invention describes a biomaterial containing a gelatin-based hydrogel carrier system. The method for preparing the composition involves the following steps: It includes: i. Selection of gelatin-based biopolymers and Gelatin A and / or Gelatin B Preparation of gelatin solution by dissolving it in an aqueous medium, ii. the molecular weight and functional characteristics of the gelatin solution in question acidic hydrolysis, enzymatic degradation to adjust its properties and / or modified gelatin is obtained by subjecting it to ozonization processes. being done, iii. The carboxylic acid groups in the gelatin structure are ethylenediamine (EDA) and ethyl- Amination using 3-(3-dimethylaminopropyl)carbodiimide HCl (EDC) Obtaining cationized gelatin by subjecting it to a process, iv. dispersing the prepared gelatin solution in the fat phase. the formation of the microdroplet structure and the dispersion in question gelatin-based gel produced by gelling at low temperature (4°C) obtaining microspheres, v. The resulting gel microspheres are subjected to centrifugation and drying processes. transforming it into a solid microsphere form 9 vi. physical (dehydrothermal) and / or chemical effects of the microspheres in question Cross-linking processes (using glutaraldehyde) in water Obtaining insoluble but swollen hydrogel microspheres (GEL), vii. Treatment of the obtained hydrogel microspheres with antibacterial phage cocktails For the purpose of loading, phage dispersions onto dry microspheres. the liquid phase of the microspheres is obtained by adding and incubating at 4°C. ensuring that it is absorbed, viii. Lyophilization of phage-loaded hydrogel microspheres stabilization and storage in solid / powder form, ix. In the application stage, the blood taken from the patient is centrifuged. Obtaining platelet-rich fibrin (PRF) phase, x. The resulting PRF phase is mixed with phage-loaded hydrogel microspheres. creation of a biohybrid structure, xi. molding and / or injection of the biohybrid structure in question shaping into the desired form. 1. BIOACTIVE AGENTS 1.1. Target pathogenic bacteria and antibacterial phages The invention describes different pathogens that may be encountered, for example, in PRF applications. pathogenic bacterial species assessed at various levels (including antibiotic-resistant strains) and Examples of strains include Staphylococcus (especially Staphylococcus aureus), Streptococcus. (especially Group A streptococci), Escherichia coli (especially Extraintestinal) Pathogenic E. coli – ExPEC), Proteus (especially Preus mirabilis) and Pseudomonas (specifically identified as Pseudomonas aeruginosa), the bacteria in question Effective phage cocktails are used against these species; however, this invention... not limited to bacteria and phages, but varying depending on demands and requirements. Modified phages are created using pathogenic bacteria and their specific effective phages. It is also possible to improve their formulations. 1.2. Bacterial multiplication Different culture media for the cultivation of bacterial strains used within the scope of the invention. It is used, for example, “Tryptic Soy Broth” (TSB) and “Tryptic Soy Agar” (TSA) The culture media are incubated for 24 hours at 37°C under aerobic conditions. After incubation, the culture medium is centrifuged at 4000 g to obtain a bacterial pellet. The pellet is then recycled in phosphate buffer (PBS, pH 7.2). It is washed three times by dispersing, then the bacterial concentration is reduced. For the purpose of determination, the classical dilution method was applied, with 1:10 consecutive steps. Bacterial suspensions are prepared at different concentrations through dilutions, 0.1 mL of the suspensions were spread onto TSA agar and incubated at 37°C for 24 hours. The resulting colonies are counted and the formula CFU / mL = N × (1 / SF) × (1 / V) is used to calculate the result. k 8 The number of bacteria per unit volume is calculated, and with this method, 10⁻¹² CFU / mL Bacterial suspensions can be obtained at concentrations of and above, for short periods. Bacteria during use are stored at 4°C, while for long-term storage, 30% (v / v) is used. They are stored in glycerol-containing culture media at -80°C. 1.3. Phage amplification The phages used in this invention are at a concentration