Composite gel scaffold as well as preparation method and application thereof

Abstract

The invention discloses a composite gel scaffold as well as a preparation method and application thereof. The preparation method of the composite gel scaffold comprises the following steps: dissolving calcium-deficient hydroxyapatite and recombinant collagen in water, adding a cross-linking agent, stirring and standing to prepare the composite gel scaffold; the preparation method of the calcium-deficient hydroxyapatite comprises the following steps: S1, calcining natural hydroxyapatite powder for 2 to 6 hours at the temperature of 700 to 900 DEG C; s2, soaking the calcined hydroxyapatite in a modifier solution to supplement phosphorus element; wherein the modifier is a salt containing a phosphorus element; and S3, drying the soaked hydroxyapatite, calcining at 500-700 DEG C for 0.5-3 hours to reduce the crystallinity, and cooling to prepare the calcium-deficient hydroxyapatite. The composite scaffold not only has the soft elastic function of gel, but also has the rigid supporting performance of particles.

Description

Technical Field

[0001] The present application relates to the technical field of medical filling materials, and in particular to a composite gel scaffold, a preparation method and use thereof, and further to calcium-deficient hydroxyapatite cross-linked collagen, a preparation method thereof, and use thereof in a calcium-deficient hydroxyapatite cross-linked collagen composite gel scaffold. Background Art

[0002] Hydroxyapatite (HAP) is the main inorganic component of human bones and teeth, and its chemical composition is (Ca 10 (PO4)6(OH)2), with a calcium / phosphorus ratio of 1.67, which is highly similar to natural bone mineral. It can form chemical bonds with bone tissue and promote bone cell attachment and growth, so it is often used in the field of bone repair.

[0003] Although hydroxyapatite has drawbacks such as unnatural facial expressions and slow degradation rate due to its hard particles, and difficulty in handling once adverse reactions (such as nodules and infection) occur, which are rarely reported in the field of facial filling, its advantages such as long maintenance time and good biocompatibility have attracted many researchers to continue to try to use it for facial injection filling, that is, to combine hydroxyapatite with other facial filling materials. On the basis of meeting clinical needs, hydroxyapatite can be used to prevent the entire filling material from shifting and spreading due to its strong supporting and plastic properties. It can also serve as a cell growth scaffold in the body, providing a platform for cell growth, stimulating the body to accelerate the production of collagen, thereby forming a long-term filling effect to solve the common problems of looseness, sagging, and depression caused by facial aging.

[0004] Calcium-deficient hydroxyapatite (CDHA) is a variant of hydroxyapatite with a lower calcium content than the ideal stoichiometric ratio of hydroxyapatite (calcium / phosphorus ratio of 1.67). Literature studies have shown that the Ca / P ratio of hydroxyapatite directly affects the material's surface properties, ionic reactivity, and ultimately its biological performance. Compared to HAP's relatively high crystallinity and stability, CDHA has higher biodegradability and degrades faster in the body, making it more advantageous in certain clinical application scenarios.

[0005] At present, artificially synthesized calcium-deficient hydroxyapatite is generally used in the field of facial filling. However, the physical and chemical properties of artificially synthesized calcium-deficient hydroxyapatite and polymer materials are quite different, resulting in poor interface compatibility and poor bonding between polymer components and calcium-deficient hydroxyapatite, which affects the biological activity and stability of the composite material and is not well adapted to clinical applications.

[0006] Therefore, there is an urgent need in this field to develop a new composite gel scaffold containing calcium-deficient hydroxyapatite based on natural hydroxyapatite to better meet practical application needs. Summary of the Invention

[0007] Based on this, it is necessary to provide at least one composite gel scaffold and its preparation method and use.

[0008] In a first aspect of the present application, a method for preparing a composite gel scaffold is provided, the method comprising:

[0009] The calcium-deficient hydroxyapatite and recombinant collagen were dissolved in water, a cross-linking agent was added, the mixture was stirred and then allowed to stand to prepare a composite gel scaffold;

[0010] Wherein, the preparation of the calcium-deficient hydroxyapatite comprises the following steps:

[0011] S1: calcining natural hydroxyapatite powder at 700℃~900℃ for 2 h~6 h;

[0012] S2: soaking the calcined hydroxyapatite in a modifier solution to supplement phosphorus; wherein the modifier includes a salt containing phosphorus; and

[0013] S3: drying the soaked hydroxyapatite, calcining it at 500°C to 700°C for 0.5 h to 3 h to reduce crystallinity, and cooling it to prepare calcium-deficient hydroxyapatite.

[0014] In some embodiments, in the step of adding a cross-linking agent, stirring, and then allowing to stand, the allowing to stand comprises:

[0015] After standing at 25℃~60℃ for 8 h~16 h, stand at 2℃~8℃ for 12 h~24 h.

[0016] In some embodiments, the recombinant collagen satisfies one or more of the conditions A1 to A2:

[0017] A1. The recombinant collagen comprises one or more of type I collagen and type III collagen; and,

[0018] A2. The molecular weight of the recombinant collagen is 20,000 Da to 40,000 Da.

[0019] In some embodiments, the preparation method satisfies one or more of the following conditions B1 to B5:

[0020] B1. The crosslinking agent is selected from the group consisting of a combination of 1-ethyl-3-dimethylaminopropylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) (EDC·NHS), 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM), adipic acid dihydrazide (ADH), and glutaraldehyde; wherein the molar ratio of EDC to NHS in the EDC·NHS is (4.5-7):1;

[0021] B2. The cross-linking agent is used in an amount of 2% to 8% of the total weight of the calcium-deficient hydroxyapatite and the recombinant collagen;

[0022] B3. Stirring conditions include: pH 5-7, 5°C-40°C, 50-70 min;

[0023] B4. The weight ratio of the calcium-deficient hydroxyapatite to the recombinant collagen is 1: (1 to 10); and,

[0024] B5. The drying temperature in step S3 is 40°C to 80°C.

[0025] In some embodiments, step S2 satisfies one or more of the following conditions C1 to C3:

[0026] C1. The concentration of the modifier is 10 mg / mL~100 mg / mL;

[0027] C2. The modifier comprises a phosphate; and,

[0028] C3. The modification temperature is 25℃~60℃, and the modification time is 12 h~36 h.

