Self-solidifying composite bone implant based on essential amino acids for human body and preparation method thereof

Abstract

This invention relates to a self-curing composite bone implant based on essential amino acids and its preparation method, belonging to the field of bone repair materials. Specifically, the self-curing composite bone implant based on essential amino acids comprises 20-50 parts by weight of amino acid calcium, 10-30 parts by weight of organic calcium phosphate, and 30-50 parts by weight of calcium sulfate. The amino acids are essential amino acids. The amino acid calcium is obtained by reacting essential amino acids with organic calcium compounds under suitable conditions for generating amino acid calcium, and the organic calcium phosphate is obtained by reacting organic phosphorus compounds with organic calcium compounds under suitable conditions for generating organic calcium phosphate. This self-curing composite bone implant based on essential amino acids is safe and non-toxic, fully degradable, highly active, and easily moldable. It can promote bone tissue repair and reconstruction and be completely replaced by bone tissue during bone tissue repair and reconstruction.

Description

Technical Field

[0001] This invention relates to a self-coagulating composite bone implant based on essential amino acids for the human body and its preparation method, belonging to the field of bone repair materials. Background Technology

[0002] Human bone tissue is formed through DNA regulation, where amino acids form collagen. During collagen formation, calcium and phosphorus are fixed to the collagen molecules in a specific ratio and arrangement, creating bone tissue with a specific physiological structure. Therefore, bone originates from small amino acids forming collagen, which, under DNA expression, is absorbed and fixed by osteoblasts to form initial bone tissue. Mature bone tissue is then reconstructed by osteoclasts. The human body's calcium absorption rate is related to various factors, generally ranging from 15% to 45%, and is difficult to increase further. Experiments have shown that the absorption rates of various calcium supplements are roughly the same. Vitamin D has a strong effect on promoting calcium absorption; therefore, adding an appropriate amount of vitamin D to calcium supplements can increase calcium absorption. Calcium gluconate can only be absorbed at 7-9%, and calcium carbonate at only 40%. Amino acid calcium has an absorption rate of over 80%, with the unabsorbed portion excreted in feces. Amino acid calcium is recommended by the American medical community as the best calcium supplement.

[0003] On the other hand, after bone tissue injury or surgical repair, the repair and reconstruction begin at the damaged wound surface (not from the surface of the implanted material). Through the physiological and physicochemical reactions of the bone tissue and the interaction with the surface of the implanted material, the components of the material surface are gradually absorbed by the bone tissue and deposited on the bone tissue surface to form new bone. As the surface components are gradually absorbed, the material gradually degrades and peels off in molecular and ionic form; the material size gradually decreases until it disappears completely; the bone tissue completes its repair and becomes a whole. During this process, DNA expression enables osteoclasts to remodel the newly formed bone, forming mature bone tissue, thus completing the comprehensive repair of the injury and defect and restoring the physiological function of the bone tissue. From this perspective, the material only plays a temporary role in providing space, support, and repair components, and needs to maintain a corresponding degradation and decomposition rate in line with the bone tissue repair and reconstruction.

[0004] Therefore, only materials that can be degraded and absorbed can complete bone tissue regeneration and reconstruction. If the material cannot be completely absorbed and degraded by the tissue, it will remain in the damaged center of the bone tissue for a long time, forming an area inconsistent with the composition and function of the bone tissue, and the physiological function of the bone tissue will not be fully restored. Most bone repair materials currently used in clinical practice, except for calcium sulfate, are of this type, which is a "half-finished project" situation. That is, part of the material implanted on the inner surface of the bone tissue is absorbed and degraded to form bone tissue, while the other part is wrapped in the middle and remains in the bone tissue for a long time. There is a physical interface between the material and the new bone, and its physicochemical and physiological composition is inconsistent with the bone tissue.

[0005] To address this "unfinished project" situation, the applicant of this invention previously invented a series of polyamino acid bone repair materials. However, due to the high polymerization temperature of amino acids, the varying molecular weights, and the lack of strict restrictions on the selection of amino acids, these materials contain a large number of non-α amino acids. Their degradation performance requires strict control over their molecular weight and molecular weight distribution, as well as strict control over the temperature and conditions for their combination with calcium phosphate salts. In other words, the entire process is difficult to control and not easy to achieve, which is not conducive to industrialization. Summary of the Invention

[0006] To address the aforementioned deficiencies, this invention provides a self-coagulating composite bone implant based on essential amino acids and its preparation method. Mature bone tissue is a functional composite material composed of proteins (collagen) made up of amino acids and bone-like apatite arranged according to a specific physiological structure. Its origin and process are not bone-like apatite or hydroxyapatite. This invention designs a self-coagulating composite bone implant with essential amino acids as the main component, based on the origin and process of bone tissue composition. It consists of essential amino acids reacting with organic calcium to form a calcium source easily absorbed by bone tissue, organic energetic and informational phosphorus compounds as a phosphorus source and nutrient component, and hemihydrate or anhydrous calcium sulfate as a rapid coagulation component. This solid phase is mixed with deionized water or water for injection in a quantitative ratio to form a self-coagulating composite bone implant product based on essential amino acids. The mass ratio of essential amino acids: organic energetic and informational phosphorus compounds: calcium sulfate is 20-50:10-30:30-50. The solid-to-liquid phase ratio is 100:30-60 (g / ml). This self-coagulating composite bone implant containing essential amino acids is safe, non-toxic, fully degradable, highly active, and easily shaped. It can promote bone tissue repair and reconstruction and be completely replaced by bone tissue during the bone tissue repair and reconstruction process.

[0007] Specifically, the present invention provides a self-coagulating composite bone implant based on essential amino acids, comprising 20-50 parts by weight of amino acid calcium, 10-30 parts by weight of organic calcium phosphate and 30-50 parts by weight of calcium sulfate, wherein the amino acids are essential amino acids.

