StemBor

Abstract

This invention relates to a bioactive hydrogel material capable of enhancing cell proliferation by combining hexagonal boron nitride (h-BN) nanoparticles with mesenchymal stem cells (MSCs). This hydrogel is hyaluronic acid (HA) based, reinforced with h-BN nanoparticles and / or boron nitride nanosheet (BNNS) particles, and stabilized by cross-linking with 1,4-Butanediol diglycidyl ether (BDDE). Within the scope of this invention, mesenchymal stem cells are cultured under controlled conditions to maintain their biocompatibility and proliferation potential, and then integrated with the resulting hydrogel structures. The prepared hybrid hydrogel material has been developed for use in biomedical applications for tissue engineering and regenerative therapy purposes. This invention provides an innovative biomaterial that supports cell viability and proliferation by offering a unique combination of material ratios, processing conditions, and cell culturing methods used in the preparation of the hydrogel.

Description

1 TARIFF StemBor Technical Area 5 The invention relates to a biomaterial that can be used in the treatment of joint and bone diseases. The invention will be used specifically in clinical trials for the treatment of joint and bone diseases. Hexagonal boron nitride (h-BN) nanoparticles integrated with mesenchymal stem cell therapy a product that can monitor the treatment process through biomaterials and fiber optic biosensors will be. 10 State of the Art Kütahya is one of the richest provinces in Türkiye in terms of boron minerals. Especially Emet and The Hisarcık district is home to significant boron reserves on a global scale. These regions... The extracted boron minerals are used in many industries such as glass, ceramics, detergents, agriculture and nuclear energy. It is used in this field. Boron also has environmentally friendly properties and can be transformed into high value-added products. thanks to its potential, it is important for both economic development and sustainable industrial production. It is of great importance. This natural resource of Kütahya is a strategic asset for the country's economy. It creates. 20 Osteoarthritis (OA), a disease of the joints and bones, is commonly referred to as "calcification". It is a known disease characterized by the wear and tear of cartilage tissue in the joint. However, OA It's not just a simple process of "wear and tear," but also inflammation in the joint. It is a disease that develops as a result of a complex interaction of factors [1, 2]. The most prominent feature of this disease is the deterioration of cartilage tissue. The process of cartilage loss is called synovitis. 25 (inflammation in the joint), subchondral bone changes (bone thickening, collapse, cyst formation), It is also associated with factors such as ligament and meniscus degeneration and thickening of the joint capsule, and These play an important role in the development of OA. Cartilage loss typically begins in a specific area and progresses progressively with variations in loading. This process proceeds as follows. This process involves complex mechanical, biological, biochemical, molecular and enzymatic processes. This occurs under the influence of nutrient cycles, which in turn affect chondrocytes (cartilage cells). It leads to morphological and metabolic changes [3]. Although joint and bone diseases are often associated with old age, they can affect individuals of all ages. However, women are more affected by this disease than men. Worldwide According to World Health Organization data, 18% of women over 60 and 10% of men over 35 He / She has limited mobility due to osteoarthritis. 2 Joint and bone diseases initially cause intermittent pain, but as they progress, they become irreversible. causing non-structural damage and loss of function, significantly affecting the quality of life. It lowers it. The FDA considers joint and bone diseases to be a serious illness for which there is no need for treatment. The draft guidelines, which state this, were published in August 2018. The ultimate goal of the treatments to be applied is 5 to prevent complications of joint dysfunction and the need for joint replacement (implant) or to significantly reduce the aim is to delay the process to a certain extent and also to reduce increasing functional loss and worsening pain. It indicates. In the EU, the IMI2 SRA Innovative Medicines Initiative (2014-2020) focuses on joint and bone diseases. He emphasized and designated it as a priority area. Joint and joint problems, which are widespread in the world, and 10 There is no clear statistical data available in Türkiye regarding bone diseases. The data are regional, and studies have shown that the prevalence of joint and bone diseases is not reflected in global health reports. This calculation was made using organizational data and population figures. Accordingly, the population aged 50 and over... It is stated that 13-15% of people are estimated to have joint and bone diseases [5]. Artificial joint Although the effect and duration of the liquids vary from person to person after application, it can last from 3 to 15 years. This provides relief to the person and postpones the surgery. In recent years, hexagonal boron nitride (hBN) has gained popularity due to its unique chemical and physical properties. Thanks to its structural properties, hBN is attracting significant interest in engineering fields. such as high oxidation resistance, chemical inertness, low thermal expansion, and high thermal conductivity. While offering advantages, it also possesses electrical insulation properties. 20 This material, with its excellent solid lubricant properties, is available in powder or raw product form for electrical applications. from the electronics sector to the maritime industry, from medical applications to the cosmetics field It is preferred in a wide range of uses. hBN's different structures, such as nanoparticles, nanosheets, and nanotubes, are subjected to chemical analyses. 25 for use in biomarkers, biosensor technologies, drug delivery systems and medicine. It has been the subject of extensive research