[0029] In some embodiments, the phosphate includes one or more of sodium dihydrogen phosphate, disodium hydrogen phosphate, and potassium dihydrogen phosphate.

[0030] In some embodiments, the preparation method further comprises:

[0031] Wash and sieve granules.

[0032] In some embodiments, the washing and sieving granulation satisfy one or more of the following conditions D1 to D2:

[0033] D1. In the washing step, the composite gel scaffold is dialyzed; the number of dialysis cycles may be, for example, 6 to 10 times; and,

[0034] D2. Use a sieve with a mesh size of 50-300 mesh to sieve and granulate.

[0035] In some embodiments, the preparation method further comprises low-temperature dehydration at 40° C. to 60° C. to achieve an ideal collagen concentration.

[0036] In some embodiments, the ideal collagen concentration is 10 mg / mL to 50 mg / mL.

[0037] In a second aspect of the present application, a composite gel scaffold is provided, which is prepared using the calcium-deficient hydroxyapatite and recombinant collagen as defined above.

[0038] In some embodiments, the composite gel scaffold is prepared according to the preparation method described above.

[0039] In a third aspect of the present application, a product for facial filling is provided, comprising the composite gel scaffold as described in the second aspect.

[0040] This application proposes, for the first time, the preparation of calcium-deficient hydroxyapatite based on naturally extracted hydroxyapatite, ensuring its safety while enabling improved applications in facial augmentation. The prepared calcium-deficient hydroxyapatite, due to its altered surface properties, is more conducive to protein adsorption. Therefore, it is compounded and cross-linked with recombinant collagen. By allowing the recombinant collagen to undergo self-crosslinking and adsorption simultaneously, a composite gel scaffold suitable for facial augmentation is produced.

[0041] This composite scaffold has both the soft elastic function of gel and the rigid support performance of particles. The two have a synergistic effect. On the one hand, the addition of CDHA particles can increase the high-temperature resistance of recombinant collagen. On the other hand, the compounding of recombinant collagen can further increase human compatibility, improve the safety of the composite scaffold, and make it more suitable for the field of facial filling. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the technical solutions in the implementation methods and examples of this application and to more completely understand the application and its beneficial effects, the following briefly introduces the drawings required for the description of the implementation methods or examples. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in explaining the present application.

[0043] Figure 1 This is an XRD pattern of hydroxyapatite (Example 1) prepared according to one embodiment of the present application.

[0044] Figure 2This is an XRD pattern of hydroxyapatite (Comparative Example 1) prepared according to one embodiment of the present application.

[0045] Figure 3A This is a SEM image (resolution of 2 μm) of an untreated natural hydroxyapatite sample in one embodiment of the present application. Specifically, the untreated natural hydroxyapatite sample is taken from the natural hydroxyapatite in step 1 of Example 1 of the present application.

[0046] Figure 3B This is a SEM image (resolution of 2 μm) of the hydroxyapatite sample prepared in one embodiment (Example 1) of the present application.

[0047] Figure 3C This is a SEM image (resolution of 2 μm) of an artificial calcium-deficient hydroxyapatite sample in one embodiment of the present application. Specifically, the artificial hydroxyapatite sample is taken from the calcium-deficient hydroxyapatite prepared in step 1 of comparative example 7 of the present application. DETAILED DESCRIPTION

[0048] To facilitate understanding of the present application, a more comprehensive description of the present application will be provided below with reference to the accompanying drawings. The accompanying drawings illustrate preferred embodiments of the present application. However, the present application may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the disclosure of the present application.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0050] In this application, unless otherwise specified, "one or more" refers to any one of the listed items or any combination of the listed items. Similarly, "one or more" and other similar expressions that refer to "one or more" are also understood in the same way unless otherwise specified.

[0051] The terms "combination thereof", "any combination thereof", "any combination thereof" and the like used in this application include all suitable combinations of any two or more of the listed items.

[0052] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method", etc. shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0053] In this application, the terms "further," "further," "particularly," "for example," "such as," "example," and "for example" are used for descriptive purposes to indicate that the preceding and following technical solutions are related in terms of the content covered, but should not be construed as limiting the preceding technical solution or the scope of protection of this application. In this application, unless otherwise specified, A (such as B) means that B is a non-limiting example of A, and it is understood that A is not limited to B.

[0054] In this application, the terms "optionally," "optional," and "optional" mean optional, that is, they refer to either option selected from the two parallel options of "yes" or "no." If multiple "options" appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "optional" is independent. Unless otherwise specified, the descriptions "optionally include," "optionally include," etc. in this application, taking "optionally include" as an example, mean "may include or not include."

[0055] As used herein, the terms "comprising," "including," and "include" are synonymous and are inclusive or open-ended, not excluding additional, unrecited members or features. Examples of members or features include materials or components, structures, elements, and instruments. Non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, and states.

[0056] In this application, the technical features or technical solutions described in open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0057] In this application, exemplary descriptions such as "in some embodiments (or examples)" and "in one embodiment (or example)" may include but are not limited to the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0058] In this application, the terms "first," "second," "third," "fourth," etc. in "the first aspect," "the second aspect," "the third aspect," "the fourth aspect," etc. are used for descriptive purposes only and should not be understood as indicating or implying relative importance or quantity, nor should they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc. serve only for the purpose of non-exhaustive enumeration and description, and should be understood not to constitute a closed-ended limitation on quantity.

[0059] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered to be continuous and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when a numerical interval only refers to an integer in the numerical interval, including the two endpoint integers of the numerical range and each integer between the two endpoints, is equivalent to directly enumerating each integer. When multiple numerical ranges are provided to describe a feature or characteristic, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical range disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0060] In this application, if a method flow involves multiple steps, unless otherwise specified herein, there is no strict order restriction on the execution of these steps, and they can be executed in an order other than the order described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages do not necessarily need to be completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating, or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0061] This study proposes a new approach. By modifying naturally extracted hydroxyapatite, calcium-deficient hydroxyapatite is prepared to improve its surface properties and enhance its adsorption capacity for proteins. Then, recombinant collagen is added for compound cross-linking to prepare a natural, additive-free composite scaffold that can be better used in the field of facial filling. This scaffold has good compatibility with the human body and can be completely degraded. When used in the field of facial filling, it can first fill the concave parts of the face with materials, and secondly, it can stimulate cell activity more quickly. During the degradation process of the scaffold, it stimulates the in situ regeneration of collagen at the filling site, thickening the dermis. The filled area can be replaced by new autologous tissue, extending the action time of the filler, and has a relatively broad application prospect.