[0008] Further, the amino acid calcium is obtained by reacting essential amino acids with an organic calcium compound under suitable conditions for generating amino acid calcium. Suitable conditions for generating amino acid calcium can be readily determined by those skilled in the art; for example, the reaction may include reacting at a pH of 7.0-9.0, preferably 7.5-8.5, under ultrasonic dispersion and / or stirring. Further, the reaction also includes filtering the reaction product to obtain a precipitate, which is then washed and dried to obtain the amino acid calcium. Further, the stoichiometry of the reaction between essential amino acids and the organic calcium compound is not strictly limited, as both the selected essential amino acids and the organic calcium compound can provide osteogenic elements and nutrients for the formation of new bone in bone tissue. For example, the ratio of essential amino acids to organic calcium compounds can be 1.5-3:1 (mol), preferably 1.75-2.25:1.

[0009] Furthermore, the essential amino acids are selected from one or more of lysine, tryptophan, phenylalanine, methionine, threonine, isoleucine, leucine, valine, and histidine. For example, the essential amino acids may be a mixture of two or more of the aforementioned essential amino acids. For example, the essential amino acids may be a mixture of two or more of the aforementioned essential amino acids in equimolar amounts.

[0010] Further, the organic calcium phosphate is obtained by reacting an organophosphorus compound with an organocalcium compound under suitable conditions for generating organic calcium phosphate. Suitable conditions for generating organic calcium phosphate can be readily determined by those skilled in the art; for example, the reaction may include reacting at a pH of 6.5-8.5, preferably 7.0-7.5, under ultrasonic dispersion and / or stirring. Further, the reaction also includes filtering the reaction product to obtain a precipitate, which is then washed and dried to obtain the organic calcium phosphate. Further, the stoichiometry of the reaction between the organophosphorus compound and the organocalcium compound is not strictly limited, as both the selected calcium source and the organophosphorus can provide osteogenic elements and nutrients for bone tissue to form new bone. For example, the ratio of the organocalcium compound to the organophosphorus compound can be 0.9-2.0:1 (mol), preferably 1.0-1.5:1.

[0011] Furthermore, the organophosphorus compound is selected from one or more of adenosine-5'-triphosphate disodium salt, adenosine-5'-triphosphate dipotassium salt, adenosine-5'-diphosphate sodium salt, adenosine-5'-diphosphate potassium salt, and adenosine-3',5'-cyclic monophosphate sodium hydrate.

[0012] Furthermore, the organic calcium compound includes calcium glycoside or small-molecule organic acid calcium.

[0013] Furthermore, the small molecule organic acid calcium includes calcium levulinate, calcium malate, calcium acetate, and calcium propionate.

[0014] Furthermore, the calcium sulfate is selected from anhydrous calcium sulfate or calcium sulfate hemihydrate.

[0015] This invention also provides a method for preparing a self-coagulating composite bone implant based on essential amino acids as described herein, comprising the following steps:

[0016] (1) Essential amino acids for the human body are reacted with organic calcium compounds under suitable conditions to produce amino acid calcium, thus obtaining amino acid calcium.

[0017] (2) An organophosphorus compound is reacted with an organocalcium compound under suitable conditions to produce organocalcium phosphorus, yielding organocalcium phosphorus.

[0018] (3) Mix 20-50 parts by weight of the amino acid calcium from step (1) and 10-30 parts by weight of the organic calcium phosphorus from step (2) with 30-50 parts by weight of calcium sulfate, and perform hydrothermal treatment at 120-200℃ and 0.1-1.0 MPa for 2-8 hours. After cooling, a self-coagulating composite bone implant based on essential amino acids for the human body is obtained.

[0019] Furthermore, the suitable conditions for generating amino acid calcium in step (1) can be readily determined by those skilled in the art. For example, the reaction may include reacting at a pH of 7.0-9.0, preferably 7.5-8.5, under ultrasonic dispersion and / or stirring. Further, the reaction may also include filtering the reaction product to obtain a precipitate, which is then washed and dried to obtain the amino acid calcium. Therefore, step (1) may include reacting essential amino acids with an organic calcium compound under ultrasonic dispersion and / or stirring at a pH of 7.0-9.0, preferably 7.5-8.5, filtering to obtain a precipitate, and washing and drying to obtain the amino acid calcium.

[0020] Furthermore, the stoichiometry of the reaction between essential amino acids and organic calcium compounds is not strictly limited, because both the selected essential amino acids and organic calcium compounds can provide osteogenic elements and nutrients for the formation of new bone in bone tissue. For example, the ratio of essential amino acids to organic calcium compounds can be 1.5-3:1 (mol), preferably 1.75-2.25:1.

[0021] Furthermore, the conditions for generating organocalcium phosphate in step (2) can be readily determined by those skilled in the art. For example, the reaction may include reacting at a pH of 6.5-8.5, preferably 7.0-7.5, under ultrasonic dispersion and / or stirring. Further, the reaction may include filtering the reaction product to obtain a precipitate, which is then washed and dried to obtain the organocalcium phosphate. Therefore, step (2) may include reacting an organophosphorus compound with an organocalcium compound at a pH of 6.5-8.5, preferably 7.0-7.5, under ultrasonic dispersion and / or stirring, filtering to obtain a precipitate, and washing and drying to obtain the organocalcium phosphate.

[0022] Furthermore, the stoichiometry of the reaction between the organophosphorus compound and the organocalcium compound is not strictly limited, because both the selected calcium source and the organophosphorus compound can provide osteogenic elements and nutrients for the formation of new bone in bone tissue. For example, the ratio of the organocalcium compound to the organophosphorus compound can be 0.9-2.0:1 (mol), preferably 1.0-1.5:1. Furthermore, in step (3), hydrothermal treatment is preferably carried out at 130-160°C and 0.3-0.6 MPa for 3-6 hours.

[0023] Furthermore, the method further includes further pulverizing the self-coagulating composite bone implant based on essential amino acids obtained in step (3) to obtain a product with a target particle size. Further, the pulverization includes ball milling the self-coagulating composite bone implant based on essential amino acids in a dry atmosphere at room temperature for 2-6 hours, and then vacuum drying it at 60°C for 12 hours after it reaches the target particle size (e.g., a particle size distribution of less than 200 mesh).