of approximately 10⁸ CFU / mL. Mix with the prepared target bacterial suspensions and leave at room temperature for 15 minutes. incubated, then transferred to TSB medium supplemented with calcium and magnesium. Transferred and incubated for 6 hours at 37°C with a shaking speed of 200 rpm. The process involves purifying the amplified phages by passing the medium through a 0.22 µm filter. The phages are passed through and centrifuged at 13,600 g to precipitate them, then stored in PBS buffer. It is re-disperseeded, and this process is repeated three times, for a short period. Storage is carried out at 4°C, while for long-term storage, SM buffer and 30% Storage is done at -80°C using glycerol, phage concentration PFU / mL = Np is determined by the formula Np × (1 / SF) × (1 / V) and also by spot test (culture on agar) The effectiveness of phages is evaluated by assessing their lysis effects on target bacteria using a phage test. is being analyzed. 2. MAIN SUPPORTING ELEMENTS 2.1. Biopolymers used in matrix preparation The invention utilizes gelatin as the primary carrier, and its isoelectric point... Gelatin A, with an isoelectric point of approximately 9.0, and Gelatin B, with an isoelectric point of approximately 5.0, are preferred. It is being investigated whether the pH-dependent charge properties of these gelatins are related to bioactive agents. It plays a critical role in terms of loading and release, and also gelatins undergo acidic hydrolysis. modified by enzymatic degradation and ozonation methods, different Gelatins with molecular weights of 20-25 kDa and below are obtained. along with ethylene diamine (EDA) and ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl (EDC) 11 Gelatin amination is performed using these methods, thus producing cationized gelatins. By obtaining these, the positive charge density is increased, and selective growth factors are used. Interaction levels and consequently release rates from the matrix are controlled, and Cross-linking capacity is enhanced with glutaraldehyde. 2.2. Production and characterization of gelatin-based microsphere gels Within the scope of this invention, gelatin-based microspheres are produced through a two-stage process. and in the first stage prepared with different molecular weights and concentrations Gelatin solutions are dissolved at appropriate temperatures, dispersed in the oil phase, and Gel microspheres are obtained by achieving gelation in a cold acetone environment. Then, solid microspheres are prepared through centrifugation and drying processes, and these The size of the microspheres is generally in the range of 1-100 µm; in the second stage microspheres can be formed physically (dehydrothermally) and chemically (using glutaraldehyde) They are cross-linked and thus insoluble in water but can swell in an aqueous environment. Hydrogel microspheres (GEL) are obtained, and the swelling degrees of these GEL microspheres are determined. Water content (%) is determined by the formula: {(M – M ) / M} × 100, also swollen, dry, puffed up Microsphere dimensions are analyzed using an optical microscope, and surface charges (zeta) are determined. It is characterized by its potential measurements. 2.3. Phase loading and release The invention involves aseptic application of antibacterial phage cocktails onto GEL microspheres. The process involves loading under specific conditions and adding phage dispersions onto dry microspheres. They are then incubated at 4°C until the liquid phase of the microspheres is completely absorbed. This ensures 100% loading efficiency, and the loaded phages are released through PBS. In the environment, samples are taken at specific intervals and measured in PFU / mL, and over time... Emission profiles are obtained, and the emission in question is usually rapid at the beginning. This process is completed within 24-48 hours, and involves cross-linking density and matrix analysis. It varies depending on the type. 2.4. Lyophilization and sterilization Within the scope of this invention, gelatin-based GEL microspheres are processed into solid / powder form by lyophilization. are transformed into this form, and during this process the microspheres are mixed with liquid nitrogen. It is frozen and sublimated under vacuum at approximately -30°C. This is carried out, and then the temperature is raised to room temperature in a controlled manner. 