[0062] In this application, hydroxyapatite is modified and naturally extracted hydroxyapatite is calcined at high temperature to remove immunogenicity, thereby preventing other substances from affecting the subsequent use effect. In this process, the crystallinity of hydroxyapatite is greatly improved; then, it is soaked in a modifier to achieve the supplementation of P element, and a second low-temperature calcination is performed to reset it. On the basis of allowing the P element to be more firmly bound to the hydroxyapatite, its crystallinity is reduced to obtain porous calcium-deficient hydroxyapatite that can be used for facial filling.

[0063] The above technology is a method of modifying naturally extracted hydroxyapatite to prepare calcium-deficient hydroxyapatite suitable for facial filling, and then compounding it with recombinant collagen to prepare a facial filling composite scaffold. The key points are as follows:

[0064] ①. A method for preparing calcium-deficient hydroxyapatite by modifying hydroxyapatite with phosphoric acid or phosphate as a modifier and calcining it in two steps at different temperatures is more conducive to protein adsorption due to changes in surface properties.

[0065] ②. In one aspect of the present application, the calcium-deficient hydroxyapatite prepared is simultaneously compounded and cross-linked with recombinant collagen to produce a composite gel scaffold suitable for facial filling through chemical cross-linking and physical adsorption, and can better stimulate the regeneration of the body's own collagen. The prepared composite scaffold is more uniform and stable.

[0066] In a first aspect of the present application, a method for preparing a composite gel scaffold is provided, the method comprising:

[0067] The calcium-deficient hydroxyapatite and recombinant collagen were dissolved in water, a cross-linking agent was added, the mixture was stirred and then allowed to stand to prepare a composite gel scaffold;

[0068] Wherein, the preparation of the calcium-deficient hydroxyapatite comprises the following steps:

[0069] S1: calcining natural hydroxyapatite powder at 700℃~900℃ for 2 h~6 h;

[0070] S2: soaking the calcined hydroxyapatite in a modifier solution to supplement phosphorus; wherein the modifier includes a salt containing phosphorus; and

[0071] S3: drying the soaked hydroxyapatite, calcining it at 500°C to 700°C for 0.5 h to 3 h to reduce crystallinity, and cooling it to prepare calcium-deficient hydroxyapatite.

[0072] Unless otherwise specified, the term "natural hydroxyapatite" in this application refers to a calcium phosphate mineral that is widely present in organisms, such as the bones and teeth of vertebrates (e.g., pigs and cattle). Natural hydroxyapatite can be extracted from animal bones through appropriate treatment (e.g., defatting, deproteinization, etc.).

[0073] In some embodiments, the natural hydroxyapatite is derived from cancellous bone of a vertebrate, for example, limb bones, pelvic bones, and the like.

[0074] In some embodiments, the natural hydroxyapatite is derived from at least one of porcine limb bones, porcine pelvic bones, bovine limb bones, and bovine pelvic bones.

[0075] Without wishing to be bound by any theory, it is believed that in the steps of adding a cross-linking agent, stirring, and then allowing the mixture to stand, the interaction between calcium-deficient hydroxyapatite and recombinant collagen mainly occurs during the standing process, and the interaction includes, for example, chemical cross-linking and physical adsorption.

[0076] In some embodiments, the standing period can be divided into two stages.

[0077] For example, the standing condition of the first stage is: standing at 25°C to 60°C for 8 h to 16 h; the standing condition of the second stage is: standing at 2°C to 8°C for 12 h to 24 h.

[0078] The resting temperature in the first stage can be, for example, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, or a range or value between any two values. The resting time can be adjusted at least according to the temperature within 8 hours to 16 hours, for example, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, or a range or value between any two values. In some embodiments, the resting condition in the first stage is 37°C for 10 hours.

[0079] Without wishing to be bound by any theory, it is believed that when the standing temperature in the first stage is too low (e.g., the temperature is lower than 25°C) and / or the standing time is short (e.g., the standing time is less than 8 h), the recombinant collagen may not be sufficiently firmly connected to the calcium-deficient hydroxyapatite, and when the standing temperature in the first stage is high (e.g., higher than 60°C) and / or the standing time is long (e.g., higher than 16 h), it may be detrimental to the stability of the composite gel scaffold.

[0080] The resting temperature in the second stage can be, for example, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, or any range or value between any two values. The resting time can be adjusted at least according to the temperature within 12 hours to 24 hours, for example, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, or any range or value between any two values.

[0081] Without wishing to be bound by any theory, it is believed that when the standing temperature of the second stage is too low (for example, below 2°C) and / or the standing time is short (for example, below 12 h), it may affect the firm restoration of the physical force between the recombinant collagen and the calcium-deficient hydroxyapatite. When the standing temperature of the second stage is high (for example, above 8°C), the physical force between the recombinant collagen and the calcium-deficient hydroxyapatite may be easily weakened. When the standing time of the second stage is long (for example, above 24 h), the composite performance of the recombinant collagen and the calcium-deficient hydroxyapatite will not be improved. On the contrary, the preparation efficiency of the composite gel scaffold will be affected due to the extended preparation time.

[0082] Without wishing to be bound by any theory, it is believed that the second stage of low temperature standing serves at least to strengthen physical adsorption.

[0083] In some embodiments, the resting may be only one stage.

[0084] For example, the mixture is allowed to stand at 25°C to 60°C for 8 to 40 hours. For example, the mixture is allowed to stand at 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, or a range or value between any two values. The standing time is exemplarily 8 hours, 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, or a range or value between any two values. In some embodiments, the mixture is allowed to stand at 37°C for 34 hours.

[0085] Without wishing to be bound by any theory, it is believed that the performance of the composite gel scaffold prepared after two stages of standing is more stable than after only one stage of standing.

