A tissue repair and filling composite gel, its preparation method and application

Abstract

This invention provides a tissue repair and filling composite gel, its preparation method, and its application, relating to the field of biomedical materials technology. The composite gel is mainly prepared by freeze-thaw physical cross-linking of hyaluronic acid and biodegradable microspheres, wherein: the hyaluronic acid has a molecular weight of 800,000 to 3,000,000, and the biodegradable microspheres are hydroxyapatite microspheres. The tissue repair and filling composite gel of this application consists of a hyaluronic acid matrix with a porous three-dimensional network structure obtained by freeze-thaw physical cross-linking and uniformly dispersed hydroxyapatite microspheres. During the filling and repair process, the hyaluronic acid provides an immediate filling effect, while the hydroxyapatite microspheres stimulate collagen regeneration. This tissue repair and filling composite gel effectively alleviates the technical problems of poor safety associated with existing hyaluronic acid gels obtained by cross-linking agents for in vivo filling, and the inconvenience of injecting regenerated microsphere fillers.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a tissue repair and filling composite gel, its preparation method, and its application. Background Technology

[0002] Hyaluronic acid (HA) is widely used in soft tissue filling due to its excellent biocompatibility and moisturizing properties. Traditional HA fillers often use chemical cross-linking agents (such as 1,4-butanediol glycidyl ether, divinyl sulfone, etc.) to improve their durability in vivo. However, chemical cross-linking may bring the following problems: (1) residual cross-linking agents may cause allergic or inflammatory reactions; (2) the chemical cross-linking process is complex and difficult to control in terms of quality; (3) it is prone to efflorescence. Therefore, exploring new cross-linking methods is of great research significance.

[0003] In recent years, there have been a few reports on the preparation of hyaluronic acid gels using non-chemical crosslinking methods. For example, patent CN117137828A discloses a hyaluronic acid gel membrane using physical crosslinking. However, this method requires acidic conditions and needs to be combined with other support materials. Furthermore, it cannot be sterilized by moist heat and cannot be used for in vivo filling. Patent CN118165310A designed a sodium hyaluronate gel crosslinked by freeze-thaw and BDDE. This method uses an alkaline reaction system and also adds a crosslinking agent, still relying primarily on chemical crosslinking, resulting in residual toxic crosslinking agents. Moreover, the filling effect of the above patents only lasts for 6-12 months, a relatively short duration.

[0004] Therefore, it is both necessary and urgent to research and develop a long-lasting hyaluronic acid tissue repair and filling composite gel prepared by pure physical cross-linking for application in medical aesthetic filling products. Summary of the Invention

[0005] The primary objective of this invention is to provide a tissue repair and filling composite gel prepared using a freeze-thaw physical cross-linking method, which eliminates the need for chemical cross-linking agents, reduces the risk of allergies, and achieves greater safety. Simultaneously, the porous three-dimensional network structure of hyaluronic acid network formed by freeze-thaw in the tissue repair and filling composite gel encapsulates hydroxyapatite microspheres, effectively preventing microsphere aggregation and optimizing degradation compatibility.

[0006] The second objective of this invention is to provide a method for preparing a tissue repair and filling composite gel.

[0007] A third objective of this invention is to provide an application of a tissue repair and filling composite gel.

[0008] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0009] The present invention provides a tissue repair and filling composite gel, which is mainly prepared by freeze-thaw physical cross-linking of hyaluronic acid (HA) and biodegradable microspheres;

[0010] The hyaluronic acid is hyaluronic acid with a molecular weight of 800,000 to 3 million, and the biodegradable microspheres are hydroxyapatite microspheres.

[0011] Furthermore, the particle size of the hydroxyapatite microspheres is 10~100μm, preferably 20~50μm.

[0012] Furthermore, the hyaluronic acid is hyaluronic acid with a molecular weight of 2 million.

