A composition containing a calcium salt and a method for its preparation

Abstract

The present application relates to a kind of calcium-containing salt composition and its preparation method, belong to the field of bioengineering technology.The composition of the present application has good mechanical properties, and has broad application prospect.

Description

Technical Field

[0001] This invention relates to a calcium salt-containing composition and its preparation method, belonging to the field of bioengineering. Background Technology

[0002] Cosmetic procedures that use drugs, surgery, medical devices, and other invasive or irreversible medical techniques to repair and reshape a person's appearance and the shape of various parts of the body have become efficient and widely accepted cosmetic methods in today's fast-paced life. Injecting absorbable biological materials (fillers) into a certain part of the body to improve and modify skin wrinkles, soft tissue defects, and contours, thereby improving local or overall shape, has the advantages of convenient operation, delaying aging, obvious cosmetic effects, and short recovery period, and has been widely used in clinical practice.

[0003] Hyaluronic acid and collagen are both major components of the extracellular matrix in the human body. Hyaluronic acid is a natural polysaccharide composed of the following repeating units: N Acetyl-D-glucosamine and D-glucuronic acid. Hyaluronic acid binds and retains moisture, playing a supporting role in skin hydration. Collagen, accounting for one-third of human protein, controls cell growth and differentiation. Both hyaluronic acid and collagen are biodegradable and have good biocompatibility, making them suitable as primary biomaterials in medical tissue engineering for applications such as artificial skin, artificial blood vessels, and filler repair. Calcium plays a crucial role in regulating the normal division and differentiation of epidermal cells, sebum synthesis, and skin barrier function regeneration. Calcium salts in the body are lost more rapidly with age, exacerbating skin aging. Supplementing the skin with calcium can be extremely beneficial for repairing damaged skin barriers and alleviating signs of aging such as dryness and fine lines. However, excessively high calcium ion concentrations can damage nerves and cause muscle spasms.

[0004] In applications, to fully and comprehensively leverage the effects of various filler materials in improving skin aging, there is a desire for filler materials to possess both good injectability and the ability to function as scaffolds in vivo, controlling cell growth and differentiation as well as providing support. However, hyaluronic acid and collagen have limited structures and functions. Hydrogels made with hyaluronic acid or collagen as active components have limited functions, weak mechanical properties, and rapid degradation, thus limiting their applications. Therefore, multi-component composites have higher application value and more potential applications. Currently, commonly used materials / formulations in clinical practice include BDDE-crosslinked hyaluronic acid, glutaraldehyde-crosslinked collagen, hydroxyapatite microspheres, or their composites. These composites containing crosslinking agents require strict control of crosslinking agent residues; otherwise, safety accidents or adverse reactions can easily occur, and production risks are also increased. Simply mixing multiple filler materials makes it difficult to fully utilize the effects of each component, let alone achieve synergistic effects.

[0005] Therefore, developing a safer, more effective, injectable, and convenient calcium salt composition with multiple functions such as controlling cell growth and differentiation and providing filling support is a clinical need and expectation. Summary of the Invention

[0006] Based on the existing problems and needs, the present invention provides a calcium salt-containing composition, which has excellent injectability, can undergo a phase transition in vivo to become a gel, and has multiple functions such as nutrition, control of cell growth and differentiation, and filling and support.

[0007] According to the present invention, a calcium salt composition comprises or its preparation raw materials include: collagen, hyaluronic acid or hyaluronic acid salt, hydroxyapatite microparticles, calcium lactate, inorganic salts and water.

[0008] According to the present invention, the mass ratio of collagen to hyaluronic acid or hyaluronic acid salt in the composition or its preparation raw materials can be (1:0.2)-(1:4). Within this ratio range, it is beneficial for the composition to fully exert the effects of each component and further enhance the effects of each component, such as increasing the mechanical properties of the composition, and also for the composition to be injected, stabilized, and prepared. In some embodiments, the mass ratio of collagen to hyaluronic acid or hyaluronic acid salt in the composition or its preparation raw materials is 1:0.5 or 1:1. In some embodiments, the mass ratio of collagen to hyaluronic acid or hyaluronic acid salt in the composition or its preparation raw materials is 1:2 or 3:4, which is beneficial for obtaining a composition with high mechanical properties and suitable for use. The above-mentioned mass ratio of collagen to hyaluronic acid or hyaluronic acid salt in the composition or its preparation raw materials is beneficial for obtaining a composition with high mechanical properties and better opacity. In the composition, hyaluronic acid or hyaluronic acid salt needs to be controlled within a suitable range. Too much or too little will make it difficult to obtain a composition with high elastic modulus and make the composition difficult to prepare. It will also be difficult to obtain a composition that is in a gel state at 35°C to 38°C. The ratio described above is beneficial to obtaining a composition with high elastic modulus and capable of phase change, which is beneficial to improving the performance of the composition.