[0086] For example, in step S1, the calcination temperature may be 700° C., 750° C., 800° C., 850° C., 900° C., or a range or value between any two values. The calcination time may be, for example, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 5.5 h, 6 h, or a range or value between any two values.

[0087] Without wishing to be bound by any theory, it was found that when the calcination temperature in step S1 is lower than 700°C, it is insufficient to remove impurities in the biological bone, affecting subsequent use; when the calcination temperature is higher than 900°C (e.g., 1400°C), hydroxyapatite is converted into tricalcium phosphate and tetracalcium phosphate, the Ca / P ratio increases, and the purity is poor.

[0088] In some embodiments, step S1 includes calcining natural hydroxyapatite powder at 800° C. for 3 h.

[0089] In some embodiments, step S1 is performed in a muffle furnace.

[0090] The recombinant collagen may be conventional recombinant collagen in the art, for example, a recombinant collagen with a molecular weight of 20,000 Da to 40,000 Da.

[0091] In some embodiments, the recombinant collagen includes one or more of type I collagen and type III collagen.

[0092] In some embodiments, the cross-linking agent may be a conventional cross-linking agent in the art, for example, a combination of 1-ethyl-3-dimethylaminopropylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) (EDC·NHS), 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM), adipic acid dihydrazide (ADH), glutaraldehyde, and the like.

[0093] In some embodiments, the crosslinking agent is EDC·NHS. Without wishing to be bound by theory, it has been found that the activator EDC·NHS has at least one advantage: it is absent (or minimally present) in the final product, with the product consisting solely (or primarily) of calcium-deficient hydroxyapatite and collagen; both of these raw materials are naturally occurring in the human body, resulting in a composite gel scaffold with high biocompatibility.

[0094] The relative amounts of EDC and NHS used may be conventional amounts in the art, for example, a molar ratio of (4.5-7):1.

[0095] In some embodiments, the relative amounts of EDC and NHS are 4.5:1, 5:1, 6:1, 7:1, or any range or value between any two values.

[0096] In some embodiments, in the preparation method, the amount of the crosslinking agent used may be 2% to 8% of the total weight of the calcium-deficient hydroxyapatite and the recombinant collagen. For example, it may be 2%, 3%, 4%, 5%, 6%, 7%, 8%, or a range or value between any two values.

[0097] In some embodiments, the stirring conditions include: pH 5-7, and stirring at 5° C.-40° C. for 50 min-70 min.

[0098] For example, the stirring temperature may be 5° C., 15° C., 20° C., 25° C., 30° C., 35° C., 40° C., or a value or range between any two values. The stirring time may be 50 min, 55 min, 60 min, 65 min, 70 min, or a value or range between any two values.

[0099] In some embodiments, the stirring conditions include pH 5-7, stirring at 37° C. for 60 min.

[0100] In some embodiments, the weight ratio of the calcium-deficient hydroxyapatite to the recombinant collagen is 1:(1-10). Without wishing to be bound by any theory, it is believed that when the weight ratio of the two is outside this range, the compounding effect of the calcium-deficient hydroxyapatite and the recombinant collagen may be poor, and the optimal binding rate may not be achieved.

[0101] Without wishing to be bound by any theory, it is believed that drying the hydroxyapatite soaked in step S2 is necessary before calcining at 500°C to 700°C in step S3. This is due to at least two reasons: calcining the soaked hydroxyapatite directly at 500°C to 700°C may cause the hydroxyapatite to agglomerate; and calcining at 500°C to 700°C in a well-sealed muffle furnace may cause smoke to form due to excessive moisture.

[0102] In some embodiments, the drying temperature in step S3 is 40° C. to 80° C. For example, it is 40° C., 45° C., 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., 80° C., or a range or value between any two values.

[0103] In some embodiments, step S3 is performed in a muffle furnace. For example, in step S3, the calcination temperature can be 500°C, 550°C, 600°C, 650°C, 700°C, or any range or value between any two values. Without wishing to be bound by any theory, it has been found that when the calcination temperature is lower than 500°C (e.g., 400°C), unreacted phosphoric acid cannot be completely removed, resulting in the sample containing multiple forms of calcium phosphate components. The resulting hydroxyapatite, despite having a low Ca / P ratio, is of poor purity, making it unsuitable for subsequent clinical applications. When the calcination temperature is higher than 700°C (e.g., 800°C), the Ca / P ratio of the sample rises to 1.67, and the sample's crystallinity increases.

[0104] Without wishing to be bound by any theory, it is believed that the role of the modifier in step S2 is at least to supplement phosphorus (P) to the hydroxyapatite obtained after high-temperature calcination. It is understood that the modifier includes but is not limited to substances containing phosphorus, such as phosphate.

[0105] Phosphates that can be used in the modifier include, but are not limited to, sodium dihydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and other conventional phosphates in the art.

[0106] The concentration of the modifier can be adjusted according to actual needs during the experiment. Without wishing to be bound by any theory, it is believed that using a higher concentration of the modifier can replenish the phosphorus element in the hydroxyapatite obtained after high-temperature calcination relatively faster; vice versa.

[0107] In some embodiments, the concentration of the modifier is 10 mg / mL to 100 mg / mL, for example, 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, or any range or value between any two values.

[0108] The modification temperature can be illustratively between 25°C and 60°C, and the modification time can be between 12 hours and 36 hours. Without wishing to be bound by any theory, it has been found that when the modification temperature is lower than 25°C or higher than 60°C, the P element cannot be effectively replenished, resulting in a high Ca / P ratio in the hydroxyapatite, which affects subsequent operations.

[0109] For example, the modification temperature may be 25° C., 30° C., 35° C., 40° C., 45° C., 50° C., 55° C., 60° C., or a range or value between any two values. The modification time may be, for example, 12 h, 16 h, 20 h, 24 h, 28 h, 32 h, 36 h, or a range or value between any two values.

[0110] In some embodiments, the concentration of the modifier is 30 mg / mL, and the modification temperature is 40° C. and the modification time is 24 h.

[0111] In some embodiments, the preparation method further comprises:

[0112] Wash and sieve granules.