12 A porous dry structure is obtained, in which case sucrose, soluble low molecular weight, is added. Phage stability is achieved using preservative agents such as gelatin and various pH buffers. It is being increased and remains above 80% during storage for up to 12 months at 4°C to 25°C. Activity preservation is ensured, and sterilization processes are carried out using autoclave and gamma ray tubes. This is carried out using radiation and ozonation methods, and these processes... Optimization by controlling the effects on the swelling and structural properties of microspheres. is being done. 3. PRF / GEL Biohybrids 3.1. Justification The platelet-rich fibrin (PRF) used in this invention is derived from the patient's own natural protein. a biomass obtained from blood and rich in growth factors It is widely used in tissue regeneration, but has limited mechanical strength. rapid degradation, uncontrolled release, and a short-term decrease in biological activity. Because it has disadvantages, GEL was developed to overcome these limitations. By developing biohybrid structures together with microsphere hydrogels, the functional properties of PRF were improved. The aim is to improve its mechanical and biological properties. 3.2. Preparation The invention specifically involves the use of "injectable" PRF (iPRF) for low speed and short-duration applications. The fibrinogen-rich liquid phase obtained by prolonged centrifugation is called dry GEL. mixed with microspheres in specific proportions and using suitable molds They are shaped so that after hardening, biohybrids are formed into the desired geometries. structures are obtained and the mechanical strength of these structures is increased and controlled It is shown that the emission characteristics have been improved. iPRF, as well as the invention, are included within the scope of the invention. Different centrifugation speeds and durations, and in different centrifuge tubes, can produce different results. For clinical applications, L-PRF (Leukocyte-PRF); A-PRF (Advanced-PRF); and T-PRF are available. Other types of PRF, such as titanium PRF, are also prepared and used to produce biohybrids. 3.3. Experimental results As a result of in vitro and in vivo studies carried out within the scope of the invention, GEL PRF biohybrids with added components increase cell viability, especially platelets and leukocytes. It prolongs their viability and activity, delays the release of growth factors, and phages It has been determined that it controls the release, on a rabbit model. 13 In the experiments conducted, biohybrids were observed in infected and non-infected wound areas. The effectiveness of the discs was compared, and the gelatin microspheres lasted approximately 14 days. They have been observed to be biodegradable. 3.4. Implementation protocol The invention includes two alternative methods for the application of PRF / GEL biohybrids. It is anticipated that, in the first method, the PRF phase obtained from the blood taken from the patient... by adding pre-prepared sterile lyophilized phage-loaded GEL microspheres biohybrid materials are being prepared, and in the second method, dry GEL microspheres are used. It is used in conjunction with PRF by adding sterile phage dispersion to it, and the resulting product... Biomass is applied directly; these biohybrid structures are used for open wounds and chronic conditions. It can be used in various clinical situations, including infections, application The frequency and phage dose can be adjusted depending on the infection status. It is stated. Industrial Applicability of the Invention The invention relates to the biological and functional effects of blood concentrates such as platelet-rich fibrin (PRF). hydrogel in the form of gelatin-based microspheres for the improvement of its properties biohybrids using these structures and their preparation and application methods. It is suitable for industrial application. The invention is not limited to the above descriptions, and a person skilled in the field can easily make further discoveries. It can demonstrate different applications of the invention. These are the claims and demands of the invention. It should be evaluated within the scope of the protection granted. 14