[13] . Intra-articular hyaluronic acid (HA) is currently used due to its successful clinical results. The injection's mechanism of action is cartilage-preserving in the treatment of joint and bone diseases. Different views have been put forward to explain this. The negatively charged (HA) is positively charged. The complex they form by binding with growth factors stimulates the secretion of these factors and 30 that it increases their activity, which is responsible for rapid and regular tissue healing. It is known

[19] Mesenchymal stem cells (MSCs) are found in the periosteum, trabecular bone, adipose tissue, synovium, and skeletal muscle. It is commonly found in other tissues including tendons, lungs, and primary teeth. MSC paracrine effects, Antiapoptosis, angiogenesis, growth and differentiation of stem and progenitor cells, 35 3 trophic (nutritive) categories in terms of immunomodulation, antiscaling and chemoattraction separable. Information derived from these paracrine mechanisms offers innovative solutions for the treatment of joint and bone diseases. They can bring solutions [20,21]. MSCs, thanks to their anti-inflammatory properties, can improve joint and bone health. It is a good candidate to address the diseases and the challenges encountered in their treatment. Damaged 5 It can repair tissues or reduce inflammation in joint and bone diseases. They can provide immunomodulatory functions. There are many studies and patent applications regarding the effect of hyaluronic acid on wound healing. There are applications. One of them, application number EP3191133A1, concerns hyaluronic acid or derivatives; borate compounds (e.g., boric acid, sodium tetraborate), various metal ions (Ca²⁺, Sr²⁺, 10 By combining monomers (Mg²⁺, etc.) and optional additional monomers, different pharmaceutical formulations can be created. These compounds have been developed to treat cuts, lacerations, bone fractures, and surgical or traumatic injuries. It has been stated that it can be used in repairs. To date, there have been various studies on the use of hyaluronic acid and its derivatives in wound healing processes. Formulations have been developed and patent applications have been filed. In these techniques, borate compounds are used with metal 15 Mixtures enriched with ions and polymeric components are effective in tissue repair. It has been shown. These formulations are mostly limited to local effects, and do not address systemic inflammation or chronic tissue problems. sufficiently complex pathophysiologies such as injury or joint degeneration It cannot offer a solution. 20 In this context, joint diseases such as osteoarthritis, which are characterized by the wear and tear of cartilage tissue, are particularly relevant. In diseases where current treatments cannot provide long-term improvement and implants are needed It is only known to be able to delay things. As highlighted in the FDA's 2018 guidelines, for joint and bone diseases... The treatments that have been developed are still among the unmet clinical needs. Also, Kütahya 25 The potential of boron-rich regions like [mention specific areas] in this field has not yet been effectively utilized. It has not been evaluated. Boron's eco-friendly nature and its capacity to be transformed into value-added products are not only economical. not only in terms of development, but also in terms of developing sustainable and effective treatment approaches. It presents a strategic opportunity. 30 Therefore, it is supported by advanced technological materials such as hexagonal boron nitride (hBN), biocompatible, anti-inflammatory and cell regeneration-promoting next-generation biomaterials There is a need. 35 Brief Description of the Invention 4 The present invention meets the aforementioned requirements while eliminating all disadvantages. and boron components that treat joint and bone diseases, bringing some additional advantages. It relates to biomaterials in which mesenchymal stem cells have been synthesized. The invention addresses the limitations of current treatment methods for joint and bone diseases. By overcoming obstacles, we aim to develop innovative approaches for biomaterial-assisted stem cell therapy. 5 The main purpose of the invention is to use boron, boric acid, and boron nitride derivatives together as active ingredients. It is made into a hydrogel by combining it with mesenchymal stem cells, for the treatment of joint and bone diseases. It is concerned with the formulation and synthesis of biomaterial compounds that promote healing. Another aim of the invention is to combine mesenchymal stem cells (MSCs) with hexagonal boron nitride (h-BN). 