[0113] The cleaning method can be a conventional cleaning method in the art, such as dialysis.

[0114] In some embodiments, the washing step comprises: dialyzing the composite gel scaffold; the number of dialysis can be, for example, 6 to 10 times, illustratively, for example, 8 times.

[0115] In some embodiments, a sieve with a mesh size of 50 to 300 is used for sieving and granulating. For example, the sieve size can be 50, 60, 70, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, or a range or value between any two values. In some embodiments, a sieve with a mesh size of 120 is used for sieving and granulating.

[0116] In some embodiments, the preparation method further comprises low-temperature dehydration at 40° C. to 60° C. to achieve an ideal collagen concentration.

[0117] In some embodiments, the ideal collagen concentration may be, for example, 10 mg / mL to 50 mg / mL, for example, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, or a range or value between any two values.

[0118] In some embodiments, the preparation method comprises:

[0119] Step 1: calcining natural hydroxyapatite powder in a muffle furnace at 800°C for 3 h.

[0120] Step 2: Prepare a 30 mg / mL modifier solution and add the calcined hydroxyapatite powder to it, soaking and stirring at 40°C for 24 hours. The modifier solution can be a phosphoric acid solution or an acid solution of a phosphorus-containing salt such as sodium dihydrogen phosphate, disodium hydrogen phosphate, or potassium dihydrogen phosphate.

[0121] Step 3: Take out the soaked hydroxyapatite, remove excess soaking liquid, dry it at 60°C, and calcine the dried sample at low temperature, calcine it at 600°C for 2 hours, and then cool it to 25°C-35°C to obtain calcium-deficient hydroxyapatite.

[0122] Step 4: Dissolve calcium-deficient hydroxyapatite and recombinant type III collagen in water at a ratio of 1:5, then add 1-ethyl-3-3-dimethylaminopropyl-carbodiimide (EDC) and N-hydroxy-succinic acid amide (NHS) crosslinker, where the molar ratio of EDC to NHS is 5:1, and the total amount of EDC and NHS added is 5% of the total amount of calcium-deficient hydroxyapatite and recombinant collagen added. Adjust the pH to 5-7 and stir at 37°C for 60±10 minutes to mix evenly.

[0123] Step 5: Place the composite gel obtained in step 4 in a water bath, adjust the temperature to 37°C, let it stand for 10 hours, transfer it to a refrigerator at 2-8°C and continue to stand for 24 hours to obtain a calcium-deficient hydroxyapatite cross-linked recombinant collagen composite gel scaffold.

[0124] Step 6: The gel obtained in step 5 was repeatedly dialyzed against a buffer solution to remove the cross-linking agent and modifier residues. The number of washes was 8 times.

[0125] Step 7: Sieve the gel obtained in step 6 to prepare a composite scaffold gel with uniform particle size distribution, wherein the mesh size of the sieve is 120 meshes.

[0126] Step 8: Dehydrate the composite scaffold prepared in step 7 at low temperature until the collagen concentration reaches 15 mg / mL.

[0127] Step 9: Fill the composite gel scaffold prepared in step 8 into a syringe, and then sterilize it with high temperature and moist heat at 121°C for 12 minutes.

[0128] In a second aspect of the present application, a composite gel scaffold is provided, which is prepared using the calcium-deficient hydroxyapatite and recombinant collagen as defined above.

[0129] In some embodiments, the composite gel scaffold is prepared according to the preparation method described above.

[0130] In a third aspect of the present application, a product for facial filling is provided, comprising the composite gel scaffold as described in the second aspect.

[0131] In a fourth aspect of the present application, a method for filling the face is provided, comprising injecting the composite gel scaffold as described in the second aspect into the face of a subject in need.

[0132] Some examples are provided below.

[0133] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods for which the conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and may also be based on the experimental manuals or conventional conditions in this area, or on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0134] Recombinant collagen: purchased from Baihong Synthetic Biotechnology (Yantai) Co., Ltd.

[0135] The naturally extracted hydroxyapatite is derived from animal cancellous bone, and the animal cancellous bone is selected from one of pig's limb bones or pelvic bones and cattle's limb bones or pelvic bones.

[0136] Example 1

[0137] This embodiment specifically includes the following steps:

[0138] Step 1: calcining natural hydroxyapatite powder in a muffle furnace at 800°C for 3 h.

[0139] Step 2: Prepare a 30 mg / mL phosphoric acid solution, add the calcined hydroxyapatite powder into it and soak and stir at 40°C for 24 h.

[0140] Step 3: Take out the soaked hydroxyapatite, remove the excess soaking liquid, dry it at 60°C, calcine the dried sample at low temperature, calcine it at 600°C for 2 hours, and then cool it to 30°C to obtain calcium-deficient hydroxyapatite.

[0141] Step 4: Dissolve calcium-deficient hydroxyapatite and recombinant type III collagen with a molecular weight of 35,000 Da in water at a ratio of 1:5, then add 1-ethyl-3-3-dimethylaminopropyl-carbodiimide (EDC) and N-hydroxy-succinic acid amide (NHS) cross-linker, where the molar ratio of EDC to NHS is 5:1, and the total amount of EDC and NHS added is 5% of the total amount of calcium-deficient hydroxyapatite and recombinant collagen added. Adjust the pH to 5-7 and stir at 37°C for 60±10 min to mix evenly.

[0142] Step 5: Place the composite gel obtained in step 4 in a water bath, adjust the temperature to 37°C, let it stand for 10 hours, transfer it to a refrigerator at 2-8°C and continue to stand for 24 hours to obtain a calcium-deficient hydroxyapatite cross-linked recombinant collagen composite gel scaffold.

[0143] Step 6: The gel obtained in step 5 was dialyzed repeatedly to remove the cross-linking agent and modifier residues, and the number of washing times was 8 times.

[0144] Step 7: Sieve the gel obtained in step 6 to prepare a composite scaffold gel with uniform particle size distribution, wherein the mesh size of the sieve is 120 meshes.

[0145] Step 8: Dehydrate the composite scaffold prepared in step 7 at low temperature until the collagen concentration reaches 25 mg / mL.

[0146] Step 9: Fill the composite gel scaffold prepared in step 8 into a syringe, and then sterilize it with high temperature and moist heat at 121°C for 12 minutes.