[0009] The molecular weight of the hyaluronic acid or hyaluronic acid salt can be between 1.2 million and 3 million Daltons. Hyaluronic acid or hyaluronic acid salts within this molecular weight range contribute to the filling and supporting properties of the composition, resistance to enzymatic hydrolysis, and ease of composition preparation. In some embodiments, the molecular weight of the hyaluronic acid or hyaluronic acid salt is between 1.5 million and 2.8 million Daltons. In some embodiments, the molecular weight of the hyaluronic acid or hyaluronic acid salt is between 2 million and 2.6 million Daltons. In some embodiments, the molecular weight of the hyaluronic acid or hyaluronic acid salt is between 2.6 million and 2.8 million Daltons. In some embodiments, the molecular weight of the hyaluronic acid or hyaluronic acid salt is 2.2 million, 2.3 million, 2.4 million, or 2.5 million Daltons. If the molecular weight of hyaluronic acid or hyaluronic acid salt is too low, it will degrade faster in the human body and maintain its effect for a shorter time; if the molecular weight is too high, it will be difficult to stir and dissolve, and difficult to prepare a suitable solution or compound solution. Hyaluronic acid or hyaluronic acid salt in the above molecular weight range can make the composition have a longer degradation time and is suitable and easy to prepare into a suitable solution, so that the composition has suitable degradation performance and rheological properties.

[0010] In the composition, the concentration of hyaluronic acid or hyaluronic acid salt can be 6 mg / mL to 20 mg / mL. This concentration of hyaluronic acid or hyaluronic acid salt is beneficial for the uniform dispersion of microparticles and for the suitable viscoelasticity and ease of extrusion of the composition. In some embodiments, the concentration of hyaluronic acid or hyaluronic acid salt in the composition is 10 mg / mL to 20 mg / mL. In some embodiments, the concentration of hyaluronic acid or hyaluronic acid salt in the composition is 10 mg / mL, 15 mg / mL, or 18 mg / mL.

[0011] In some embodiments, the concentration of hyaluronic acid or hyaluronic acid salt in the composition is 6 mg / mL to 30 mg / mL, and the molecular weight is 1.2 million Daltons to 2.8 million Daltons. In some embodiments, the concentration of hyaluronic acid or hyaluronic acid salt in the composition is 10 mg / mL to 20 mg / mL, and the molecular weight is 1.5 million Daltons to 2.8 million Daltons. In some embodiments, the concentration of hyaluronic acid or hyaluronic acid salt in the composition is 5 mg / mL to 10 mg / mL, and the molecular weight is 1.5 million Daltons to 2.6 million Daltons. In some embodiments, the concentration of hyaluronic acid or hyaluronic acid salt in the composition is 10 mg / mL or 20 mg / mL, and the molecular weight is 2 million Daltons, 2.2 million Daltons, or 2.6 million Daltons.

[0012] Different types of collagen can result in mixtures with hyaluronic acid or hyaluronic acid salts that exhibit different properties. According to the present invention, the collagen is extracted collagen, or a mixture of extracted collagen and reconstituted collagen.

[0013] According to the present invention, the collagen is preferably collagen with a triple helix structure, or collagen with at least most of a triple helix structure, or a combination thereof. The collagen may be collagen with a molecular weight greater than 200,000 Daltons, or a combination of collagen with a molecular weight greater than 200,000 Daltons and collagen with a molecular weight less than 100,000 Daltons.

[0014] In some embodiments, based on the total mass of collagen, at least 60% of the collagen is triple-helical collagen or collagen with a molecular weight greater than 200,000 Daltons. In some embodiments, based on the total mass of collagen, at least 70%, 80%, or 90% of the collagen is triple-helical collagen or collagen with a molecular weight greater than 200,000 Daltons. In some embodiments, based on the total mass of collagen, at least 95% of the collagen is triple-helical collagen or collagen with a molecular weight greater than 200,000 Daltons. In some embodiments, based on the total mass of collagen, at least 98% or 99% of the collagen is triple-helical collagen or collagen with a molecular weight greater than 200,000 Daltons.

[0015] In some embodiments, the collagen is preferably extracted from yak collagen, which is more conducive to phase transformation into a gel with higher mechanical properties at 35℃-38℃ compared to bovine collagen extracted from ordinary cattle.

[0016] In some embodiments, the molecular weight of the collagen is not less than 300,000 Daltons. In some embodiments, the molecular weight of the collagen is between 300,000 and 400,000 Daltons.

[0017] In some embodiments, the collagen is a combination of collagen with a molecular weight of not less than 300,000 Daltons and collagen with a molecular weight of less than 50,000 Daltons. In some embodiments, the collagen is a combination of collagen with a molecular weight of not less than 300,000 Daltons and collagen with a molecular weight of less than 10,000 Daltons. The combination of high and low molecular weight collagen, due to the different degradation properties and functions of collagen with different molecular weights, can exert a synergistic effect, which is beneficial for early collagen filling and replenishment, early stimulation of collagen regeneration, and can maintain the stimulation of collagen regeneration for a longer period of time.

[0018] In the composition, the concentration of collagen is not less than 3 mg / mL, and can be between 3 mg / mL and 30 mg / mL. This concentration of collagen is beneficial for the uniform dispersion of microparticles and for the composition to have suitable viscoelasticity, extrusion force and opacity.

[0019] In some embodiments, the concentration of collagen in the composition is 4 mg / mL to 20 mg / mL. In some embodiments, the concentration of collagen in the composition is 5 mg / mL to 15 mg / mL. In some embodiments, the concentration of collagen in the composition is 5 mg / mL to 10 mg / mL. In some embodiments, the concentration of collagen in the composition is 10 mg / mL to 30 mg / mL. In some embodiments, the concentration of collagen in the composition is 10 mg / mL to 20 mg / mL. In some embodiments, the concentration of collagen in the composition is 20 mg / mL to 30 mg / mL. In some embodiments, the concentration of collagen in the composition is 5 mg / mL to 7.5 mg / mL. In some embodiments, the concentration of collagen in the composition is 5 mg / mL, 7.5 mg / mL, 10 mg / mL, 16 mg / mL, 20 mg / mL, 24 mg / mL, or 28 mg / mL.