[0024] Furthermore, the method also includes mixing and blending the self-coagulating composite bone implant based on essential amino acids obtained in step (3) with a curing liquid at a solid-liquid ratio of 100:30-60 (g:ml) to obtain bone cement, which can be loaded into a mold to make a test sample or shaped into the required shape, or can be directly injected into the required wound or defect site through an injection device.

[0025] Furthermore, the curing solution is selected from one or a mixture of several of water for injection, deionized water, and physiological saline.

[0026] This invention also provides a method for applying a self-curing composite bone implant based on essential amino acids as described herein, which includes mixing and blending the self-curing composite bone implant based on essential amino acids as described herein with a curing liquid at a solid-liquid ratio of 100:30-60 (g:ml) to obtain bone cement, which can be loaded into a mold to make a test sample or shaped into the desired shape, or can be directly injected into the desired wound or defect site through an injection device.

[0027] Furthermore, the curing solution is selected from one or a mixture of several of water for injection, deionized water, and physiological saline.

[0028] Furthermore, the application method includes thoroughly mixing the self-curing composite bone implant based on essential amino acids as described herein with a curing liquid to form a paste, continuing to mix until it reaches a dough-like state, and then implanting it into the wound or defect site using a syringe or by shaping it into the desired form. The curing time is 5-45 minutes, with 15-30 minutes generally considered the optimal surgical time. It can also be pre-formed into particles of different diameters (φ2-10mm) or molded into blocks and cylinders (size determined based on actual needs, such as φ3*10mm, 20*10*5mm) for use as a filler during surgery.

[0029] The present invention also provides a bone cement comprising a self-curing composite bone implant based on essential amino acids as described herein, with a solid-liquid ratio of 100:30-60 (g:ml), and a curing liquid. The bone cement can be loaded into a mold to form a test sample or shaped as desired, or can be directly injected into the desired wound or defect site via an injection device. Further, the curing liquid is selected from one or a mixture of several of water for injection, deionized water, and physiological saline.

[0030] This invention also provides the use of self-coagulating composite bone implants based on essential amino acids as described herein for promoting bone tissue repair and reconstruction and complete replacement by bone tissue during bone tissue repair and reconstruction.

[0031] The beneficial effects of this invention are:

[0032] The self-coagulating composite bone implant provided by this invention contains essential amino acids (the core components of collagen formation), as well as osteogenic elements organic calcium and phosphorus. It is self-coagulating, pH neutral, and fully degradable, capable of gradual degradation from the side in contact with bone tissue. It provides components throughout the entire process of bone tissue repair and reconstruction from the wound site, promoting tissue repair and reconstruction. Its cured strength provides early defect support, gradually degrades and integrates with tissue, and is absorbed and replaced by tissue. Simultaneously, it provides various organic and inorganic components that promote bone tissue regeneration, enhancing tissue regeneration and reconstruction. Its degradation rate can be controlled according to its composition, matching bone tissue regeneration and reconstruction. It can be widely used in the repair and reconstruction of bone trauma and defects. Its most significant feature is that it can provide some of the energy needed by the tissue during tissue formation, accelerating tissue repair and reconstruction. Detailed Implementation

[0033] This invention, starting from the perspective of small molecules and based on the origin and processes of human bone tissue, designs a self-coagulating composite bone implant with essential amino acids as the main component. It consists of essential amino acids reacting with organic calcium to form a calcium source easily absorbed by bone tissue, organic energetic and informational phosphorus compounds as a phosphorus source and nutrient component, and hemihydrate or anhydrous calcium sulfate as a rapid coagulation component. This solid phase is then mixed with deionized water or water for injection in a quantitative ratio to form a self-coagulating composite bone implant product based on essential amino acids. As an example only, and to aid in understanding this invention, the composition of the self-coagulating composite bone implant based on essential amino acids can be illustrated as follows:

[0034]

[0035] Based on the absorption efficiency of calcium in the body, the first step of this invention is to select essential amino acids as carriers for calcium. There are 20 types of amino acids, which can be broadly classified into three categories: essential amino acids, semi-essential amino acids, and non-essential amino acids. Histidine has been found to be essential for infants; therefore, infants require nine essential amino acids (the remaining eight are essential). These amino acids are all crucial and must be obtained through food; these are called essential amino acids. Furthermore, the human body's ability to synthesize arginine and histidine is insufficient to meet its needs, requiring some to be obtained from food; these are called semi-essential amino acids. The remaining ten amino acids can be synthesized by the human body and do not require dietary supplementation; these are called non-essential amino acids. Based on this principle, the amino acids involved in this invention are nine in total.

[0036] In formula (I), m1 (i.e., amino acid calcium) includes component (1), which represents essential amino acids for the human body. Its R change is essential amino acids, which refer to amino acids that the human body cannot synthesize or whose synthesis rate is far from meeting the body's needs and must be supplied by dietary protein. These amino acids are called essential amino acids. There are 9 in total: (1) Lysine (2) Tryptophan (3) Phenylalanine (4) Methionine (5) Threonine (6) Isoleucine (7) Leucine (8) Valine (9) Histidine

[0037] In formula (I), m1 also includes component (2): an organic calcium compound. Unlike inorganic calcium compounds such as calcium phosphate, calcium silicate, and calcium carbonate, which are difficult to be completely absorbed and degraded by bone tissue, organic calcium compounds mainly consist of calcium gluconate and calcium compounds using small-molecule organic acids, such as calcium levulinate (calcium fructate). Molecular weight 270.29; calcium malate Molecular weight 172.15; calcium acetate Molecular weight 158.17; calcium propionate Molecular weight: 186.22;

[0038] Among them, m1 represents a quantitative component that needs to be prepared from component (1) and component (2) through a chemical reaction. First, the essential amino acids for the human body are... It dissolves in deionized water to form a solution with a concentration of 10-60 wt%, preferably 20-50 wt%, and the amino acid can be one or more of the essential amino acids for the human body. Then, an organic calcium compound is added, mainly calcium gluconate and small molecule organic acid calcium, such as calcium levulinate, calcium malate, calcium acetate, calcium propionate, etc.