[0147] Example 2

[0148] The difference from Example 1 is that in step 5, the composite gel obtained in step 4 is placed in a 37° C. water bath and allowed to stand for 34 h.

[0149] Comparative Example:

[0150] Comparative Example 1

[0151] The difference from Example 1 is that step 2 is not performed, and in step 3, the hydroxyapatite calcined at high temperature is calcined at low temperature, and the remaining steps are the same.

[0152] Comparative Example 2

[0153] The difference from Example 1 is that the low-temperature calcination process in step 3 is no longer performed, and the remaining steps are the same.

[0154] Comparative Example 3

[0155] The difference from Example 1 is that the calcination temperature in step 1 is 1400°C.

[0156] Comparative Example 4

[0157] The difference from Example 1 is that the calcination temperature in step 3 is 400°C.

[0158] Comparative Example 5

[0159] The difference from Example 1 is that the calcination temperature in step 3 is 800°C.

[0160] Comparative Example 6

[0161] The difference from Example 1 is that steps 1 to 3 are not performed, and in step 4, the natural hydroxyapatite powder and recombinant collagen in step 1 are directly subjected to the same subsequent operations.

[0162] Comparative Example 7

[0163] The difference from Example 1 is that steps 1 to 3 are not performed, and in step 4, the recombinant collagen and the calcium-deficient hydroxyapatite prepared by the following method are subjected to the same subsequent operations.

[0164] (1) 500 g of calcium hydrogen phosphate dihydrate and 145 g of calcium carbonate were mixed under mechanical stirring to obtain mixed powder A; 6.5 g of 1% by mass polyvinyl alcohol was then added to obtain mixed powder B. The mixed powder B was heated to 1000°C and kept at this temperature for 1 h. After that, the mixed powder B was calcined in a furnace at 1400°C for 2 h and then taken out and rapidly cooled under a blast environment to obtain α-TCP powder.

[0165] (2) α-TCP powder was ball-milled and passed through a 500-mesh sieve to obtain a particle size range of 1-30 μm;

[0166] (3) Prepare a phosphate solution with a pH of 9. Add the powder to the solution at a mass ratio of 1:4 during stirring. Continue stirring for 2 h after addition, and then let it stand for 24 h to allow for complete phase transformation. The product is filter-pressed, dried at 80 °C, and crushed to obtain calcium-deficient hydroxyapatite.

[0167] 2. Performance Test

[0168] 1. Ca / P ratio of hydroxyapatite

[0169] The XRD patterns of the hydroxyapatite samples in Example 1 and Comparative Example 1 were obtained according to the X-ray diffraction acquisition method in GB / T 23101.3-2023 "Hydroxyapatite for surgical implants Part 3: Chemical analysis and characterization of crystallinity and phase purity", as shown in Figures 1 and 2. Figure 1 and Figure 2 The atomic ratios of Ca and P (i.e., Ca / P ratios) in the hydroxyapatite samples obtained in Example 1 and Comparative Examples 1-5 were determined according to the method for determining the calcium-phosphorus atomic ratio (Ca:P) in GB / T 23101.3-2023, "Hydroxyapatite for Surgical Implants - Part 3: Chemical Analysis and Characterization of Crystallinity and Phase Purity." Specific data are shown in Table 1. As can be seen from the data in Table 1, natural calcium-deficient hydroxyapatite was successfully obtained after steps one through three.

[0170] Table 1. Ca / P ratios of hydroxyapatite samples obtained in Example 1 and Comparative Examples 1-5

[0171]

[0172] From the data in Table 1, it can be seen that the method of the present application can indeed process natural hydroxyapatite into calcium-deficient hydroxyapatite, and Figure 1 It can be seen that the crystallization peak position of the calcium-deficient hydroxyapatite prepared in Example 1 is the same as that of the standard hydroxyapatite, but the crystallinity is lower, and the Ca / P ratio is 1.55, indicating that it is indeed calcium-deficient hydroxyapatite.

[0173] When step 2 is not performed (Comparative Example 1), the natural bone block can be calcined into standard hydroxyapatite by first calcining at high temperature and then calcining at low temperature. Figure 2 As shown, there are no impurities in the standard hydroxyapatite after two-step calcination, the Ca / P ratio is 1.70, and its crystallinity is close to that of the standard hydroxyapatite. It can be seen that adding phosphoric acid and treating according to the preparation method of the present application is more likely to successfully prepare pure calcium-deficient hydroxyapatite.

[0174] When step 2 is performed without step 3 (Comparative Example 2) or the calcination temperature in step 3 is low (Comparative Example 4), the unreacted phosphoric acid cannot be completely removed, resulting in the sample containing multiple forms of calcium phosphate. In this case, although the Ca / P ratio of the hydroxyapatite produced is relatively low, its purity is also poor, which is not conducive to future clinical applications. When the calcination temperature in step 3 is higher (Comparative Example 5), the Ca / P ratio of the sample rises to 1.67, and the crystallinity of the sample is improved.

[0175] It should also be noted that the calcination temperature in step 1 needs to be within an appropriate range. If the temperature is too low, it will not be sufficient to remove impurities in the biological bone, affecting subsequent use. However, when the temperature is too high (Comparative Example 3), hydroxyapatite will be converted into tricalcium phosphate and tetracalcium phosphate, the Ca / P ratio will increase, and the purity will be poor.

[0176] From the above, it can be seen that the temperature of the two calcination processes in this application is very important and needs to be strictly controlled.

[0177] 2. Collagen composite properties of composite gel scaffolds

[0178] Testing method: Approximately 0.3 g (accurate to 0.1 mg) of the composite gel scaffold product prepared in each of the above examples and comparative examples was accurately weighed, diluted to 10 mL with purified water, and shaken until uniform. The solution was poured into a centrifuge tube and centrifuged at 10,000 rpm for 15 minutes. 0.5 mL of the supernatant was collected as the test sample solution. Collagen content was then assayed using the BCA assay.