[0020] In some embodiments, the collagen in the composition is a mixture of collagen with a molecular weight of not less than 300,000 Daltons and collagen with a molecular weight of less than 100,000 Daltons, at a concentration of 4 mg / mL to 30 mg / mL.

[0021] In some embodiments, the concentration of collagen in the composition is 5 mg / mL to 30 mg / mL, and it is collagen with a triple helix structure or collagen with a molecular weight of not less than 300,000 Daltons, which is beneficial to obtaining a composition with better mechanical properties.

[0022] In some embodiments, the collagen concentration in the composition is 5 mg / mL to 30 mg / mL, and based on the total mass of collagen, at least 60% of the collagen is triple-helix collagen or at least 60% of the collagen has a molecular weight of 300,000 to 400,000 Daltons, which is beneficial for obtaining a composition with better mechanical properties. In some embodiments, the collagen concentration in the composition is 5 mg / mL to 15 mg / mL, and based on the total mass of collagen, at least 60% of the collagen is triple-helix collagen or at least 60% of the collagen has a molecular weight of 300,000 to 400,000 Daltons, which is beneficial for more conveniently obtaining a composition with better mechanical properties. In some embodiments, the collagen concentration in the composition is 7.5 mg / mL to 24 mg / mL, and based on the total mass of collagen, at least 60% of the collagen is triple-helix collagen or at least 60% of the collagen has a molecular weight of 300,000 to 400,000 Daltons, which is beneficial for obtaining a composition with high mechanical properties and high opacity. In some embodiments, the collagen concentration in the composition is 5 mg / mL to 7.5 mg / mL, and based on the total mass of collagen, at least 60% of the collagen is triple-helix collagen or at least 60% of the collagen has a molecular weight of 300,000 to 400,000 Daltons, which is beneficial for conveniently obtaining a composition with high mechanical properties.

[0023] The hydroxyapatite microparticles are spiky spherical particles with multiple protrusions or spikes on their surface. This structure gives them a large specific surface area, which is more conducive to stimulating collagen regeneration. The particle size of the hydroxyapatite microparticles can be 20 micrometers to 80 micrometers, preferably 20 micrometers to 50 micrometers.

[0024] The concentration of the hydroxyapatite can be 3 mg / mL to 12 mg / mL. In some embodiments, the concentration of the hydroxyapatite is 5 mg / mL to 10 mg / mL.

[0025] The concentration of calcium lactate can be between 1 mg / mL and 10 mg / mL. At this concentration, calcium lactate is beneficial for both calcium supplementation and safety (excessive calcium ion concentration can lead to cell poisoning). In some embodiments, the concentration of calcium lactate is between 5 mg / mL and 10 mg / mL. In some embodiments, the concentration of calcium lactate is between 1 mg / mL and 5 mg / mL.

[0026] The inorganic salt is at least one selected from disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, and potassium chloride. In some embodiments, the inorganic salt is disodium hydrogen phosphate, sodium dihydrogen phosphate, and sodium chloride. In some embodiments, the inorganic salt is dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and potassium chloride.

[0027] In some embodiments, the composition or its preparation materials may further include a pH adjuster to adjust the pH to a suitable range for use. The pH adjuster may be any one or more of hydrochloric acid, sodium hydroxide, and potassium hydroxide.

[0028] According to the present invention, the pH of the composition can be 6.0-7.6. In some embodiments, the pH is preferably 6.8-7.4, which is more conducive to the injection use of the composition. In some embodiments, the pH is 6.9, 7.0, 7.1 or 7.2, which is more suitable for the stability of the composition and injection use.

[0029] The aforementioned buffer salts and / or pH adjusters can give the composition a suitable osmotic pressure and / or pH value for injection use, thereby facilitating injection use and contributing to composition stability.

[0030] According to the present invention, the osmotic pressure of the composition can be 200 mOsm / L-400 mOsm / L, preferably 250 mOsm / L-350 mOsm / L. In some embodiments, the osmotic pressure of the composition is 270 mOsm / L-330 mOsm / L. In some embodiments, the osmotic pressure of the composition is 300 mOsm / L-330 mOsm / L.

[0031] According to an embodiment of the present invention, the composition has the following characteristics: its elastic modulus is higher than the sum of the elastic moduli of its raw materials collagen and hyaluronic acid or hyaluronic acid salt; the elastic modulus is measured at 0.1 Hz, at the same concentration, and under the same detection method and detection conditions.

[0032] According to embodiments of the present invention, in some implementations, the composition has the following characteristics: it is a flowable suspension at ambient temperatures below 25°C, and can transform into a gel at temperatures between 35°C and 38°C.

[0033] According to embodiments of the present invention, in some embodiments, the composition has the following characteristics: its elastic modulus is higher than the sum of the elastic moduli of its raw materials collagen and hyaluronic acid or hyaluronic acid salt; and the composition is a flowable suspension at ambient temperatures below 25°C and can transform into a gel at 35°C to 38°C.