[0013] The present invention provides a method for preparing the above-mentioned tissue repair and filling composite gel, the preparation method comprising:

[0014] (A) Dissolve hyaluronic acid in a buffer solution, then add biodegradable microspheres and mix well to obtain a mixed solution;

[0015] (B) The mixed solution is subjected to freeze-thaw physical cross-linking to form an intermediate gel;

[0016] (C) After the intermediate gel is cut into pieces and homogenized, the mobile phase hyaluronic acid and excipients are added and mixed to obtain the tissue repair and filling composite gel.

[0017] Furthermore, in the mixed solution of step (A), the content of hyaluronic acid is 1.5-5.0%, preferably 3%;

[0018] And / or, in the mixed solution of step (A), the content of biodegradable microspheres is 10-20%;

[0019] And / or, the buffer solution in step (A) includes one of phosphate buffer, tris(hydroxymethyl)aminomethane, tris(hydroxymethyl)aminomethane hydrochloride or 4-hydroxyethylpiperazine ethanesulfonic acid, preferably phosphate buffer.

[0020] Furthermore, step (B) freeze-thaw physical crosslinking includes:

[0021] The mixed solution is subjected to 2-8 freeze-thaw cycles;

[0022] Preferably, each cycle includes: freezing at -80°C to -60°C for 1 to 6 hours, preferably 4 hours, followed by thawing at 15 to 30°C.

[0023] Furthermore, in step (C), the mass ratio of the homogenized intermediate gel to the mobile phase hyaluronic acid is 4~9:1~9.

[0024] Furthermore, the excipients in step (C) include: local anesthetics and / or nutrients;

[0025] Preferably, the local anesthetic is lidocaine or a pharmaceutically acceptable salt thereof;

[0026] More preferably, the local anesthetic accounts for 3% of the tissue repair and filling composite gel;

[0027] Preferably, the nutrients include at least one of amino acids and vitamins;

[0028] More preferably, the proportion of the nutrients in the tissue repair and filling composite gel is 0.005~0.8%;

[0029] Furthermore, the preparation method further includes:

[0030] (D) The step of performing moist heat sterilization on the tissue repair filling composite gel;

[0031] Preferably, the F0 of the moist heat sterilization is 8.

[0032] The present invention relates to the application of the tissue repair and filling composite gel provided above in the preparation of medical aesthetic filling products.

[0033] Preferably, the cosmetic filler product is an injectable regenerative cosmetic filler.

[0034] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0035] This invention provides a tissue repair and filling composite gel, which is mainly prepared by freeze-thaw physical cross-linking of hyaluronic acid and biodegradable microspheres, wherein: the hyaluronic acid is hyaluronic acid with a molecular weight of 800,000 to 3,000,000, and the biodegradable microspheres are hydroxyapatite microspheres.

[0036] This tissue repair and filling composite gel is prepared using a freeze-thaw physical cross-linking method, eliminating the need for chemical cross-linking agents, reducing the risk of allergies, and achieving greater safety. Simultaneously, the porous three-dimensional network structure of hyaluronic acid formed by freeze-thaw interaction encapsulates hydroxyapatite microspheres, effectively preventing microsphere aggregation and optimizing degradation compatibility. This tissue repair and filling composite gel consists of a porous three-dimensional network structure of hyaluronic acid matrix and uniformly dispersed hydroxyapatite microspheres. During the filling and repair process, hyaluronic acid provides an immediate filling effect, while the hydroxyapatite microspheres stimulate collagen regeneration. This tissue repair and filling composite gel effectively alleviates the poor safety issues associated with existing hyaluronic acid gels relying on cross-linking agents for in vivo filling, as well as the inconvenience of injecting regenerated microsphere fillers. This tissue repair and filling composite gel can be widely used in the preparation of cosmetic filling products.

[0037] This invention provides a method for preparing a tissue repair and filling composite gel. The method involves first preparing a mixed solution of hyaluronic acid and biodegradable microspheres; then, subjecting the mixed solution to freeze-thaw physical cross-linking to form an intermediate gel; subsequently, homogenizing the intermediate gel and adding the mobile phase of hyaluronic acid and excipients, mixing thoroughly to obtain the tissue repair and filling composite gel. This preparation method has the advantages of simple processing and ease of operation.