[0179] BCA method: based on the protein molecules in alkaline solution to Cu 2+ Reduction to Cu + , 2,2'-biquinoline-4,4'-dicarboxylic acid (BCA) and Cu + The complex forms a purple color, the color of which is proportional to the protein concentration within a certain range. A standard curve is drawn using a protein reference solution, and the protein content in the test sample is determined by colorimetry. A series of recombinant type III collagen standard solutions are prepared according to Table 2.

[0180] Table 2. Concentration of recombinant type III collagen (RHC III) standard solution series

[0181]

[0182] Accurately measure 0.5 mL of the test sample and set aside for each test tube of the standard solution series. Slowly add 10.0 mL of copper-BCA test solution to each tube, mix immediately, and place in a 37°C water bath for 30 min. After cooling to 30°C, according to the UV-visible spectrophotometry method, use tube 0 as the blank, and immediately measure the absorbance of each standard tube and sample tube at a wavelength of 562 nm.

[0183] An absorbance-concentration curve (coefficient of determination, R² ≥ 0.99) was constructed using the test data from the standard tubes. The concentration of recombinant type III collagen in the sample tubes was determined from the standard curve based on the absorbance of the sample tubes. This concentration was then converted to the free collagen content of each group based on the dilution factor. The experimental results are shown in Table 3.

[0184] Table 3. Free collagen content in each sample in Examples 1-2 and Comparative Examples 1-7

[0185]

[0186] From the data in Table 2, it can be seen that the natural calcium-deficient hydroxyapatite prepared in the examples of this application has a good composite performance with recombinant collagen. On the one hand, after two steps of calcination, the surface impurities of the natural hydroxyapatite are completely removed, and a porous structure can be formed on the surface (see Figure 3B ), which makes hydroxyapatite bind more tightly to recombinant collagen; in contrast, untreated natural hydroxyapatite (see Figure 3A ) and synthetic calcium-deficient hydroxyapatite (see Figure 3C ) has a relatively dense structure and a relatively small specific surface area, which is not conducive to contact and complexation with recombinant collagen. On the other hand, the calcium-deficient hydroxyapatite prepared through the above-mentioned treatment steps exhibits superior adsorption performance for recombinant collagen. Compared to Comparative Examples 6 and 7, it can adsorb more recombinant collagen at the same addition amount. Specifically, after the calcium-deficient hydroxyapatite is blended with the recombinant collagen, the crosslinking and adsorption processes proceed simultaneously, resulting in stable performance of the composite gel scaffold prepared in Example 1. After dilution and centrifugation, the concentration of free recombinant collagen is even lower.

[0187] According to the data of Example 2, if the calcium-deficient hydroxyapatite and recombinant type III collagen are only placed at 37°C for a sufficiently long time, the concentration of free recombinant collagen in the obtained composite gel scaffold is relatively high. It is speculated that this may be due to the long-term temperature of 37°C, which will affect the physical adsorption of calcium-deficient hydroxyapatite on recombinant type III collagen, resulting in the weakening of the composite performance between calcium-deficient hydroxyapatite and recombinant type III collagen. The performance of the composite gel scaffold is slightly inferior to that of the composite gel scaffold prepared in Example 1. After dilution and centrifugation, the concentration of free recombinant type III collagen is higher.

[0188] In Comparative Examples 1, 3, and 5, improper processing resulted in the composite gel scaffolds having a Ca / P ratio of hydroxyapatite as high as 1.67 or above. This composite gel scaffold may have poor adsorption and cross-linking properties between hydroxyapatite and recombinant type III collagen, resulting in a high concentration of free recombinant type III collagen after dilution and centrifugation, which is not conducive to the efficient function of the composite gel scaffold. Similarly, the composite gel scaffold prepared in Comparative Example 6 had a high concentration of free recombinant type III collagen after dilution and centrifugation.

[0189] In the composite gel scaffolds prepared in Comparative Examples 2 and 4, although the Ca / P ratio of hydroxyapatite is relatively low, due to the high content of P element, the purity of calcium-deficient hydroxyapatite is poor, and it exists in the form of various types of calcium phosphate, which in turn causes the adsorbent cross-linking performance between hydroxyapatite and recombinant type III collagen to be poor, and the concentration of free recombinant type III collagen is high, which is not conducive to the efficient functioning of the composite gel scaffold.

[0190] 3. Stability test of composite gel scaffold

[0191] Testing method: Referring to YY / T 0681.1-2018 "Test Methods for Sterile Medical Device Packaging Part 1: Guide to Accelerated Aging Tests", "Guidelines for Registration and Application Materials for Shelf Validity Period of Passive Implantable Medical Devices (2017 Revised Edition)" and other methods, the composite gel scaffolds prepared in the above comparative examples and embodiments were placed in a stability test chamber, and the temperature was adjusted to 40°C ± 2°C and the relative humidity was 75% ± 5%. Under these conditions, samples were taken after 1 week, 4 weeks, 12 weeks, 24 weeks, and 36 weeks, and then their appearance was comprehensively evaluated. The uniformity of the solution was graded according to the evaluation results, and the uniformity of the solution was graded from 0 to 3, where grade 0 represents that the uniformity of the solution is basically unchanged, grade 1 represents that the solution begins to become uneven, but there is no visible stratification, grade 2 represents that the solution begins to stratify, and grade 3 represents that the solution is completely stratified. The experimental results are shown in Table 4 below:

[0192] Table 4 Test results of uniform stability of composite gel scaffolds in Examples 1-2 and Comparative Examples 1-7

[0193]

[0194] As can be seen from Table 4, the method provided in the examples of the present application can ensure entanglement between the recombinant collagen and the calcium-deficient hydroxyapatite, strengthen the force of the solution network, improve the stability of the system, and ensure the overall uniformity and stability of the composite solution. The system remains stable after 6 months of acceleration.

[0195] 4. Activity detection of composite gel scaffold

[0196] Detection method: Human fibroblasts were revived in DMEM medium containing 10% animal serum (FBS) and cultured at 37°C in a saturated humidity of 5% CO2. The cells were passaged 2-3 times until they were in the logarithmic growth phase. The cells were digested with trypsin, collected, and the cell concentration was adjusted to 1×10 5 Cell experiments were performed with 10 cells / mL.