[0034] In some embodiments, the composition can transform into a gel state at 35°C to 38°C, exhibiting relatively higher viscoelasticity. In some embodiments, the composition is a flowable suspension at ambient temperatures below 34°C and a gel state at 35°C to 38°C.

[0035] According to the present invention, in some embodiments, the composition can continuously and stably transform from a relatively free-flowing suspension to a gel state at 35°C-38°C. In some embodiments, at 37°C, the composition can transform from a suspension to a gel state within 30 minutes. In some embodiments, at 37°C, the composition can transform from a suspension to a gel state within 20 minutes. In some embodiments, at 37°C, the composition can transform from a suspension to a gel state within 15 minutes. In some embodiments, at 37°C, the composition can transform from a suspension to a gel state within 10 minutes. In some embodiments, at 37°C, the composition can transform from a suspension to a gel state within 5 minutes or within 3 minutes. The composition can undergo a phase transition to form a gel state in a short time. The phase transition process is stable, has good mechanical properties, is easy to shape, and is more conducive to medical applications, such as nourishing the skin, resisting aging, filling and shaping, and improving the health or shape of the skin.

[0036] According to the present invention, in some embodiments, after the composition undergoes a phase transition to a gel, both its elastic modulus and viscous modulus increase. At 37°C and 0.1Hz, its elastic modulus or viscous modulus is at least 1.2 times, 1.5 times, 2 times, 2.5 times, 3 times, 4 times, or 5 times that of its pre-phase transition elastic modulus or viscous modulus at 25°C and 0.1Hz. In some embodiments, after the composition undergoes a phase transition to a gel, at 37°C and 0.1Hz, its elastic modulus or viscous modulus is at least 3 times, at least 4 times, or at least 5 times that of its pre-phase transition elastic modulus or viscous modulus at 25°C and 0.1Hz.

[0037] In the composition, as the collagen content increases and / or the amount of hydroxyapatite microparticles increases, the light transmittance decreases and the opacity of the composition increases. Low light transmittance implies high opacity, which is beneficial in reducing or mitigating the Tyndall effect. According to the present invention, the light transmittance of the composition is less than 50% under conditions not exceeding 34°C and 400nm. In some embodiments, the light transmittance of the composition is less than 50%, or less than 30%, or less than 25% under conditions not exceeding 34°C and 250nm-350nm. In some embodiments, the light transmittance of the composition is less than 50% under conditions not exceeding 34°C and 250nm-350nm. In some embodiments, the light transmittance of the composition is less than 40% under conditions not exceeding 34°C and 250nm-350nm. In some embodiments, the light transmittance of the composition is less than 25% under conditions not exceeding 34°C and 250nm-350nm. In some embodiments, the transmittance of the composition is less than 20% or 15% under conditions not exceeding 34°C and 250nm-350nm.

[0038] The composition provided by this invention does not contain cross-linking agents or other components with potential safety risks, exhibiting high safety, good stability, and good biocompatibility. It maintains a stable, flowable suspension state and relatively high viscoelasticity for extended periods at low temperatures. Due to the specific selection and unique compounding method of collagen, hyaluronic acid or hyaluronic acid salts, and calcium-containing substances, the composition exhibits a synergistic effect. Compared to simple mixtures of these substances, the composition demonstrates higher viscoelasticity, better gelling properties, improved resistance to enzyme degradation, and enhanced mechanical properties. This facilitates moisture retention, cell proliferation, diffusion, and migration, making it ideal for filling, shaping, and supporting soft tissues, joint cavities, and other areas requiring these functions.

[0039] The composition provided by this invention, in some embodiments, can undergo a phase transition to form a stable gel with a certain strength under relatively high temperature conditions (e.g., 37°C); therefore, it can be smoothly and conveniently injected into the body at relatively low temperature conditions (e.g., not exceeding 30°C) using a syringe (e.g., a syringe with a 30G needle). After injection into the human body, it forms a solid gel, which can play a filling and supporting role; and compared with a single component, it has higher viscoelasticity, improved resistance to enzyme degradation, mechanical properties, etc., which is beneficial for moisturizing and water retention, cell proliferation, diffusion and migration, and is more conducive to application.

[0040] On the other hand, the present invention also provides a method for preparing the aforementioned composition. A method for preparing the aforementioned composition includes: preparing a solution with pH 6.0-7.6 by dissolving hyaluronic acid or hyaluronic acid salt in an aqueous solution of inorganic salts; then adding no more than half of calcium lactate and stirring until homogeneous; then adding hydroxyapatite and the remaining calcium lactate, stirring and mixing for 0.1-2 hours, and then allowing it to stand at 15°C-25°C for 0.5-8 hours to obtain a composite solution; preparing a solution with pH 6.0-7.6 by dissolving collagen in an aqueous solution of inorganic salts at 0°C-10°C; then mixing the collagen solution with the aforementioned composite solution and stirring until homogeneous. Then place at 15℃-25℃ for 0.5 hours to 6 hours; optionally adjust the pH to 6.0-7.6 using hydrochloric acid, sodium hydroxide, or potassium hydroxide to obtain a suspension; optionally sterilize to obtain the composition; the pH of the aqueous solution of the inorganic salt is 6.0-7.6; in the composition, the concentration of hyaluronic acid or hyaluronic acid salt is 6 mg / mL-20 mg / mL, the concentration of collagen can be 4 mg / mL-30 mg / mL, the concentration of hydroxyapatite is 1 mg / mL-12 mg / mL, and the concentration of calcium lactate is 1 mg / mL-10 mg / mL.