[0039] Furthermore, the mixture is dispersed or stirred using ultrasound at a speed greater than 120 rpm. This ensures that the added organic calcium compound reacts fully with essential amino acids. The pH is adjusted using sodium hydroxide and calcium hydroxide solutions, with a final pH of 7.0-9.0, preferably 7.5-8.5. The reaction slurry is then filtered and washed 5-6 times with deionized water. The resulting powder is then vacuum-dried at 80°C for 8 hours.

[0040] The reaction is illustrated as follows:

[0041]

[0042] This reaction is the process of forming essential amino acids (calcium salts) for the human body. Strict stoichiometry is not required because both the selected essential amino acids and organic calcium sources can provide osteogenic elements and nutrients for the formation of new bone in bone tissue. Preferably, the ratio of essential amino acids to organic calcium compounds is 1.5-3:1 (mol), with 1.75-2.25:1 being the most preferred.

[0043] The second step of this invention involves preparing sustainably degradable and absorbable organophosphorus and calcium sources for the components, providing osteogenic components for bone injuries and surgical sites. Since bone formation requires sufficient calcium and phosphorus, this invention selects organophosphorus compounds. It provides osteogenic phosphorus and energy and nutrients to tissues. Y represents an organic active, soluble phosphoric acid energy and information phosphorus source, which provides phosphorus for bone tissue formation and can provide energy and nutrients.

[0044] Component m3 in formula (I) is prepared by the following reaction diagram:

[0045]

[0046] in The optional range is: (1) adenosine-5'-triphosphate disodium salt Molecular weight: 551.14; (2) Potassium adenosine-5'-triphosphate Molecular weight 583.36; (3) Sodium adenosine-5'-bisphosphate Molecular weight: 451.2; (4) Potassium adenosine-5'-bisphosphate Molecular weight: 501.32; (5) Adenosine-3',5'-cyclic sodium monophosphate hydrate Molecular weight 351.19.

[0047] The reaction does not proceed in a completely stoichiometric manner; its calcium source and organic phosphorus can provide osteogenic elements and nutrients for the formation of new bone in bone tissue.

[0048] Specifically, organophosphorus compounds are used under ultrasonic dispersion or stirring conditions (stirring speed greater than 120 r / m). Examples of suitable organic calcium compounds include adenosine-5′-triphosphate disodium salt, adenosine-5′-triphosphate dipotassium salt, adenosine-5′-diphosphate sodium salt, adenosine-5′-diphosphate potassium salt, and adenosine-3′,5′-cyclic monophosphate sodium hydrate. These compounds are completely dissolved in deionized water or pure water, and then an organic calcium compound is added. This organic calcium compound is primarily calcium gluconate and small-molecule organic acid calcium compounds, such as calcium levulinate, calcium malate, calcium acetate, and calcium propionate. The ratio of organic calcium compound to organic phosphorus compound is 0.90-2.0:1 (mol), preferably 1.0-1.5:1. The pH is adjusted using sodium hydroxide or calcium hydroxide solution, with a final pH of 6.5.0-8.5 as the endpoint, preferably 7.0-7.5.

[0049] The reaction slurry was further filtered, washed 1-2 times with deionized water, and the resulting powder was vacuum dried at 80°C for 12 hours.

[0050] The third step of the invention is to select hemihydrate calcium sulfate or anhydrous calcium sulfate as a rapid solidification component, which enables each component to solidify in a relatively short time during the hydration reaction, and has good plasticity and good mechanical strength after plasticization in a short time.

[0051] In formula (I), component m2 represents the rapidly solidifying components: hemihydrate calcium sulfate and anhydrous calcium sulfate.

[0052] Specifically, the obtained components m1, m3 and calcium sulfate (m2) are mixed in a mass ratio of 20-50:10-30:30-50. The powder is then hydrothermally treated at 120-200℃ and 0.1-1.0MPa for 2-8 hours, preferably at 130-160℃ and 0.3-0.6MPa for 3-6 hours, and then allowed to cool naturally to room temperature to obtain a self-coagulating composite bone implant based on essential amino acids.

[0053] The obtained self-coagulating composite bone implant based on essential amino acids was further ball-milled in a dry atmosphere at room temperature for 2-6 hours until its particle size distribution reached less than 200 mesh. Then, it was vacuum-dried at 60°C for 12 hours to obtain the application product of the self-coagulating composite bone implant based on essential amino acids.

[0054] The fourth step of this invention involves mixing and homogenizing a self-coagulating composite bone implant based on essential amino acids with a curing solution in a specific ratio, then filling the mixture into a mold to create a test sample or shaping it into the desired form. The curing solution can be (1) water for injection, (2) deionized water, (3) physiological saline, or (4) a mixture of the above. The solid-liquid ratio is (g:ml) 100:30-60.

[0055] Furthermore, the self-curing composite bone implant based on essential amino acids is thoroughly mixed with a curing liquid to form a paste. After continuing to mix until it reaches a dough-like consistency, it is implanted into the wound or defect site using a syringe or mold to create the desired shape. The curing time is 5-45 minutes, with 15-30 minutes generally considered the optimal surgical time. Alternatively, it can be pre-formed into granules of different diameters (φ2-10mm) or molded into blocks and cylinders (size determined based on actual needs, such as φ3*10mm, 20*10*5mm) for use as filler during surgery.

[0056] The fifth step of the present invention involves shaping, degrading, evaluating the mechanical and biological properties of the self-coagulating composite bone implant containing essential amino acids for the human body, and obtaining characteristics such as safety and non-toxicity, complete degradation, high activity, and easy shaping. It can promote bone tissue repair and reconstruction during the bone tissue repair and reconstruction process and be completely replaced by bone tissue, etc. in vitro.