[0038] The tissue repair and filling composite gel provided by this invention can be widely used in the preparation process of cosmetic filling products. Attached Figure Description

[0039] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0040] Figure 1 SEM image of the tissue repair and filling composite gel prepared in Example 1 of Experimental Example 2 of this invention;

[0041] Figure 2 This is a SEM image of the tissue repair and filling composite gel prepared in Comparative Example 1, provided in Experimental Example 2 of the present invention.

[0042] Figure 3 Cell activity analysis diagram of the tissue repair and filling composite gel prepared in Example 1 and Comparative Example 1, which are experimental examples of the present invention. Detailed Implementation

[0043] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0044] It should be noted that physical crosslinking technology constructs a three-dimensional network structure of hyaluronic acid through non-covalent interactions (such as hydrogen bonds, ionic bonds, hydrophobic interactions, molecular entanglement, and physical embedding), thereby fundamentally solving key problems associated with chemical crosslinking, such as residual crosslinking agents, potential toxicity, and uncontrollable inflammatory reactions. Hyaluronic acid gels prepared using physical crosslinking, without completely avoiding the use of chemical crosslinking agents, not only achieve instant gel molding and injectability, but also allow for customized design of the material's mechanical properties, degradation cycle, and pore structure by precisely controlling the crosslinking density (such as freeze-thaw cycles, ion concentration, and pH value).

[0045] Meanwhile, existing regenerative microsphere fillers used for cosmetic filling have certain limitations, such as: (1) requiring multiple injections (usually more than 3 times) and slow to take effect (3-6 months); (2) easily forming nodules or granules when used alone, affecting aesthetics; (3) requiring high injection technique, and improper operation may lead to uneven distribution.

[0046] According to one aspect of the present invention, the present invention provides a tissue repair and filling composite gel, the tissue repair and filling composite gel being mainly prepared by freeze-thaw physical cross-linking of hyaluronic acid and biodegradable microspheres, wherein: the hyaluronic acid is hyaluronic acid with a molecular weight of 800,000 to 3,000,000, and the biodegradable microspheres are hydroxyapatite microspheres.

[0047] The tissue repair and filling composite gel provided in this application is prepared using a freeze-thaw physical cross-linking method, eliminating the need for chemical cross-linking agents, reducing the risk of allergies, and achieving greater safety. Simultaneously, the porous three-dimensional network structure of hyaluronic acid formed by freeze-thaw interaction encapsulates hydroxyapatite microspheres, effectively preventing microsphere aggregation and optimizing degradation compatibility. This tissue repair and filling composite gel consists of a porous three-dimensional network structure of hyaluronic acid matrix and uniformly dispersed hydroxyapatite microspheres. During the filling and repair process, hyaluronic acid provides an immediate filling effect, while the hydroxyapatite microspheres stimulate collagen regeneration. This tissue repair and filling composite gel effectively alleviates the poor safety issues associated with existing hyaluronic acid gels relying on cross-linking agents for in vivo filling, as well as the inconvenience of injecting regenerated microsphere fillers. This tissue repair and filling composite gel can be widely used in the preparation of cosmetic filling products.

[0048] It should be noted that the freeze-thaw physical crosslinking method of the present invention induces the self-assembly of hyaluronic acid molecules to form a three-dimensional network structure through a controllable low-temperature phase transition process: during the freezing stage, the formation of ice crystals forces the hyaluronic acid molecular chains to concentrate and approach each other in the gaps between the ice crystals, promoting the formation of a large number of intermolecular hydrogen bonds between carboxyl and hydroxyl groups; during the subsequent thawing process, the selective escape of water molecules when the ice crystals melt causes the hyaluronic acid network to elastically shrink and lock the structure. After 3-5 freeze-thaw cycles, a physical crosslinking network with gradient pore size distribution and dynamic reversible crosslinking points is finally formed. This process completely avoids the use of chemical crosslinking agents and preserves the natural conformation of HA.