[0041] In some embodiments, the method for preparing the aforementioned composition includes: preparing a solution with pH 6.8-7.6 by dissolving hyaluronic acid or hyaluronic acid salt in an aqueous solution of inorganic salts; then adding a portion of calcium lactate, stirring, dissolving, and mixing evenly; then adding hydroxyapatite and the remaining calcium lactate, stirring and mixing for 0.1-1 hour, and then allowing it to stand at 15-25°C for 1-8 hours to obtain a composite solution; preparing a solution with pH 6.8-7.6 by dissolving collagen in an aqueous solution of inorganic salts at 0-10°C; then mixing the collagen solution with the aforementioned composite solution, stirring and mixing evenly, and then allowing it to stand at 15-25°C for 0.5-6 hours; optionally adjusting the pH to 6.8-7.6 using hydrochloric acid, sodium hydroxide, or potassium hydroxide to obtain a suspension; optionally sterilizing to obtain the composition; wherein the pH of the aqueous solution of inorganic salts is 6.8-7.6.

[0042] In some embodiments, the concentration of hyaluronic acid or hyaluronic acid salt in the composition is 6 mg / mL-15 mg / mL, the concentration of collagen is 5 mg / mL-30 mg / mL, the concentration of hydroxyapatite is 5 mg / mL-12 mg / mL, and the concentration of calcium lactate is 5 mg / mL-10 mg / mL.

[0043] In some embodiments, the concentration of hyaluronic acid or hyaluronic acid salt in the composition is 6 mg / mL-15 mg / mL, the concentration of collagen is 5 mg / mL-20 mg / mL, the concentration of hydroxyapatite is 1 mg / mL-5 mg / mL, and the concentration of calcium lactate is 1 mg / mL-5 mg / mL.

[0044] In some embodiments, the concentration of hyaluronic acid or hyaluronic acid salt in the composition is 6 mg / mL-15 mg / mL, the concentration of collagen is 5 mg / mL-20 mg / mL, the concentration of hydroxyapatite is 5 mg / mL-12 mg / mL, and the concentration of calcium lactate is 5 mg / mL-10 mg / mL.

[0045] In some embodiments, in a method for preparing the aforementioned composition, the molecular weight of hyaluronic acid or hyaluronic acid salt is 1.2 million to 2.8 million Daltons; the molecular weight of collagen is 300,000 to 400,000 Daltons. In some embodiments, in a method for preparing the aforementioned composition, the molecular weight of hyaluronic acid or hyaluronic acid salt is 2 million to 2.6 million Daltons; the collagen is a combination of collagen with a molecular weight of 300,000 to 400,000 Daltons and collagen with a molecular weight of less than 100,000 Daltons, or based on the total mass of the protein, at least 60% of the collagen is collagen with a triple helix structure or with a molecular weight of not less than 300,000 Daltons.

[0046] The composition can be prepared using sterile raw materials / materials under aseptic production conditions, or the raw materials or solutions / composite solutions can be sterilized by irradiation, such as using cobalt-60 at an irradiation dose not exceeding 15 kGy at -70°C to 0°C. In some embodiments, the raw materials or solutions are irradiated with cobalt-60 at an irradiation dose not exceeding 12 kGy at -70°C to -20°C. In some embodiments, the raw materials or solutions are irradiated with cobalt-60 at an irradiation dose of 5 kGy to 8 kGy at -70°C to -20°C. Detailed Implementation

[0047] The terms “comprising” or “including” are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0048] In this invention, "composed of the following components" or "consistently composed of the following components" means that it may also contain unavoidable impurities and / or water.

[0049] In this invention, "optional" means that it may or may not be present.

[0050] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "some implementations," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0051] In this invention, room temperature refers to ambient temperature, which is between 20℃ and 30℃, or between 22℃ and 28℃, or 25℃.

[0052] In this invention, the molecular weight of hyaluronic acid or hyaluronic acid salt is expressed as viscosity-average molecular weight.

[0053] Hyaluronic acid salts are pharmaceutically acceptable salts, including but not limited to one or more of sodium, potassium, calcium, and zinc salts.

[0054] In this invention, a flowable suspension refers to a suspension in which flow deformation is observable to the naked eye when its container is inverted; a gel refers to a composition in which no flow deformation is observed to the naked eye when its container is inverted. In this invention, phase change or gelation refers to a change in the state of the composition from a flowable liquid to a gel. The completion of the phase change or gelation is indicated by the absence of flow deformation when the composition sample is inverted and observed to the naked eye.

[0055] In the composition, the concentration / content of hyaluronic acid is calculated based on the total mass of hyaluronic acid or hyaluronic acid salts. If the sample contains hyaluronic acid, it is calculated as hyaluronic acid; if the sample contains sodium hyaluronate, it is calculated as sodium hyaluronate; if the sample contains both hyaluronic acid and sodium hyaluronate, it is calculated as the total amount of both.

[0056] In the following experiments / examples, the PBS solution was prepared by mixing 0.01 mol / L of disodium hydrogen phosphate and sodium dihydrogen phosphate aqueous solution with 0.8% (mass fraction) sodium chloride, with a pH of 7.2.