[0057] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0058] Example 1

[0059] (1) Preparation of component m1: In a 2L three-necked flask, add 73g of L-lysine, 75g of L-methionine, and 83g of L-phenylalanine. Then, under nitrogen protection, add 1000ml of deionized water while stirring. Stir until completely dissolved, and then add calcium levulinate. 205g, adjust the pH to 7.5 with calcium hydroxide solution, stir for 3 hours, and measure the pH of the supernatant to be 7.5.

[0060] The product was then filtered to obtain a white solid product. The powder was dried under vacuum at 80°C for 8 hours to obtain 258g of white product (m1).

[0061] (2) Preparation of component m3: In a 2L beaker, add 1000ml of deionized water and add adenosine-5'-triphosphate disodium salt under ultrasonic dispersion. 138g, add calcium levulinate after it is completely dissolved. 90g of the solution was adjusted to pH 7.0 using a saturated calcium hydroxide solution. The reaction slurry was filtered through a No. 4 glass frit funnel, and the resulting white powder was vacuum dried at 80°C for 12 hours. 135g of white powder product (m³) was obtained.

[0062] (3) Preparation of three-component composite product: Weigh 35g of component m1 (human essential amino acid calcium) obtained in step (1), 25g of component m3 (organic calcium phosphorus) obtained in step (2), and 45g of calcium sulfate dihydrate (equivalent to 40g of calcium sulfate hemihydrate) and mix them. The powder is hydrothermally treated at 145℃ and 0.40MPa for 6 hours and then naturally cooled to room temperature to obtain a self-coagulating composite bone implant based on human essential amino acids.

[0063] (4) The self-coagulating composite bone implant based on essential amino acids obtained in step (3) is further ball-milled until its particle size distribution reaches less than 200 mesh, and then vacuum-dried at 80°C for 12 hours to form the final product.

[0064] The experimental and testing methods used in the examples of this invention are as follows:

[0065] (1) Sample preparation: Take 20g of the product, add 10ml of water for injection, stir for 2 minutes, press into a porous polytetrafluoroethylene mold with a pore diameter of 6mm and a height of 12mm, eject after 5 minutes, and allow to dry naturally in an environment with a temperature of 25℃ and a humidity of 60%. Determine its curing time (the curing time of the sample is determined according to ISO 9597-2008 standard using a Vicat apparatus); start the calculation from the time the curing liquid is added to the sample powder, and end when the final setting needle (0.5mm) of the Vicat apparatus can no longer leave a mark on the sample surface. The time calculated in this way is the final setting time of the bone cement; each sample should be measured at least 5 times.

[0066] (2) Compression test method: Loading speed 1 mm / min:

[0067] Test the compressive strength after one day (1D), three days (3D), and five days (5D): After the sample (diameter 6mm, height 12mm) has been cured in air for 3 days, the mechanical strength of the sample is measured using a universal mechanical testing instrument. Each sample is measured at least 5 times.

[0068] (3) Degradation test method: The injection-molded material was subjected to degradation test in PBS solution. The PBS preparation method is as follows: accurately weigh KH2PO4 (0.544g), Na2HPO4·12H2O (7.16g), NaCl (16g) and KCl (0.402g) and dissolve them in 2L of deionized water, and make up to volume with a volumetric flask. The pH measurement range is 7.2-7.4. Put the sample into a centrifuge tube and add a certain amount of PBS solution. The volume ratio of PBS solution to sample mass is 1g / 30mL. Then place the centrifuge tube containing the sample in a constant temperature shaking incubator at 37℃ and 80rpm / min. Take out the sample at 1 (1D), 1w (7D), 4w, 8w, 12w and 24w, weigh it and calculate the weight loss rate. Three control groups were set up for each sample.

[0069] (4) pH measurement: pH meter was used to measure the supernatant of the sample. PBS was replaced every 7 days.

[0070] (5) Cytotoxicity and cell proliferation rate test: The extract was prepared in accordance with the provisions of T16886 concerning biological materials, and the proliferation rate was calculated by comparing the standard extract with the blank.

[0071] Test results:

[0072] Setting time: 30 min. Compressive strength: 1D: 15 MPa; 3D: 24 MPa; 5D: 32 MPa. Degradation results: 1D: 12.5%; 1w: 20.6%; 4w: 35.5%; 8w: 56.2%; 12w: 80.5%; 24w: 99.5%.

[0073] pH changes: 1 day: 7.05, 1 week: 6.96; 4 weeks: 7.20, 8 weeks: 7.35; 12 weeks: 7.30; 24 weeks: 7.35. Cytotoxicity and cell proliferation rate: Grade 0;

[0074] Compared with the blank, the standard extract had a proliferation rate of 120% and a proliferation rate of 135% after being diluted by half. Compared with the blank, i.e. the culture medium, the self-coagulating composite bone implant extract containing essential amino acids can provide more nutrition, and promote cell differentiation and proliferation.

[0075] Example 2 (different from Example 1 in terms of amino acid types, but both are essential amino acids for the human body)

[0076] (1) Preparation of component m1: In a 2L three-necked flask, add 73g of L-lysine, 75g of L-methionine, 83g of L-phenylalanine and 65.6g of leucine; then add 1000ml of deionized water while stirring under nitrogen protection, stir until completely dissolved, then add 270g of calcium levulinate (molecular weight: 270.29), adjust the pH to 7.5 with calcium hydroxide solution, stir for 3 hours, and measure the pH of the supernatant to be 7.5.

[0077] The product was then filtered to obtain a white solid product. The powder was dried under vacuum at 80°C for 8 hours to obtain 330 g (ml) of white product.

[0078] (2) Preparation of component m3: exactly the same as in Example 1. Steps (3) to (4) are exactly the same as in Example 1.

[0079] Test results:

[0080] Setting time: 40 min. Compressive strength: 1D: 9 MPa; 3D: 21 MPa; 5D: 33 MPa. Degradation results: 1D: 12.3%; 1w: 21.2%; 4w: 32.5%; 8w: 51.2%; 12w: 82.3%; 24w: 100%.

