Treatment of bone necrosis lesions with injectable hydrogel containing angiogenic peptides

Abstract

Osteonecrosis is the destruction of bone and its supporting structure in which bone cells die due to disruption of proper blood supply. The rate of progression is high, causing the bone to collapse within a few months. Clinical treatments such as bone grafting and non-surgical methods such as the administration of non-steroidal anti-inflammatory drugs have been investigated, but none have been definitive treatments for the permanent resolution of the condition. However, new therapies based on tissue engineering offer new prospects for improving this disease. A key pillar of tissue engineering is the design of appropriate scaffolds. Among them, hydrogels are considered the best choice due to their unique features, including sufficient flexibility, biocompatibility, and tunable mechanical properties compared to other scaffolds. Natural polymers, especially polysaccharides, have received much attention due to their similarity to the extracellular matrix. Therefore, in this invention, an injectable hydrogel based on the polysaccharide carboxymethyl chitosan and carboxymethyl cellulose has been designed, which has appropriate strength due to the open shift bond they form together, and a commercially available angiogenic peptide was used to stimulate angiogenesis.

Description

Description of the invention Title of the invention (as stated in the declaration) Treatment of bone necrosis lesions with injectable hydrogel containing angiogenic peptides Treatment of bone necrosis defects by injectable hydrogel containing angiogenic peptide Technical background of the relevant invention Bone necrosis, also known as avascular necrosis (AVN), osteonecrosis, or bone infarction, is a type of bone tissue death caused by loss of blood supply. In this disease, hematopoietic cells are most sensitive to low oxygen levels and are the first cells to die after the blood supply is reduced or eliminated, usually within 12 hours. Experimental evidence suggests that bone cells (osteocytes, osteoclasts, osteoblasts, etc.) die within 12 to 48 hours, and bone marrow fat cells die within 5 days. With reperfusion, bone repair occurs in two stages. First, angiogenesis occurs, followed by the movement of undifferentiated mesenchymal cells from adjacent living bone tissue into the dead marrow space. Then macrophages, which degrade dead cell debris and fat, enter the fray. Second, cellular differentiation of mesenchymal cells into osteoblasts or fibroblasts occurs. Unfortunately, today in developing countries, including Iran, invasive methods and complex surgeries are used to treat this disease. Techniques include bone grafting from healthy parts of the body, osteotomy or cutting the bone and changing its alignment to eliminate stress on the bone, complete joint replacement or removing the damaged joint and replacing it with an artificial joint, internal decompression by removing part of the inner part of the bone to reduce pressure and create new blood vessels, and vascularized bone grafting using blood vessel-rich bone from another part of the body, such as the fibula, are among the cases that are used to treat this lesion, and the use of minimally invasive tissue engineering alternatives such as hydrogels and angiogenesis drug carriers is not common. However, numerous studies have shown that patients with severe avascular necrosis lesions require a scaffold containing angiogenic drugs for treatment. A suitable scaffold for the treatment of avascular necrosis should be able to stimulate angiogenesis and natural bone tissue regeneration, reduce the time required for healing, and prevent infections in the bone marrow area. On the other hand, this scaffold should be available and economically affordable for the patient. Carboxymethyl chitosan polymer, due to its biocompatibility and good degradation rate, can be a suitable carrier for drugs and growth factors, and also has mechanical properties equivalent to trabecular bone. Also, hydrogels based on carboxymethyl chitosan are most widely used for bone tissue engineering due to properties such as gelation capacity, low toxicity, antibacterial properties, high availability, and low cost. The inherent crosslinking and ionic nature of this polymer ensures its injectable nature. Another material considered in this invention is the use of carboxyl methyl cellulose (CMC). CMC is soluble in water and becomes completely transparent after dissolution. This material has surface activity, thermoplastic properties and thickening properties. It is also stable against heat, hydrolysis and oxidation. Low price, biodegradability, high viscosity, non-toxicity and non-allergenicity are important features of CMC, which have led to its diverse applications. Having numerous hydroxyl and carboxyl groups has made it capable of absorbing water. Another important factor that is considered in the treatment of avascular necrosis and is considered in this design is angiogenesis. After selecting a hydrogel that is designed to suit the desired tissue, the need for angiogenesis is felt in the treatment of this disease. The peptide selected in this invention has high thermal stability and retains its functional structure well in water, while its potential in therapeutic angiogenesis has also been confirmed in many studies. It has also been shown that this peptide can bind to and phosphorylate VEGFR-1,2 receptors on endothelial cells, subsequently promoting capillary proliferation and formation. Therefore, in this invention, in order to design and manufacture a suitable and efficient alternative, the above-mentioned advantages have been considered. To date, different scaffolds have been designed to treat this condition, each of which has advantages and disadvantages. In this invention, the intention is to design and manufacture a minimally invasive method for the treatment of avascular necrosis using an injectable hydrogel carrying an angiogenesis-stimulating peptide. Technical problem and stating the objectives of the invention Avascular necrosis has caused many problems for patients due to its pain and inability to