[0197] The sample extraction diluent was prepared according to the recommended method specified in GB / T 16886.12 10.3.2. MEM culture medium containing 10% serum was used as the extraction diluent. The sample was extracted at a ratio of 0.2 g / mL at 37°C for 72 h. The test sample: extraction diluent = 1 mL: 5 mL.

[0198] The above cell suspension was added to a 96-well plate, 100 μL per well, for a total of 1×10 4 Cells were cultured at 37°C, 5% CO₂ for 24 h. After incubation, the culture medium in the culture plate was discarded, and the extract (four dose groups: 100%, 50%, 25%, and 12.5%) was added to each plate, along with a negative control, a blank control, and a positive control. Each plate had 6 wells, and the cells were cultured in a 37°C, 5% CO₂, saturated water vapor incubator.

[0199] After 24 hours, the culture medium was aspirated, washed with PBS, and the PBS discarded. The MTT cytotoxicity assay was performed. 50 μL of MTT (1 mg / mL) solution was added to each test well and incubated in a 37°C, 5% CO2 saturated humidity incubator for 2 hours. After incubation, the plate was discarded, and 100 μL of isopropanol solution was added to each well. Mix thoroughly in the dark for 30 minutes. The plates were then placed in a microplate reader and absorbance was measured at a detection wavelength of 570 nm and a reference wavelength of 650 nm. The relative cell proliferation rate (%) was calculated based on the absorbance: average absorbance of the test sample / average absorbance of the blank group × 100%. The experimental data are shown in Table 5.

[0200] Table 5. Relative cell proliferation rate (%) of the composite gel scaffolds prepared in Examples 1-2 and Comparative Examples 1-7

[0201]

[0202] As shown in Table 5, the composite gel scaffold prepared by the method provided in the examples of the present application has the highest activity. The naturally extracted calcium-deficient hydroxyapatite and recombinant collagen, when combined, can provide a scaffold for cells and promote cell proliferation. However, the artificially synthesized calcium-deficient hydroxyapatite has lower activity (Comparative Example 7) and cannot promote cell proliferation. In addition, the composite gel scaffolds prepared in Comparative Examples 1-6 have poor activity, which may be due to the poor binding performance between hydroxyapatite and recombinant collagen in the composite gel scaffold, resulting in its inability to fully exert its effect. Therefore, it can be demonstrated that the composite gel prepared in the present application has higher tissue compatibility when used for filling, and can provide a scaffold for fibroblasts and promote collagen regeneration.

[0203] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0204] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art could make various modifications and improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the patent in this application shall be determined by the appended claims, and the specification and drawings shall serve to interpret the claims.

Claims

1. A method for preparing a composite gel scaffold, characterized in that: The preparation method comprises: The calcium-deficient hydroxyapatite and recombinant collagen were dissolved in water, a cross-linking agent was added, the mixture was stirred and then allowed to stand to prepare a composite gel scaffold; Wherein, the preparation of the calcium-deficient hydroxyapatite comprises the following steps: S1: calcining natural hydroxyapatite powder at 700℃~900℃ for 2 h~6 h; S2: soaking the calcined hydroxyapatite in a modifier solution to supplement phosphorus; wherein the modifier includes a salt containing phosphorus; and S3: drying the soaked hydroxyapatite, calcining it at 500°C to 700°C for 0.5 h to 3 h to reduce crystallinity, and cooling it to prepare calcium-deficient hydroxyapatite.

2. The preparation method according to claim 1, wherein In the step of adding a cross-linking agent, stirring and then allowing to stand, the allowing to stand comprises: After standing at 25℃~60℃ for 8 h~16 h, stand at 2℃~8℃ for 12 h~24 h.

3. The preparation method according to claim 1, wherein The recombinant collagen satisfies one or more of the conditions A1 to A2: A1. The recombinant collagen comprises one or more of type I collagen and type III collagen; and, A2. The molecular weight of the recombinant collagen is 20,000 Da to 40,000 Da.

4. The preparation method according to claim 1, wherein The preparation method satisfies one or more of the following conditions B1 to B5: B1. The crosslinking agent is selected from the group consisting of a combination of 1-ethyl-3-dimethylaminopropylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) (EDC·NHS), 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM), adipic acid dihydrazide (ADH), and glutaraldehyde; wherein the molar ratio of EDC to NHS in the EDC·NHS is (4.5-7):1; B2. The cross-linking agent is used in an amount of 2% to 8% of the total weight of the calcium-deficient hydroxyapatite and the recombinant collagen; B3. Stirring conditions include: pH 5-7, 5°C-40°C, 50-70 min; B4. The weight ratio of the calcium-deficient hydroxyapatite to the recombinant collagen is 1: (1 to 10); and, B5. In step S3, the prepared calcium-deficient hydroxyapatite is dried at 40°C to 80°C.

5. The preparation method according to any one of claims 1 to 4, characterized in that Step S2 satisfies one or more of the following conditions C1 to C3: C1. The concentration of the modifier is 10 mg / mL~100 mg / mL; C2. The modifier comprises a phosphate; optionally comprising one or more of sodium dihydrogen phosphate, disodium hydrogen phosphate and potassium dihydrogen phosphate; and, C3. The modification temperature is 25℃~60℃, and the modification time is 12 h~36 h.

6. The preparation method according to any one of claims 1 to 4, characterized in that The preparation method further comprises: Wash and sieve granules.

7. The preparation method according to claim 6, wherein The washing and sieving granulation meet one or more of the following conditions D1 to D2: D1. In the washing step, the composite gel scaffold is dialyzed; the number of dialysis is optionally 6 to 10 times; and, D2. Use a sieve with a mesh size of 50-300 mesh to sieve and granulate.

8. The preparation method according to claim 7, wherein The preparation method further comprises low-temperature dehydration at 40°C to 60°C to an ideal collagen concentration; The ideal collagen concentration may be 10 mg / mL to 50 mg / mL.

9. A composite gel scaffold, characterized in that: It is prepared using calcium-deficient hydroxyapatite and recombinant collagen as defined in claim 1; Optionally, the composite gel scaffold is prepared according to the preparation method according to any one of claims 1 to 8.

10. A product for facial filling, characterized in that: It comprises the composite gel scaffold as claimed in claim 9.

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