[0081] pH changes: 1D: 7.0-2.9; 1w: 6.90; 4w: 7.21, 8w: 7.15; 12w: 7.32; 24w: 7.30. Cytotoxicity and cell proliferation: Grade 0; the standard extract showed a proliferation rate of 128% compared to the blank, and 135% after a 1-fold dilution.

[0082] Example 3 (different from Example 1 in terms of amino acid types)

[0083] (1) Preparation of component m1: In a 2L three-necked flask, add 73g of L-lysine, 75g of L-methionine, 83g of L-phenylalanine, 65.6g of leucine and 58.5g of valine. Then, under nitrogen protection, add 1000ml of deionized water while stirring. Stir until completely dissolved, then add 337g of calcium levulinate. Adjust the pH to 7.5 using calcium hydroxide solution. Stir for 3 hours and measure the pH of the supernatant to be 7.5.

[0084] The product was then filtered to obtain a white solid product. The powder was dried under vacuum at 80°C for 8 hours to obtain 400 g (ml) of white product.

[0085] (2) Preparation of component m3: exactly the same as in Example 1. Steps (3) to (4) are exactly the same as in Example 1.

[0086] Test results:

[0087] Setting time: 38 min. Compressive strength: 1D: 11 MPa; 3D: 23 MPa; 5D: 36 MPa. Degradation results: 1D: 11.9%; 1w: 20.5%; 4w: 31.6%; 8w: 50.5%; 12w: 80.6%; 24w: 98.9%.

[0088] pH changes: 1D: 6.9-8.9; 1w: 6.9; 4w: 7.25, 8w: 7.21; 12w: 7.31; 24w: 7.43. Cytotoxicity and cell proliferation rate: Grade 0; the standard extract showed a proliferation rate of 118% compared to the blank, and 136% after a 1-fold dilution.

[0089] Example 4 (same as Example 1, except the organic calcium compound is different from that in Example 1, it is calcium propionate).

[0090] (1) Preparation of component m1: In a 2L three-necked flask, add 73g of L-lysine, 75g of L-methionine and 83g of L-phenylalanine. Then, under nitrogen protection, add 1000ml of deionized water while stirring. Stir until completely dissolved, then add 140g of calcium propionate. Adjust the pH to 7.5 using calcium hydroxide solution. Stir for 3 hours and measure the pH of the supernatant to be 7.5.

[0091] The product was then filtered to obtain a white solid product. The powder was dried under vacuum at 80°C for 8 hours to obtain 255 g (ml) of white product.

[0092] (2) Preparation of component m3: In a 2L beaker, 1000ml of deionized water was added. Under ultrasonic dispersion, 138g of adenosine-5'-triphosphate disodium salt was added. After complete dissolution, 82g of calcium propionate was added, and the pH was adjusted to 7.0 using a saturated calcium hydroxide solution. The reaction slurry was filtered through a No. 4 glass frosted funnel, and the resulting white powder was vacuum dried at 80℃ for 12 hours. 127g of white powder product m3 was obtained.

[0093] Steps (3) to (4) are exactly the same as in Example 1.

[0094] Test results:

[0095] Setting time: 39 min. Compressive strength: 1D: 8 MPa; 3D: 18 MPa; 5D: 29 MPa. Degradation results: 1D: 15.1%; 1w: 23.4%; 4w: 28.7%; 8w: 39.5%; 12w: 82.9%; 24w: 100%.

[0096] pH changes: 1 day: 6.9; 1 week: 7.10; 4 weeks: 7.31; 8 weeks: 7.25; 12 weeks: 7.38; 24 weeks: 7.35. Cytotoxicity and cell proliferation rate: Grade 0;

[0097] Compared with the blank, the standard extract had a proliferation rate of 120% and a proliferation rate of 145% after being diluted by half. Compared with the blank, i.e. the culture medium, the self-coagulating composite bone implant extract containing essential amino acids can provide more nutrition, and promote cell differentiation and proliferation.

[0098] Example 5 (same as Example 1, except the organophosphorus compound differs from that in Example 1; it is sodium adenosine-5'-bisphosphate). )

[0099] (1) Preparation of component m1: In a 2L three-necked flask, add 75g of L-lysine, 75g of L-methionine and 83g of L-phenylalanine. Then, under nitrogen protection, add 1000ml of deionized water while stirring. Stir until completely dissolved, then add 205g of calcium levulinate. Adjust the pH to 7.5 using calcium hydroxide solution. Stir for 3 hours and measure the pH of the supernatant to be 7.5.

[0100] The product was then filtered to obtain a white solid product. The powder was dried under vacuum at 80°C for 8 hours to obtain 258 g (ml) of white product.

[0101] (2) Preparation of component m3: In a 2L beaker, 1000ml of deionized water was added. Under ultrasonic dispersion, 90g of sodium adenosine-5'-bisphosphate was added. After complete dissolution, 90g of calcium levulinate was added, and the pH was adjusted to 7.0 using a saturated calcium hydroxide solution. The reaction slurry was filtered through a No. 4 glass frosted funnel, and the resulting white powder was vacuum dried at 80℃ for 12 hours. 89g of white powder product m3 was obtained.

[0102] Steps (3) to (4) are exactly the same as in Example 1.

[0103] Test results:

[0104] Setting time: 35 min. Compressive strength: 1D: 9 MPa; 3D: 21 MPa; 5D: 30 MPa. Degradation results: 1D: 13.5%; 1w: 22.1%; 4w: 29.9%; 8w: 41.5%; 12w: 83.4%; 24w: 100%.

[0105] pH changes: 1 day: 6.95; 1 ​​week: 7.15; 4 weeks: 7.30; 8 weeks: 7.28; 12 weeks: 7.35; 24 weeks: 7.30. Cytotoxicity and cell proliferation rate: Grade 0.

[0106] The standard extract showed a proliferation rate of 125% compared to the blank, and a proliferation rate of 139% after being diluted by one-fold.