move. Risk factors for this disease include trauma such as bone fractures and joint dislocations, which lead to interruption of blood supply and formation of blood clots, inflammation and damage to the arteries. In a relatively short period of time, the patient develops arthritis, chronic and long-term pain, bone deformation and shortening of long bones, stretching and shortening of leg muscles, severe limitation of joint range of motion, especially of the hip, and collateral damage in avascular necrosis of the femoral head to the spine and neck. Many studies have emphasized that avascular necrosis requires tissue engineering-based scaffolds for complete repair. In developing countries, the treatment of this lesion is carried out using old methods, which ultimately affects the quality of repair to a great extent. In these cases, the presence of a biocompatible, degradable scaffold that carries angiogenic drugs, prevents bone marrow infection, and regenerates necrotic bone is essential. Studies have shown that polysaccharide polymers are a good option for repairing damaged bone. The most important limitation of using any polysaccharide polymer alone as a drug carrier scaffold is its low mechanical properties and high biodegradability. On the other hand, peptides have received much attention for their ability to stimulate repair and prevent infection and antibacterial properties. The disadvantages of peptides include their solubility and rapid leakage in the physiological environment of the body. In order to overcome this limitation, it is necessary to release them slowly inside a scaffold-like environment such as a hydrogel and to release them in a controlled manner in the cell environment. Therefore, in order to create a complete scaffold that overcomes the limitations of polysaccharide polymers and peptides and is cost-effective, an injectable hydrogel was made from polysaccharide polymers and an angiogenesis-stimulating peptide. The final hydrogel product is expected to have better mechanical properties and high antibacterial properties, as well as the ability to regenerate necrotic bone tissue. Polysaccharide polymers are made soluble in water after being purchased from Sigma. Peptides are reacted with polymers after being designed, prepared, and prepared. In this invention, a suitable injectable hydrogel was made from polysaccharide polymers and angiogenesis-stimulating peptide. The product was then examined mechanically and cellularly in a laboratory environment. The effectiveness of this alternative in repairing avascular lesions was confirmed. Given the ability to localize the present technology as a product in the field of tissue engineering and biotechnology, as well as the pristine nature of the research field in this field, the present invention can undoubtedly bring many innovations and be considered a suitable platform for use in the repair of avascular necrosis lesions as well as future research. A description of the state of the prior art and the history of developments related to the claimed invention. Currently, there is no effective treatment to prevent the progression of this disease in its early stages, and the later stages can only be treated with joint reconstruction surgery. In fact, non-surgical treatments for this disease, including oral alendronate, oxygen therapy, shock therapy, and electrical stimulation, have encountered difficulties and have been reported to be of little effectiveness. Therefore, surgical interventions such as the bone head decompression technique have been investigated, which involves making one or more deep holes with a drilling tool in the femoral head from the base of the trochanter in order to reduce intraosseous pressure in that area in order to remove necrotic bone tissue and then using autologous or allogeneic bone to fill the bone defect. Unfortunately, autologous bone grafting has disadvantages such as lack of bone mass, the need for additional surgeries, longer operating time, increased blood loss and related complications, hospitalization time, and high costs.However, most of the treatments for avascular necrosis that are in the clinical trial stage are medications. Drugs such as ibuprofen, alendronate, aspirin, diclofenac, naproxen, Voltaren, Fosamax, Ketorolac, asparagine, etc., are largely prescribed in the early stages of the disease and cause severe complications for patients. In 1993, the first comprehensive review article on tissue engineering-based therapies was published in Science, and in 2014, the first review article on the use of bone marrow-derived mesenchymal stem cells was published. However, in 2017, the FDA approved the first tissue engineering product derived from bone marrow-derived mesenchymal stem cells and osteoblasts, OSSGROW®, the first stem cell-based therapy by Regrow Biosciences. The product was developed in a laboratory at Apollo Hospitals, India. Another stem cell-based therapy using adipose-derived stem cells is also in clinical trials, but has not yet been approved by the FDA. Nowadays, the use of tissue engineering, especially in the field of scaffolds for the repair of avascular necrosis lesions, has received attention. One of the main limitations of using natural polymers alone to make hydrogel scaffolds is the very low mechanical properties of these polymers and their very high degradation rate. So that it is difficult to work with them as engineered structures and drug carriers in the physiological environment of the body. In terms of natural polymers, they have many advantages in repair and regeneration, and if they are made together with a strong crosslinker, they show ideal mechanical properties. Meanwhile, the use of a drug or peptide that stimulates regeneration and has antibacterial properties along with these polymers creates suitable drug carriers for therapeutic purposes. For this purpose, polysaccharide polymers are loaded with peptides and can be used as an injectable hydrogel network in the body environment. Providing a solution to an existing technical problem along with an accurate, sufficient, and integrated description of the invention Today, tissue engineering technology is used to repair damaged