[0049] Furthermore, sodium hyaluronate and hydroxyapatite can achieve ionic cross-linking through electrostatic interactions and coordination chemistry: under physiological pH conditions, the carboxylate group (-COO⁻) on the HA chain interacts with the calcium ions (Ca) on the surface of hydroxyapatite. 2+ Electrostatic attraction occurs, leading to the formation of a stable five-membered ring coordination structure (Ca-OC=O); simultaneously, the hydroxyl group (-OH) of HA reacts with the phosphate group (PO4) of hydroxyapatite. 3- ) generates a hydrogen bond network through multivalent Ca 2+ The bridging effect constructs a three-dimensional cross-linked network.

[0050] Therefore, this application prepares a tissue repair and filling composite gel by freeze-thaw physical cross-linking of hyaluronic acid and biodegradable microspheres. This not only significantly improves the elasticity of the gel material and is beneficial to the mechanical strength of filling and shaping, but also maintains the bioactivity of hydroxyapatite and endows the material with pH-responsive characteristics (dissociation at pH < 5), making it particularly suitable for medical aesthetic filling applications that require osseointegration.

[0051] As an optional implementation, the molecular weight of the hyaluronic acid can be, but is not limited to, 800,000, 1,000,000, 1,500,000, 2,000,000, 2,500,000 or 3,000,000, or any value between 800,000 and 3,000,000.

[0052] As an optional implementation, the particle size of the hydroxyapatite microspheres can be, but is not limited to, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm or 90 μm, or any value between 10 and 100 μm.

[0053] In the preferred embodiment described above, the hyaluronic acid is hyaluronic acid with a molecular weight of 2 million.

[0054] According to one aspect of the present invention, a method for preparing the above-mentioned tissue repair and filling composite gel, the preparation method comprising:

[0055] (A) Dissolve hyaluronic acid in a buffer solution, then add biodegradable microspheres and mix well to obtain a mixed solution;

[0056] (B) The mixed solution is subjected to freeze-thaw physical cross-linking to form an intermediate gel;

[0057] (C) After the intermediate gel is cut into pieces and homogenized, the mobile phase hyaluronic acid and excipients are added and mixed to obtain the tissue repair and filling composite gel.

[0058] This invention provides a method for preparing a tissue repair and filling composite gel. The method involves first preparing a mixed solution of hyaluronic acid and biodegradable microspheres; then, subjecting the mixed solution to freeze-thaw physical cross-linking to form an intermediate gel; subsequently, homogenizing the intermediate gel and adding the mobile phase of hyaluronic acid and excipients, mixing thoroughly to obtain the tissue repair and filling composite gel. This preparation method has the advantages of simple processing and ease of operation.

[0059] As an optional implementation, the hyaluronic acid content in the mixed solution in step (A) can be, but is not limited to, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5% or 5.0%, or any value between 1.5% and 5.0%.

[0060] As an optional implementation, the content of biodegradable microspheres in the mixed solution in step (A) can be, but is not limited to, 10%, 12%, 14%, 16%, 18% or 20%, or any value between 10% and 20%.

[0061] As an optional implementation, the buffer solution in step (A) includes one of phosphate buffer, tris(hydroxymethyl)aminomethane, tris(hydroxymethyl)aminomethane hydrochloride, or 4-hydroxyethylpiperazine ethanesulfonic acid, preferably phosphate buffer.

[0062] In a preferred embodiment of the present invention, step (B) freeze-thaw physical crosslinking includes:

[0063] The mixed solution is subjected to 2-8 freeze-thaw cycles; each cycle includes: rapid freezing at -80℃ to -60℃ for 1-6 hours, followed by thawing at 15-30℃.

[0064] As an optional implementation, the number of freeze-thaw cycles can be, but is not limited to, 1, 2, 3, 4, 5, 6, 7, or 8.

[0065] As an optional implementation, the freezing temperature can be, but is not limited to, -80°C, -70°C, -65°C or -60°C, or any value between -80°C and -60°C.

[0066] As an optional implementation, the freezing time can be, but is not limited to, 1 hour, 3 hours, 4 hours or 6 hours, or any value between 1 and 6 hours.

[0067] As an optional implementation, the thawing temperature can be, but is not limited to, 15°C, 20°C, 25°C or 30°C, or any value between 15°C and 30°C.