[0107] Example 6 (same as Example 5, except the organophosphorus compound differs from that in Examples 1 and 5; it is adenosine-3',5'-cyclic sodium monophosphate hydrate). (Molecular weight 351.19).

[0108] Preparation of component m3: In a 2L beaker, 1000ml of deionized water was added. Under ultrasonic dispersion, 88g of adenosine-3',5'-cyclic sodium monophosphate hydrate (molecular weight: 351.19) was added. After complete dissolution, 90g of calcium levulinate (molecular weight: 270.29) was added, and the pH was adjusted to 7.0 using a saturated calcium hydroxide solution. The reaction slurry was filtered through a No. 4 glass frit funnel, and the resulting white powder was vacuum dried at 80℃ for 12 hours to obtain 70g (m3) of white product.

[0109] Test results:

[0110] Setting time: 36 min. Compressive strength: 1D: 11 MPa; 3D: 26 MPa; 5D: 38 MPa. Degradation results: 1D: 12.5%; 1w: 23.6%; 4w: 33.2%; 8w: 55.0%; 12w: 81.1%; 24w: 100%.

[0111] pH changes: 1D: 6.89; 1w: 6.95; 4w: 7.21, 8w: 7.18; 12w: 7.22; 24w: 7.26. Cytotoxicity and cell proliferation: Grade 0; the standard extract showed a proliferation rate of 132% compared to the blank, and 151% after a 1-fold dilution.

[0112] Example 7

[0113] Similar to Example 1, the content of calcium sulfate hemihydrate in m2 is different, being 35g.

[0114] Test results:

[0115] Setting time: 45 min. Compressive strength: 1D: 8 MPa; 3D: 18 MPa; 5D: 25 MPa. Degradation results: 1D: 11.1%; 1w: 19.5%; 4w: 29.6%; 8w: 49.0%; 12w: 80.5%; 24w: 98.5%.

[0116] pH changes: 1 day: 7.15; 1 week: 7.12; 4 weeks: 7.20; 8 weeks: 7.10; 12 weeks: 7.30; 24 weeks: 7.25. Cytotoxicity and cell proliferation rate: Grade 0; the standard extract showed a proliferation rate of 125% compared to the blank, and 138% after a 1-fold dilution.

[0117] Example 8

[0118] Similar to Example 5, but with a different content of calcium sulfate hemihydrate in m2: 45g.

[0119] Test results:

[0120] Setting time: 30 min. Compressive strength: 1D: 15 MPa; 3D: 28 MPa; 5D: 39 MPa. Degradation results: 1D: 11.5%; 1w: 22.3%; 4w: 29.8%; 8w: 51.2%; 12w: 88.3%; 24w: 100%.

[0121] pH changes: 1D: 6.72; 1w: 6.85; 4w: 7.10, 8w: 7.21; 12w: 7.23; 24w: 7.30. Cytotoxicity and cell proliferation: Grade 0; the standard extract showed a proliferation rate of 110% compared to the blank, and 130% after a 1-fold dilution.

[0122] Example 9

[0123] Similar to Example 1, but with a different curing solution: take 20g of the product and add 12ml of water for injection.

[0124] Test results:

[0125] Setting time: 45 min. Compressive strength: 1D: 7 MPa; 3D: 16 MPa; 5D: 24 MPa.

[0126] Degradation results: 1D: 13.9%; 1w: 26.5%; 4w: 38.9%; 8w: 53.4%; 12w: 85.1%; 24w: 100%.

[0127] pH changes: 1D: 7.0-1.9; 1w: 6.95; 4w: 7.26, 8w: 7.31; 12w: 7.25; 24w: 7.33.

[0128] Cytotoxicity and cell proliferation rate: Grade 0; the standard extract showed a proliferation rate of 122% compared to the blank, and 136% after a 1-fold dilution.

[0129] Example 10:

[0130] Similar to Example 1, but with a different curing solution ratio: take 20g of the product and add 8ml of water for injection.

[0131] Test results:

[0132] Setting time: 32 min. Compressive strength: 1D: 13 MPa; 3D: 26 MPa; 5D: 33 MPa. Degradation results: 1D: 9.5%; 1w: 16.4%; 4w (7D): 24.9%; 8w (14D): 35.1%; 12w: 67.7%; 24w: 95.5%.

[0133] pH changes: 1 day: 7.01; 1 week: 7.17; 4 weeks: 7.20; 8 weeks: 7.22; 12 weeks: 7.33; 24 weeks: 7.29. Cytotoxicity and cell proliferation: Grade 0; the standard extract showed a proliferation rate of 110% compared to the blank, and 125% after a 1-fold dilution.

[0134] Comparative Example 1: Individual amino acids

[0135] Weigh 20g of the three types of amino acids from Example 1 according to the specified ratio, add 7ml of water for injection, stir for 2 minutes to form a transparent slurry, fill a syringe with an inner diameter of 5mm, and squeeze it out through the syringe. Other operations are the same as in Example 1.

[0136] Results: Dissolved in PBS within 20 minutes, pH: 6.8. Highly acidic and prone to disintegration, it cannot be used alone as a bone repair material.

[0137] Comparative Example 2: Individual

[0138] Calcium levulinate was selected. Add 20g of the solution to 10ml of water for injection, stir for 2 minutes to form a transparent paste, and fill the paste into a syringe with an inner diameter of 5mm. Expel the paste through the syringe. Other procedures are the same as in Example 1.

[0139] Results: The material disintegrated in PBS within 20 minutes at pH 6.8. It disintegrated and cannot be used alone as a bone repair material.

[0140] Comparative Example 3: Individual

[0141] Weigh out sodium adenosine-5'-bisphosphate. Add 20g of the solution to 10ml of water for injection, stir for 2 minutes to form a transparent paste, fill a syringe with a 5mm inner diameter outlet, and squeeze it out through the syringe. Other procedures are the same as in Example 1.

[0142] Results: The material disintegrated in PBS within 20 minutes at pH 6.8. It disintegrated and cannot be used alone as a bone repair material.