tissues. Among the research conducted to repair various tissues, some products have entered the commercial arena and are used in medical centers today. In this product, polysaccharide polymers and biocompatible angiogenesis-stimulating and osteogenesis-stimulating peptides have been used to treat avascular necrosis. Polysaccharide polymers are very suitable for tissue engineering purposes and drug carriers in terms of structure and biocompatibility. These polymers are also used to make scaffolds to replace body tissues. The most important problems of using each of these polymers alone are their premature degradation and low mechanical properties, as well as their difficulty in solubility. So that these polymers are often very difficult to dissolve alone in an aqueous environment and ultimately create a weak structure. Therefore, using them as engineered scaffolds definitely requires a strong crosslinker.In this invention, in order to overcome the problems associated with using polymers alone, two polysaccharide polymers with excellent biocompatibility properties that can form strong chemical bonds with each other and form a coherent hydrogel network with injectable properties have been used. This injectable hydrogel has very high mechanical properties and elasticity, selectively allows peptides and oxygen to pass through it, and prevents the passage of unnecessary substances and enzymes into the necrotic environment. By evaluating the mechanical and physical structure of the resulting hydrogel, we achieved the optimal parameters of the structure. After manufacturing the hydrogel with the aforementioned specifications, extensive biological tests were performed and the biological parameters for its use in the body were optimized. Explanation of shapes, maps and diagrams Polysaccharide polymers were obtained from Sigma-Aldrich and each was dissolved separately in an amount of 0.1 g in 1 ml of water on a stirrer and then mixed together for 1 to 2 minutes under sterile conditions to obtain the desired hydrogel network. The formation of the hydrogel network after the aforementioned processes was confirmed by various tests. The peptide of interest in this invention was designed and prepared and the purity of the peptide was examined using relevant tests. In the final step, the peptide is loaded onto the polymer network to create the final injectable hydrogel made of polysaccharide / peptide polymers. A clear and precise statement of the advantages of the claimed invention over prior inventions. Advantages of the manufactured product: High regenerative power Ideal mechanical properties Antibacterial properties Availability and ease of construction Affordable High biocompatibility No need for invasive surgery Long-term storage in the laboratory High impact on cell proliferation and angiogenesis Prevent infection Slow drug release and regeneration as quickly as possible Description of at least one implementation method for implementing the invention Using this alternative to treat avascular necrosis, osteomyelitis, and osteoporosis causes complete repair of necrotic bone and prevents bone marrow infection. Explicit mention of the industrial application of the invention Treatment of osteonecrosis of the hip with minimally invasive therapy Brief description of the invention Bone necrosis lesions, also known as avascular necrosis, osteonecrosis, or bone infarction, are a type of bone tissue death due to loss of blood supply. Unfortunately, today in developing countries, including Iran, invasive methods and complex surgeries are used to treat this disease. Techniques include bone grafting from healthy parts of the body, osteotomy or cutting the bone and changing its alignment to eliminate stress on the bone, total joint replacement or removing the damaged joint and replacing it with an artificial joint, internal decompression by removing part of the inner part of the bone to reduce pressure and create new blood vessels, and vascularized bone grafting using blood vessel-rich bone from another part of the body, such as the fibula, are among the cases used to treat this lesion. Currently, there is no effective treatment method to prevent the progression of this disease in its early stages, and the later stages can only be treated with joint reconstruction surgery.In fact, non-surgical treatments for this disease, including oral alendronate, oxygen therapy, shock therapy, and electrical stimulation, have encountered difficulties, and little effectiveness has been reported for these treatments. The use of minimally invasive alternative tissue engineering methods such as hydrogels and angiogenesis-promoting drug carriers is not common. However, numerous studies have shown that patients with severe avascular necrosis require a scaffold containing an angiogenic drug for treatment. In this invention, the intention is to design and manufacture a minimally invasive method for the treatment of avascular necrosis using an injectable hydrogel and an angiogenesis-promoting peptide carrier.

Claims

Claims What is claimed: Claim 1) This invention is a QK peptide conjugated to a slow-release injectable hydrogel based on derivatives of chitosan and cellulose polymers, which contains the sequence N-acetyl-KLTWQELYQLKYKGI-amide, such that this peptide caused angiogenesis and osteogenesis and repaired avascular necrosis in a rat animal model. Claim 2) The method for synthesizing the hydrogel claimed in claim (1) includes steps including: a) A solution of carboxymethyl cellulose and carboxymethyl chitosan with a volume ratio of 1 to 1 are mixed at a rotational speed of 2000-3000 m / s at a temperature of 25 to 35 degrees Celsius for 60 minutes and forms a coherent hydrogel network within (18 ± 3) seconds. 2) Angiogenic peptide is conjugated in it at a concentration of 300 micrograms per microliter (with a volume ratio of 1 to 10). Claim 3) In the injectable hydrogel of claim (1), the pore size of the final product is in the range of 100-150 microns and its dimensions are suitable for 0.5 to 5 cc syringes for injection. Claim 4) The hydrogel of claim (1) has antibacterial properties.

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