[0068] In a preferred embodiment of the present invention, in step (C), the mass ratio of the homogenized intermediate gel to the mobile phase hyaluronic acid is 4~9:1~9.

[0069] In a preferred embodiment of the present invention, the excipients in step (C) include: local anesthetic and / or nutrients;

[0070] In the preferred embodiment described above, the local anesthetic is lidocaine or a pharmaceutically acceptable salt thereof;

[0071] In the preferred embodiment described above, the local anesthetic accounts for 3% of the tissue repair and filling composite gel;

[0072] In the preferred embodiment described above, the nutrients include at least one of amino acids and vitamins;

[0073] In the preferred embodiment described above, the proportion of the nutrients in the tissue repair and filling composite gel is 0.005~0.8%;

[0074] In the preferred embodiment described above, the mass ratio of the body gel to the mobile phase hyaluronic acid is 9:1;

[0075] In a preferred embodiment of the present invention, the preparation method further includes: (D) a step of moist heat sterilization of the tissue repair filling composite gel;

[0076] Preferably, the F0 of the moist heat sterilization is 8.

[0077] According to one aspect of the present invention, the above-described tissue repair and filling composite gel is used in the preparation of cosmetic filling products.

[0078] The tissue repair and filling composite gel provided by this invention can be widely used in the preparation process of cosmetic filling products.

[0079] It should be noted that the cryo-physical cross-linking method of this application significantly improves the clinical safety of tissue repair and filling composite gel (cell survival rate > 95%), while retaining the natural bioactivity of hyaluronic acid. It shows unique advantages in the fields of medical aesthetic filling (such as long-lasting water light injection), joint lubrication (such as osteoarthritis treatment), wound dressing (such as burn repair) and drug sustained release (such as anticancer drug carrier), and represents the development direction of the next generation of biomedical materials.

[0080] The technical solution of the present invention will be further described below with reference to the embodiments.

[0081] Example 1

[0082] A method for preparing a tissue repair and filling composite gel, the method comprising:

[0083] (1) Sodium hyaluronate with a molecular weight of 2 million was dissolved in phosphate buffer solution to prepare a solution with a hyaluronic acid content of 3%. Then, hydroxyapatite microspheres were added and mixed to obtain a mixed solution.

[0084] The hydroxyapatite microspheres have a particle size of 20-50 μm, and the amount of hydroxyapatite microspheres added is 20% of the mass of the mixed solution.

[0085] (2) The mixed solution is subjected to freeze-thaw physical cross-linking to form an intermediate gel;

[0086] The specific method is as follows: the mixed solution is subjected to three freeze-thaw cycles, each cycle including: rapid freezing at -80°C for 4 hours, followed by thawing at 20°C.

[0087] (3) After cutting the intermediate gel into pieces, homogenize it. The parameters of the homogenizer are: air pressure 0.07MPa and homogenizer head 6000-8000rpm. Then add the mobile phase hyaluronic acid and excipients and mix well to obtain tissue repair and filling composite gel.

[0088] The mobile phase contained 10 mg / ml of hyaluronic acid, and the solvent was phosphate buffer.

[0089] The mass ratio of the intermediate gel to the mobile phase hyaluronic acid was 9:1.

[0090] The excipient is lidocaine, and the amount of lidocaine added is 3% of the mass of the tissue repair and filling composite gel.

[0091] (4) After filling the syringe with tissue repair filling composite gel, perform moist heat sterilization with sterilization parameters F0=8.

[0092] Example 2

[0093] Except for step (1), in which “the amount of hydroxyapatite microspheres added is 10% of the mass of the mixed solution”, the rest of this embodiment is the same as in embodiment 1.

[0094] Example 3

[0095] Except for step (1) where “the hyaluronic acid is hyaluronic acid with a molecular weight of 800,000”, the rest of this embodiment is the same as in embodiment 1.

[0096] Example 4

[0097] Except for step (1), in which "the hyaluronic acid is hyaluronic acid with a molecular weight of 3 million", the rest of this embodiment is the same as in embodiment 1.