10 using a combination of nanoparticles in the treatment of joint and bone diseases The goal is to enhance cellular regeneration. Hexagonal boron nitride (h-BN) is an amorphous and nanostructured substance with wound-healing properties. The aim is to develop a novel biomaterial containing both nanoparticles and mesenchymal stem cells. The synthesis of the aforementioned compound using an easy, economical, and repeatable production method. to provide. 15 By ensuring the resulting structure remains stable in biological solutions, the gel The goal is to make it usable in formulations. The production process is simple and low-cost. and a system that supports scarless tissue healing by offering a method suitable for industrial production. to reveal. Figures that will help understand the invention. Figure 1 shows an overview of the Mesenchymal Stem Cell Production process, which is the subject of this invention. Figure 2 shows a general overview of boron nanoparticle production. Figure 3 shows an overview of biomaterial production. Figure References 1. Mesenchymal Stem Cell Production 2. Production of Boron Nanoparticles 3. Biomaterial Production 30 Detailed Description of the Invention Mesenchymal stem cells (MSCs) to be used in applications within the scope of the invention, Available from the American Type Culture Collection (ATCC® PCS-500-011), suitable The cells are transferred to culture medium according to thawing protocols. The cells are 35% fetal bovine serum. (FBS), in DMEM medium containing 0.2% primosin (Invivogen), 1% (ml / ml) Glutamax (Sigma), In an incubator with a humid atmosphere at 37 °C, containing 5% O₂ and 5% CO₂, The cells are cultured until they adhere to the surface. Throughout the culture process, cells are monitored daily using microscopic methods, and on the surface After they establish themselves, the culture medium is changed every three days. The cells have a high confluence rate. When it reaches 70-80%, it is separated from the surface with trypsin-EDTA solution and transferred to new culture dishes in a 1:3 5 ratio. It is subcultured by passing it through at a certain rate. In experimental applications, the fifth passage Untreated cells are used. According to the MTT (methyltetrazolium) analysis results, the subject of the invention is hexagonal boron nitride (h-BN). The use of nanoparticles in combination with mesenchymal stem cells significantly increases cell proliferation. It increases to a certain level. 10 In experimental studies, hydrogels were used as a control group and a comparison group, consisting of two different molecular structures. It is prepared using hyaluronic acid (HA) with a certain weight. The hydrogel in the control group The production process involves dispersing hyaluronic acid in a phosphate-buffered saline (PBS) solution and then involves the cross-linking process. In this context, the control group hydrogels were treated with 5 mL of pH 7.4 PBS solution containing 1.6% HA. It is prepared using [method name], and the gelling process is carried out at 60 °C to obtain a homogeneous structure. with the help of a magnetic stirrer operating at a temperature not exceeding three (3) hours is being carried out. In hybrid hydrogel formulations, BNP (boron nitride particles) and BNNS (boron nitride nanosheet) are used. The powders are placed in an ultrasonic bath in pure water for 20 minutes before being mixed with hyaluronic acid. It is being dispersed. Following dispersion, BNP and BNNS particles were obtained as pure form during the second hour of the gelation process. HA is added to the hydrogel phase and mixed at a temperature not exceeding 60 °C. The process continues. At the end of the third hour, a 0.1 N NaOH solution is added for cross-linking purposes. 1,4-Butanediol diglycidyl ether (BDDE) dissolved in it is added and in this medium 40 25 It is stirred for one hour at °C temperature. During the preparation of hydrogels, all environmental and chemical conditions are kept constant, only BNP and BNNS concentrations are used as variations. The resulting hydrogels are sterile. The syringes are stored in a refrigerator at +4°C until tribological testing. They are stored under these conditions. For characterization studies, the hydrogels are lyophilized to 30°C. It is stored by being prepared. 