Claims

1. A biomaterial composition containing a gelatin-based hydrogel carrier system. Its characteristic is; - gelatin-based hydrogel microspheres, - antibacterial agents contained within the hydrogel microspheres in question bacteriophage cocktails, - platelet-rich fibrin (PRF) phase It includes.

2. The biomaterial composition according to Claim 1, and its characteristic is; as mentioned above. The difference is that PRF is "injectable" PRF (iPRF).

3. The biomaterial composition according to Claim 1 is characterized by the following: the gelatin in question. The base consists of hydrogel microspheres containing Gelatin A and / or Gelatin B.

4. According to Claim 1, the biomaterial composition is characterized by the following: the hydrogel in question. The microspheres are in lyophilized solid / powder form.

5. The biomaterial composition according to Claim 1 is characterized by the following: the gelatin in question. sucrose-based hydrogel microspheres, soluble low molecular weight gelatin and / or contain preservative agents, including various pH buffers.

6. The biomaterial composition according to Claim 1 is characterized by the following: the gelatin in question. The base consists of hydrogel microspheres containing cationized gelatin.

7. The biomaterial composition according to Claim 6, and its characteristic is; Cationized gelatin enriched with amino groups. It is the fact that.

8. According to Claim 1, the biomaterial composition is characterized by the following: the hydrogel in question. This refers to the physical cross-links between the microspheres.

9. According to Claim 1, the biomaterial composition is characterized by the following: the hydrogel in question. This refers to the fact that the microspheres contain chemical cross-links.

10. According to Claim 1, the biomaterial composition is characterized by the following: the hydrogel in question. The microspheres have sizes ranging from 1 to 100 µm.

11. The biomaterial composition according to Claim 1, and its characteristic is; Bacteriophage cocktails are specific against multiple different pathogenic bacteria. It contains phages.

12. The biomaterial composition according to Claim 1, and its characteristic is; Bacteriophage cocktails in the concentration range of 10⁴-10⁸ PFU / mL It is the fact that.

13. According to Claim 1, the biomaterial composition is characterized by the following: the hydrogel in question. The microspheres exhibit a structure that has absorbed the liquid phase within its internal structure.

14. The biomaterial composition according to Claim 1, and its characteristic is; the lyophilized material in question. hydrogel microspheres made of sucrose, low molecular weight gelatin and / or It contains preservative agents that act as various pH buffers. According to Claim 15, the biomaterial composition and its characteristic is; Platelet-rich fibrin (PRF) phase is a structure containing a fibrin matrix. According to Claim 16, the biomaterial composition and its characteristic is; Platelet-rich fibrin (PRF) phase of platelet, leukocyte and growth factors It contains biological components. According to Claim 17, the biomaterial composition and its characteristic is; Bacteriophage cocktails of Staphylococcus species, especially Staphylococcus aureus, Streptococcus species, especially Group A streptococci, Escherichia coli, especially Extraintestinal Pathogenic E. coli (ExPEC), Proteus species, especially Proteus mirabilis and Pseudomonas species, especially Pseudomonas It contains phages that are effective against aeruginosa bacteria.

18. A biomaterial containing a gelatin-based hydrogel carrier system. It is a method for preparing the composition, and its characteristic feature is; i. Selection of gelatin-based biopolymers and Gelatin A and / or Gelatin Preparation of gelatin solution by dissolving B in an aqueous medium, ii. the molecular weight and functional characteristics of the gelatin solution in question acidic hydrolysis, enzymatic to adjust its properties modified by undergoing degradation and / or ozonation processes gelatin production, iii. Carboxylic acid groups in the gelatin structure are converted into ethylenediamine (EDA) and using ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl (EDC) Cationized gelatin is obtained by subjecting it to an amination process. being done, iv. dispersing the prepared gelatin solution in the fat phase. the creation of the microdroplet structure and the aforementioned gelatin by gelling the dispersion at a low temperature (4°C) Obtaining gel-based microspheres, v. The resulting gel microspheres are subjected to centrifugation and drying processes. by being held and formed into a solid microsphere, 16 vi. physical (dehydrothermal) and / or chemical effects of the microspheres in question by undergoing cross-linking processes (using glutaraldehyde) hydrogel microspheres (GEL) that are insoluble in water but can swell are obtained. being done, vii. Treatment of the obtained hydrogel microspheres with antibacterial phage cocktails For the purpose of loading, phage dispersions onto dry microspheres. by adding and incubating at 4°C, the microspheres are dissolved in liquid. ensuring that it absorbs the phase, viii. Lyophilization of phage-loaded hydrogel microspheres stabilization and storage in solid / powder form, ix. In the application stage, the blood taken from the patient is centrifuged. Obtaining platelet-rich fibrin (PRF) phase, x. The resulting PRF phase, with GELs and phage-loaded hydrogel microspheres by mixing to form a biohybrid structure, xi. Molding and / or injection method of the biohybrid structure in question shaping into the desired form It includes the steps of the process.

19. Gelatin-based hydrogel carrier system prepared by a method according to claim 18. a biomaterial composition containing.

20. In clinical applications where there is a risk of open wounds, tissue damage, and infection. Gelatin-based hydrogel carrier systems for use in accordance with Claim 19. a biomaterial composition. 17