[0057] In the following examples, the hydroxyapatite microparticles are spiky spherical microparticles with a particle size of 20-50 micrometers.

[0058] In this invention, the viscoelasticity testing method is as follows: A rheometer is used for testing. A plate with a diameter of 20 mm is selected, and the plate spacing is set to 0.4 mm. The temperature of the unheated system is controlled at 25°C, and the temperature of the system after heating at 37°C is controlled at 37°C. Based on the linear viscoelastic region, a suitable strain is obtained, and a frequency scan of 0.01 Hz to 10 Hz is performed to measure the elastic modulus (G') and viscous modulus (G”). The viscoelasticity of the sample at 25°C and / or the viscoelasticity of the sample at 37°C after heating at 37°C for 1 hour are tested respectively, and the elastic modulus or viscous modulus value at 0.1 Hz is taken.

[0059] The following experiments / examples were conducted using sterile raw materials and materials under sterile production conditions.

[0060] Preliminary Experiment 1:

[0061] Yak collagen with a molecular weight of 300,000 (unsterilized by irradiation) was sterilized at 0°C with a cobalt-60 irradiation dose of 10 kGy to obtain irradiated raw material.

[0062] Under stirring conditions at 4℃, the extracted collagen or recombinant collagen was prepared into a 15 mg / mL solution with PBS solution, and then heated at 37℃ for 1 h. The gelation process was observed, and the viscoelasticity before and after gelation was measured.

[0063] Under stirring conditions at 4℃, yak-derived collagen extracted from yak was prepared into a solution of 19.5 mg / mL using PBS, and recombinant collagen was prepared into a solution of 10.5 mg / mL using PBS. Equal volumes of the solutions were mixed evenly to obtain a mixed solution of 15 mg / mL (mass ratio 65:35). The solution was then heated at 37℃ for 1 hour, and the gelation process was observed. The viscoelasticity before and after gelation was measured.

[0064] The viscoelastic results are shown in the table below.

[0065]

[0066] The results show that recombinant collagen is not conducive to gelation, while yak-derived collagen is relatively easier to gel.

[0067] Preliminary Experiment 2:

[0068] Sodium hyaluronate with a molecular weight of 2.6 million Daltons was prepared into a 20 mg / mL solution using PBS solution at 4°C and with stirring. Recombinant collagen with a molecular weight of 55,000 Daltons was prepared into a 15 mg / mL solution using PBS solution at 4°C and with stirring. 10 mL of sodium hyaluronate solution and 10 mL of recombinant collagen solution were taken and mixed evenly at 4°C with stirring to obtain a mixed solution. The resulting mixed solution was heated at 37°C for 1 hour, but failed to gel.

[0069] Preliminary Experiment 3:

[0070] Sodium hyaluronate was prepared into a solution of a predetermined concentration using PBS solution. Collagen was prepared into a solution of a predetermined concentration using PBS solution under stirring conditions at 4°C. The viscoelasticity of each solution was then measured.

[0071]

[0072] Example 1

[0073] Formula Table A Series:

[0074]

[0075] The raw materials include: sodium hyaluronate with a molecular weight of 2.6 million Da; and collagen (bovine source) with a molecular weight of 300,000 Da and a triple helix structure.

[0076] Formula Table B Series:

[0077]

[0078] The raw materials include: sodium hyaluronate with a molecular weight of 2.6 million Da; and collagen (from yak) with a molecular weight of 300,000 Da and a triple helix structure.

[0079] Preparation method:

[0080] According to the formula, sodium hyaluronate was mixed with PBS solution and stirred to prepare a 20 mg / mL solution; collagen was mixed with PBS solution and stirred at 4°C to prepare solutions of the concentrations set for each formula group; 10 mL each of sodium hyaluronate solution and collagen solution were mixed at 4°C and stirred for 2 hours (it was observed that the collagen concentration increased and the difficulty of stirring and mixing increased), to obtain the composition.

[0081] Formula Table C Series:

[0082]

[0083] The raw materials include: sodium hyaluronate with a molecular weight of 2.6 million Da; and collagen (from yak) with a molecular weight of 300,000 Da and a triple helix structure.

[0084] Formula Table d series:

[0085]

[0086] The ingredients include sodium hyaluronate with a molecular weight of 2 million Da. The collagen concentration in the formula is the total concentration of the two types of collagen. Specifically, from group 1-d to group 3-d, the mass ratio of yak-derived collagen to recombinant collagen is 8:2; from group 4-d to group 6-d, the mass ratio of yak-derived collagen to recombinant collagen is 6:4. The yak-derived collagen has a molecular weight of 300,000 Da and a triple helix structure; the recombinant collagen has a molecular weight of 55,000 Da and no triple helix structure.

[0087] Preparation methods for formulations c and d series:

[0088] According to the formula, sodium hyaluronate and PBS solution were mixed and stirred to prepare a 20 mg / mL solution. 10 mL of sodium hyaluronate solution was taken, and one-quarter of the calcium lactate was added. The mixture was stirred at 200-300 rpm for 1 hour. Hydroxyapatite and the remaining calcium lactate were then added and stirred at 200-300 rpm for 2 hours. The mixture was then allowed to stand at 25°C for 8 hours to obtain a composite solution. Collagen was mixed with PBS solution at 4°C and stirred to prepare solutions of the concentrations specified for each formulation group. 10 mL of collagen solution was then mixed with the aforementioned composite solution and stirred for 1 hour to ensure homogeneity. The mixture was then placed at 25°C for 4 hours to obtain the final composition.