35 6 Formulation Contents Article amount (mmol) Quantity of substance (g) Amount of solvent (ml) Available by weight. amount (%) MKH 5 22.167 0.1 - 1.0 HbN NP 5 21.929 0.1 - 1.0 Sodium Hydroxide 45 18,000 0.1 - 1.0 Boric Acid 90 55.649 1.0 - 2.0 PEG 2000 20 400.000 Oct.40 Water 300 88.7 - 57.7 Preparation Method The invention describes the interaction of hexagonal boron nitride (h-BN) nanoparticles with mesenchymal stem cells (MSCs). It relates to a bioactive material created by bringing together cells. This biomaterial contains cells 5 Proliferation-promoting, highly biocompatible, and suitable for tissue engineering and regenerative medicine. It has a hybrid hydrogel structure suitable for various applications. MSCs to be used in biomaterial production are from the American Type Culture Collection (ATCC®). The cells are supplied via PCS-500-011. The cells are supplied within the appropriate dissolution protocols. dissolved in 10% fetal bovine serum (FBS), 0.2% primosin (Invivogen), and 1% Glutamax (Sigma) 10 The mixture is transferred to DMEM (Dulbecco's Modified Eagle Medium) medium and heated to 37 °C. They are cultured in incubators with humid atmosphere conditions containing 5% O₂ and 5% CO₂. The cells are monitored daily under microscopic conditions, and after they adhere to the surface, they are monitored every three (3) days. The culture medium is changed. When the cells reach a confluence level of 70-80%, trypsin is added. It is separated from the surface using EDTA solution and transferred to new culture dishes in a 1:3 ratio. Culture processing is applied. In experimental applications, only those who have not passed the fifth passage are included. cells are used. The hydrogel phase, used as a base building block in biomaterial production, has different molecular weights. It is synthesized using hyaluronic acid (HA). This hydrogel is made with 5 mL of phosphate at pH 7.4. It is prepared by dissolving 1.6% HA in buffered saline (PBS), and the gelling process takes 20 minutes. This process is carried out for 3 hours at a temperature not exceeding 60 °C with the help of a magnetic stirrer. is provided. 7 Hexagonal boron nitride nanoparticles (BNP) and boron nitride nanosheet (BNNS) powders, hyaluronic It is dispersed in pure water using an ultrasonic bath method before being combined with the acid. The resulting dispersion should be placed at a temperature not exceeding 60 °C during the second hour of the hydrogel's gelation process. It is added to the prepared HA phase by mixing as described. At the end of the third hour, a 0.1 N NaOH solution was added to the gelled structure as a crosslinking agent. 1,4-Butanediol diglycidyl ether (BDDE) dissolved in it is added and this mixture is heated to 40 °C. It is stirred at a certain temperature for one hour. This process increases the structural stability of the hydrogel and It is suitable for long-term biological applications. Cultured MSCs are incorporated into a cross-linked HA-BN hybrid hydrogel structure at a specific rate. The biomaterial formulation is completed by integrating the components at a high density. The final biomaterial is 10 They are stored in sterile syringes at +4 °C and characterized before use. For processing, it is lyophilized and then dried. The subject of the invention is a bioactive hybrid hydrogel material; • With its effect of increasing cell proliferation, • With its biocompatible and biodegradable properties, 15 • Thanks to its high mechanical stability, • Various applications such as wound healing, tissue engineering, and cartilage and bone regeneration. Suitable for use in biomedical applications. KAYNAKLAR 20 1. Berenbaum F (2013) Osteoarthritis as an inflammatory disease (osteoarthritis is not osteoarthrosis!). Osteoarthr Cartil 21:16–21. https: / / doi.org / https: / / doi.org / 10.1016 / j.joca.2012.11.012 2. Kapoor M, Martel-Pelletier J, Lajeunesse D, et al (2011) Role of proinflammatory cytokines in the pathophysiology of osteoarthritis. Nat Rev Rheumatol 7:33–42. 25 https: / / doi.org / 10.1038 / nrrheum.2010.196 3. Martel-Pelletier J, Boileau C, Pelletier JP, Roughley PJ (2008) Cartilage in normal and osteoarthritis conditions. 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Appl Radiat 40 Isot Incl data, Instrum methods use Agric Ind Med 69:1725–1727. https: / / doi.org / 10.1016 / j.apradiso.2011.02.032 8 9. Del Turco S, Ciofani G, Cappello V, et al (2013) Cytocompatibility evaluation of glycol-chitosan coated boron nitride nanotubes in human endothelial cells. Colloids Surf B Biointerfaces 111:142–149. https: / / doi.org / 10.1016 / j.colsurfb.2013.05.031 10. Kumbhakar P, Kole AK, Tiwary CS, et al (2015) Nonlinear Optical Properties and Temperature-Dependent UV– Vis Absorption and Photoluminescence Emission in 2D 5 Hexagonal Boron Nitride Nanosheets. Adv Opt Mater 3:828–835. https: / / doi.org / doi:10.1002 / adom.201400445 11. Panchal MB, Upadhyay SH Boron nitride nanotube-based biosensor for acetone detection: molecular structural mechanics-based simulation. https: / / doi.org / 10.1080 / 08927022.2013.837906 10 12. An WZ, Han B, Li K, et al (2017) The protective study about alleviation of simvastatin on the damages of PEGBNs in mice. 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Claims