[0098] Example 5

[0099] In this embodiment, except for step (2), the specific method of freeze-thaw physical crosslinking is as follows:

[0100] The mixed solution was subjected to two freeze-thaw cycles, each cycle consisting of: rapid freezing at -80°C for 4 hours, followed by thawing at 20°C.

[0101] The difference between this embodiment and Embodiment 1 is that the mixed solution undergoes two freeze-thaw cycles.

[0102] Example 6

[0103] In this embodiment, except for step (2), the specific method of freeze-thaw physical crosslinking is as follows:

[0104] The mixed solution was subjected to 8 freeze-thaw cycles, each cycle consisting of: rapid freezing at -80°C for 4 hours, followed by thawing at 20°C.

[0105] The difference between this embodiment and Embodiment 1 is that the mixed solution undergoes 8 freeze-thaw cycles.

[0106] Example 7

[0107] In this embodiment, except for step (2), the specific method of freeze-thaw physical crosslinking is as follows:

[0108] The mixed solution was subjected to three freeze-thaw cycles, each cycle consisting of: rapid freezing at -80°C for 1 hour, followed by thawing at 20°C.

[0109] The difference between this embodiment and Embodiment 1 is that the freezing time for each cycle is 1 hour.

[0110] Example 8

[0111] In this embodiment, except for step (2), the specific method of freeze-thaw physical crosslinking is as follows:

[0112] The mixed solution was subjected to three freeze-thaw cycles, each cycle consisting of: rapid freezing at -80°C for 6 hours, followed by thawing at 20°C.

[0113] The difference between this embodiment and Embodiment 1 is that the freezing time for each cycle is 6 hours.

[0114] Example 9

[0115] Except for step (4), where the sterilization parameter for moist heat sterilization is F0=12, this embodiment is the same as in embodiment 1.

[0116] Example 10

[0117] Except for step (4), where the sterilization parameter for moist heat sterilization is F0=15, this embodiment is the same as in embodiment 1.

[0118] Example 11

[0119] Except for step (1) where “the particle size of the hydroxyapatite microspheres is 150~200μm”, this embodiment is the same as in Example 1.

[0120] Example 12

[0121] Except for step (1) where “the particle size of the hydroxyapatite microspheres is 2~8μm”, this embodiment is the same as in embodiment 1.

[0122] Comparative Example 1

[0123] A method for preparing a tissue repair and filling composite gel, the method comprising:

[0124] (1) Sodium hyaluronate with a molecular weight of 2 million was dissolved in phosphate buffer solution to prepare a solution with a hyaluronic acid content of 3%; then the mobile phase hyaluronic acid and excipients were added and mixed to obtain a tissue repair and filling composite gel.

[0125] The mobile phase hyaluronic acid and excipients are the same as in Example 1;

[0126] The mass ratio of hyaluronic acid solution to mobile phase hyaluronic acid is 9:1;

[0127] (2) After filling the syringe with tissue repair filling composite gel, perform moist heat sterilization with sterilization parameters F0=8.

[0128] The difference between this comparative example and Example 1 is that no physical cross-linking was performed.

[0129] Comparative Example 2

[0130] A method for preparing a tissue repair and filling composite gel, the method comprising:

[0131] (1) Dissolve sodium hyaluronate with a molecular weight of 2 million in phosphate buffer solution to prepare a solution with a hyaluronic acid content of 3%;

[0132] (2) The solution containing 3% hyaluronic acid in step (1) above is subjected to freeze-thaw physical cross-linking to form an intermediate gel;

[0133] The specific method is as follows: the mixed solution is subjected to three freeze-thaw cycles, each cycle including: rapid freezing at -80°C for 4 hours, followed by thawing at 20°C.

[0134] (3) Same as Example 1;

[0135] (4) Same as Example 1.

[0136] The difference between this comparative example and Example 1 is that hydroxyapatite microspheres were not added to the gel.

[0137] Comparative Example 3

[0138] This comparative example is the same as Example 1, except that the hydroxyapatite microspheres in step (1) are replaced with an equal amount of "poly-L-lactic acid microspheres with a particle size of 20~50um".