[0089] Example 2: Performance Testing

[0090] 1) Pushing force

[0091] At room temperature, the sample was pushed into the syringe plunger at a constant speed. The syringe needle was 30G and the pushing speed was 30mm / min. The pushing force was measured, and the average pushing force of each formulation group of each series was between 25N and 40N. Among them, the average pushing force of the c series samples was slightly higher than that of other series, but still did not exceed 40N.

[0092] 2) Osmotic pressure

[0093] Samples were taken separately and their osmotic pressure was measured using an osmometer, referring to the method for determination of osmolar concentration in Chinese Pharmacopoeia 2020 edition 0632. The osmotic pressure of each formulation group of series a and series b was found to be between 280 mOsmol / kg and 350 mOsmol / kg.

[0094] 3) Viscoelasticity

[0095] Following the aforementioned method, the viscoelasticity of the samples obtained according to each formulation at 25°C (before gelation) and the viscoelasticity of the samples obtained according to each formulation at 37°C after heating at 37°C for 1 hour at 37°C (after gelation) were tested respectively. The results are shown in the table below.

[0096] result:

[0097]

[0098] Before gelation, samples were inverted and observed to exhibit flow. After gelation, samples 1-a, 1-b, 1-c, and 1-d, when inverted, showed decreased flowability compared to before gelation, but still retained some flowability. Samples 1-c and 1-d exhibited even lower flowability than samples 1-a and 1-b. Samples 2-a and 2-b, when inverted, showed weak flowability (significantly weaker than before gelation). No flowability was observed in the other samples when inverted. The results indicate that the viscoelasticity of samples in each formulation group after gelation was higher than that before gelation. For some samples, the viscoelasticity after gelation was approximately 5 times or more than 5 times that before gelation (the viscoelasticity of samples in series a was approximately 1.6 times that before gelation). Furthermore, the use of yak-derived collagen with a triple helix structure, or a combination of yak collagen and recombinant collagen, resulted in even higher viscoelasticity after gelation, with a greater increase compared to before gelation, which is more conducive to obtaining products with good mechanical properties.

[0099] 4) Collagenase hydrolysis test

[0100] Collagen solution: 10 mg / mL, prepared using PBS buffer, take 0.3 g;

[0101] Weigh 0.3g of each formulation group and soak it in 100mL of 5U / mL collagenase PBS buffer at 37℃. The remaining amount of sample is estimated by measuring the hydroxyproline content in the solution, thereby evaluating the degree of degradation of the sample. The higher the hydroxyproline content in the solution, the less the remaining amount of sample, indicating faster degradation.

[0102] Results: At the same concentration, the B series formulations degraded more slowly than the A series, the C series degraded more slowly than the D series, and the C and D series formulations degraded more slowly than the A and B series. Furthermore, at the same concentration, the calcium salt-containing compositions degraded even more slowly.

[0103] 5) Stability

[0104] Collagen solution: 10 mg / mL, prepared using PBS buffer; Samples: obtained according to the aforementioned series of formulations.

[0105] The sample was stored at 4℃, and the shape of the sample was observed at 0 days and 90 days. Then the sample was placed in a 37℃ water bath, and the gelation time was recorded to test the viscoelasticity.

[0106] Results: Upon initial removal, the sample remained a free-flowing, homogeneous mixture (no stratification or aggregation observed). Upon inversion, significant flow was observed. At 37°C, gelation was observed from 3 to 30 minutes, with higher collagen concentrations resulting in faster gelation. After gelation, inversion significantly reduced flowability or eliminated flow. At the same concentration, the b-series and d-series formulations gelled faster than the a-series, the c-series faster than the d-series, and the d-series faster than the b-series. Viscoelasticity testing (using the aforementioned method) showed that the viscoelastic curve of the same sample essentially overlapped with its value at day 0 (before gelation), indicating minimal change. This demonstrates that the sample provided by this invention possesses good stability and gelation properties.

[0107] 6) Light transmittance

[0108] The samples in the c-series and d-series formulations containing hydroxyapatite microparticles showed visibly weaker light transmittance compared to the samples in the b-series and a-series formulations.

[0109] Sample: Prepared according to the corresponding formula of series b or series a; Reference: Sodium hyaluronate in PBS, 10 mg / mL.

[0110] The transmittance of each sample at 250 nm, 350 nm and 400 nm was measured using a spectrophotometer at 25 °C. The transmittance of the reference standard was set to 100%. The results for the samples are shown in the table below.

[0111]

[0112] According to the transmittance results, the sample provided by this invention can have a certain or good opacity, which is beneficial to reduce the Tyndall effect. In addition, the opacity increases with the increase of collagen concentration. Within a certain concentration range, it can achieve good viscoelasticity, high opacity, and ease of production.