REQUESTS 1. It is a bioactive hydrogel material whose characteristic is; • hexagonal boron nitride (h-BN) nanoparticles, • hyaluronic acid (HA), 5 • 1,4-Butanediol diglycidyl ether (BDDE) as a cross-linking agent, • and contains mesenchymal stem cells (MSCs) and increases cell proliferation It is prepared for that purpose.

2. It is a bioactive hydrogel material according to Claim 1, and its characteristic is that it contains 1.6% hyaluronic acid in 10 parts per million units. Obtained by dissolving in phosphate-buffered saline (PBS) solution, and gelling. The process is carried out for three hours at a temperature not exceeding 60 °C using a magnetic stirrer. It is the realization of.

3. Bioactive hydrogel material according to claim 1 or 2, with the property of being; hexagonal boron nitride (h-15 ultrasonic mixing of boron nitride nanoparticles (BN) and / or boron nitride nanosheet (BNNS) powders in pure water. By dispersing using the bath method, it is added to the hydrogel phase during the second hour of the gelling process. It is done.

4. Bioactive hydrogel material according to any of claims 1 to 3, with the characteristic of being cross-linked 20 For the binding process, BDDE dissolved in 0.1 N NaOH solution was left for three hours. Finally, it is added to the mixture and stirred for one hour at 40°C.

5. A bioactive hydrogel material according to any of claims 1 to 4, with the property of being mesenchymal. Stem cells were cultured in DMEM medium containing 10% FBS, 0.2% primosin, and 1% Glutamax at 37°C for 25 minutes. culturing under 5% O₂ and 5% CO₂ conditions and cells that have not passed the fifth passage It is the use of.

6. The material is a bioactive hydrogel according to any of claims 1 to 5, and its properties are: prepared The hydrogel material should be transferred to sterile syringes, stored at +4 °C, and 30... It is preserved by lyophilization for characterization.