[0143] Comparative Example 4

[0144] Weigh 20g of calcium sulfate hemihydrate alone, add 10ml of water for injection, stir for 2 minutes to form a transparent paste, fill a syringe with an inner diameter of 5mm, and squeeze it out through the syringe. Other operations are the same as in Example 1.

[0145] Test results:

[0146] Setting time: 5 min. Compressive strength: 1D: 12 MPa; 3D: 25 MPa; 5D: 40 MPa. Degradation results: 1D: 20.5%; 1w: 35.1%; 4w: 80.2%; 4w: 100%.

[0147] pH changes: 1 day: 4.51; 1 week: 5.15; 4 weeks: 5.40; 6 weeks: 6.50. Cytotoxicity and cell proliferation rate: Grade 1;

[0148] The standard extract showed a 60% proliferation rate compared to the blank, and an 80% proliferation rate after being diluted one-fold.

[0149] The results showed that the prepared material was too acidic and degraded too quickly.

[0150] Comparative Example 5

[0151] Same as Example 1, except that component m2 is removed and calcium sulfate hemihydrate is not included. Weigh 20g, add 10ml of water for injection, stir for 2 minutes, fill into a syringe with an inner diameter of 5mm, and squeeze out through the syringe.

[0152] Result: The system collapsed after 12 hours, making further testing impossible.

[0153] Comparative Example 6

[0154] Same as Example 1, except that component m3 is removed and it does not contain organophosphorus compounds.

[0155] Test results:

[0156] Setting time: 35 min. Compressive strength: 1D: 10 MPa; 3D: 21 MPa; 5D: 32 MPa. Degradation results: 1D: 15.3%; 1w: 30.6%; 4w: 38.2%; 8w: 62.0%; 12w: 92.1%; 24w: 100%.

[0157] pH changes: 1D: 6.95; 1w: 7; 12w: 7.20; 8w: 7.22; 12w: 7.29; 24w: 7.26. Cytotoxicity and cell proliferation rate: Grade 0; the standard extract showed a 90% proliferation rate compared to the blank, and a 108% proliferation rate after a one-fold dilution.

[0158] The results showed that cell proliferation was poor in the absence of phosphorus.

[0159] Comparative Example 7: Inorganic calcium and phosphorus comparison, using hydroxyapatite

[0160] Weigh 20g of hydroxyapatite and add 10ml of water for injection. Stir for 2 minutes to form a slurry. Fill the slurry with a syringe with an inner diameter of 5mm and squeeze it out through the syringe.

[0161] It disintegrates in water after 5 hours, and upon drying and weighing, shows no weight loss and is not degradable. After 1 day, upon drying and weighing, there is no weight loss and it is not degradable. After 7 days, upon drying and weighing, the weight increases by 2.50%, and it is not degradable. It cannot be degraded or absorbed.

[0162] It should be noted that while the preferred embodiments of the present invention are given in the specification and accompanying drawings, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. These embodiments are not intended to impose additional limitations on the content of the present invention; their purpose is to provide a more thorough and comprehensive understanding of the disclosure of the present invention. Furthermore, the above-described technical features can be combined with each other to form various embodiments not listed above, all of which are considered to be within the scope of the present invention specification. Moreover, those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A self-coagulating composite bone implant based on essential amino acids for the human body, characterized in that, The product comprises 20-50 parts by weight of amino acid calcium, 10-30 parts by weight of organic calcium phosphate, and 30-50 parts by weight of calcium sulfate, wherein the amino acid is an essential amino acid for the human body; wherein the amino acid calcium is obtained by reacting an essential amino acid with an organic calcium compound under suitable conditions for the formation of amino acid calcium, and the essential amino acid is selected from one or more of lysine, tryptophan, phenylalanine, methionine, threonine, isoleucine, leucine, valine, and histidine; the organic calcium phosphate is obtained by reacting an organic phosphorus compound with an organic calcium compound under suitable conditions for the formation of organic calcium phosphate, and the organic phosphorus compound is selected from one or more of adenosine-5'-disodium triphosphate, adenosine-5'-dipotassium triphosphate, adenosine-5'-diphosphate sodium salt, adenosine-5'-diphosphate potassium salt, and adenosine-3',5'-cyclic monophosphate sodium hydrate; the organic calcium compound includes calcium gluconate or small molecule organic acid calcium.

2. The self-coagulating composite bone implant according to claim 1, characterized in that, The small molecule organic acid calcium includes calcium levulinate, calcium malate, calcium acetate, and calcium propionate.

3. The self-coagulating composite bone implant according to claim 1, characterized in that, The calcium sulfate is selected from anhydrous calcium sulfate or hemihydrate calcium sulfate.

4. A method for preparing a self-coagulating composite bone implant based on essential amino acids as described in any one of claims 1-3, characterized in that, Includes the following steps: (1) The essential amino acids and organic calcium compounds are reacted with ultrasonic dispersion and / or stirring at a pH of 7.0-9.0, the precipitate is obtained by filtration, washing and drying to obtain the amino acid calcium. (2) The organophosphorus compound is reacted with the organocalcium compound under ultrasonic dispersion and / or stirring at a pH of 6.5-8.

5. The precipitate is filtered, washed, and dried to obtain the organocalcium phosphorus. (3) Mix 20-50 parts by weight of the amino acid calcium from step (1) and 10-30 parts by weight of the organic calcium phosphorus from step (2) with 30-50 parts by weight of calcium sulfate, and perform hydrothermal treatment at 120-200℃ and 0.1-1.0MPa for 2-8 hours. After cooling, a self-coagulating composite bone implant based on essential amino acids for the human body is obtained.

5. The method according to claim 4, characterized in that, The method further includes pulverizing the self-coagulating composite bone implant based on essential amino acids obtained in step (3) to obtain a product with a target particle size.

6. A bone cement, characterized in that, The self-curing composite bone implant based on essential amino acids for human use and the curing solution as described in any one of claims 1-3, comprising a solid-liquid ratio of 100 g: 30-60 ml.

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