[0113] Example 3: Animal Experiment

[0114] Sample group: obtained from formulation group 4-c of Example 1;

[0115] Control group: Formulation group 4-b obtained from Example 1;

[0116] Control group: physiological saline diluted 1:1;

[0117] Methods: Healthy male ICR mice, 6-8 weeks old and weighing approximately 20 grams, were selected. Temperature (21-25℃), humidity (60±5%), and light exposure (12 / 12h light / dark cycle) were controlled. Free access to standard feed and water was provided. Mice were randomly divided into groups of three. The test sample was subcutaneously injected into the same area on the back of each mouse, with an injection volume of 0.8 mL per injection point and four injection points per mouse. After injection, the mice were fed normally, then euthanized by cervical dislocation. Skin sections from the same area on the back were quickly collected and stained with hematoxylin and eosin (HE) and eosin (MASSON). Observation and data processing were performed using NDP.VIEW. All reagents used in the experiment were aseptically prepared, and all instruments were pre-sterilized.

[0118] result:

[0119] Sample group: Immediately after injection, the implant was visible to the naked eye; 21 days after injection, no obvious swelling was observed. After dissection, the implant was visible to the naked eye under the skin. HE section showed a certain amount of inflammatory infiltration (inflammatory cells surrounding the foreign body) around the microspheres. No obvious cyst phenomenon was found, indicating that there was a mild inflammation. Masson section showed obvious new collagen between the microparticles.

[0120] Control group: A small amount of new collagen was generated;

[0121] Control group: No new collagen production was observed.

[0122] The method of this invention has been described through preferred embodiments. Those skilled in the art will readily be able to modify or appropriately alter and combine the solutions or applications described herein within the scope and content of this invention to implement and apply the technology of this invention. Those skilled in the art can refer to the content herein to appropriately improve the conditions / parameters to implement and / or apply the technology of this invention. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within this invention.

Claims

1. A composition comprising, or comprising, the raw materials for its preparation: Collagen, hyaluronic acid or hyaluronic acid salt, hydroxyapatite microparticles, calcium lactate, inorganic salts and water; In the composition, the mass ratio of collagen to hyaluronic acid or hyaluronic acid salt is (1:0.2)-(1:4), the concentration of collagen is 3 mg / mL-30 mg / mL, the collagen is collagen extracted from yak, the collagen molecular weight is greater than 200,000 Daltons, the concentration of hyaluronic acid or hyaluronic acid salt is 6 mg / mL-20 mg / mL, the concentration of hydroxyapatite is 3 mg / mL-12 mg / mL, the hydroxyapatite microparticles are spiky spherical microparticles with multiple protrusions or spikes on their surface, and the concentration of calcium lactate is 1 mg / mL-10 mg / mL.

2. The composition according to claim 1, wherein, The hyaluronic acid or hyaluronic acid salt has a molecular weight of 1.2 million to 3 million Daltons.

3. The composition according to claim 1, wherein, The inorganic salt is at least one selected from disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, and potassium chloride; the pH of the composition is 6.0-7.6 or 6.8-7.4; and the osmotic pressure of the composition is 250 mOsm / L-350 mOsm / L.

4. The composition according to claim 1, wherein the hydroxyapatite has a particle size of 20 micrometers to 80 micrometers.

5. The composition according to claim 1, wherein, The concentration of the collagen is 5 mg / mL to 20 mg / mL; and / or the concentration of the hyaluronic acid or hyaluronic acid salt is 6 mg / mL to 15 mg / mL.

6. The composition according to claim 1, comprising at least one of the following conditions: i. The elastic modulus of the composition is higher than the sum of the elastic moduli of its raw materials collagen and hyaluronic acid or hyaluronic acid salt, and the elastic modulus is measured at 0.1 Hz, at the same concentration, and under the same detection method and detection conditions. ii. It exists as a flowable suspension at ambient temperatures below 25°C, and undergoes a phase transition to a gel state at temperatures between 35°C and 38°C; and iii. At 37°C, the composition changes from a suspension to a gel state within 30 minutes or less.

7. The composition according to claim 1, under conditions not exceeding 34°C and 250nm-350nm, has a transmittance of not more than 50%, not more than 30%, or not more than 25%.

8. The composition according to claim 6, after phase transformation into a gel, has an elastic modulus / or viscous modulus at 37°C and 0.1Hz that is at least 1.2 times, 1.5 times, 2 times, 2.5 times, 3 times, 4 times, or 5 times that of its elastic modulus / or viscous modulus before phase transformation at 25°C and 0.1Hz.

9. A method for preparing the composition according to any one of claims 1-8, comprising: Prepare a solution with a pH of 6.0-7.6 by mixing hyaluronic acid or hyaluronic acid salts with an aqueous solution of inorganic salts. Then add no more than half of the calcium lactate and stir well; then add hydroxyapatite and the remaining calcium lactate, stir and mix for 0.1-2 hours, and then let it stand at 15℃-25℃ for 0.5-8 hours to obtain a composite solution; under 0℃-10℃ conditions, prepare a solution with a pH of 6.0-7.6 by mixing collagen and inorganic salt aqueous solution; then mix the collagen solution with the aforementioned composite solution, stir and mix well, and then let it stand at 15℃-25℃ for 0.5-6 hours. The pH is optionally adjusted to 6.0-7.6 using hydrochloric acid, sodium hydroxide, or potassium hydroxide to obtain a suspension; optionally, sterilization is performed to obtain a composition; the pH of the aqueous solution of the inorganic salt is 6.0-7.6; the method is carried out under aseptic production conditions, or each raw material or each composite solution is sterilized by irradiation with cobalt-60 at a dose not exceeding 15 kGy at -70°C to 0°C.

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