[0139] Comparative Example 4

[0140] Except for step (1), in which "the hyaluronic acid is hyaluronic acid with a molecular weight of 200,000", the comparative example is the same as in Example 1.

[0141] Comparative Example 5

[0142] In this embodiment, except for step (2), the specific method of freeze-thaw physical crosslinking is as follows:

[0143] The mixed solution was subjected to three freeze-thaw cycles, each cycle consisting of: rapid freezing at -20°C for 4 hours, followed by thawing at 20°C.

[0144] The difference between this embodiment and Embodiment 1 is that the freezing temperature of the freeze-thaw cycle is -20°C.

[0145] Experimental Example 1

[0146] This experiment tested the performance of the tissue repair and filling composite gels prepared in Examples 1-10 and Comparative Examples 1-5. The specific testing methods are as follows:

[0147] 1. Rheological Properties: The rheological properties of the filler were determined using a rotational rheometer. Tests were conducted at 25°C using a parallel plate fixture. First, the linear viscoelastic region was determined through strain scanning (0.01%-100%, 1Hz). Then, frequency scanning (0.1-100 rad / s) was performed at selected strains (typically 0.1-1%), focusing on the storage modulus (G') and loss modulus (G") at 1Hz. Creep-recovery tests characterized the viscoelastic behavior. All tests were repeated three times to ensure data reliability.

[0148] 2. Pushing Force Test: The pushing force was tested using a texture analyzer and a 1mL syringe. The filler sample was precisely loaded into a 1mL syringe, a 26G needle was attached, and the piston rod was advanced at a constant speed of 50mm / min, simulating clinical injection. The force change throughout the pushing process was recorded in real time (sampling frequency 100Hz). The test was conducted at 23±1℃, and each group of samples was measured in parallel 6 times. The average value was taken after discarding the first measurement. Specific test results are shown in Tables 1 and 2.

[0149] Table 1. Detection results of Examples 1-12 of the present invention:

[0150]

[0151] Table 2. Detection results of Comparative Examples 1-5 of the present invention:

[0152]

[0153] As shown in Tables 1 and 2 above, the tissue repair and filling composite gels prepared in Examples 1-8 of this application possess suitable rheological properties and extrusion force. Specifically, the comparison results of Examples 1 and 2 show that the elasticity of the gel system is significantly improved after adding hydroxyapatite. This is because the increase in hydroxyapatite enhances the ability to physically crosslink with hyaluronic acid, further improving the crosslinking effect. The comparison results of Examples 1, 3, and 4 show that as the molecular weight increases, the viscoelasticity tends to increase, but the difference is not significant. The comparison results of Examples 1, 5, and 6 show that increasing the number of freezing cycles can improve the viscoelasticity of the product. The number of freezing cycles can be adjusted according to actual needs, but the elasticity results are similar between 3 cycles and 8 cycles, so 3 freezing cycles are preferable. The comparison results of Examples 1, 7, and 8 show that extending the freezing time increases the elasticity of the physically crosslinked gel, which can improve the crosslinking effect to a certain extent.

[0154] The difference between Examples 9 and 10 and Example 1 is the sterilization parameters. The results show that as the sterilization intensity increases, the viscoelasticity of the product decreases significantly. Considering all factors, the gel system of this invention is not suitable for higher sterilization intensities, and F0=8 is more reasonable.

[0155] The difference between Examples 11 and 12 and Example 1 is that the particle size of the hydroxyapatite microspheres is not within the range of 10-100 μm. Results showed that Example 11 resulted in needle bursting, excessive pushing force, and extremely high viscoelasticity. This was due to the excessively large particle size causing needle blockage and excessive pushing force, which is detrimental to clinical application. Example 12 used 2-8 μm hydroxyapatite, which had poor physical cross-linking and low viscoelasticity. Furthermore, the smaller particle size made it easily phagocytosed by macrophages after subcutaneous injection. Therefore, the particle size range of hydroxyapatite should be within a suitable range, i.e., the particle size of the hydroxyapatite microspheres should be 10-100 μm.

[0156] Comparative Example 1 did not undergo a freeze-reconstitution operation. Compared with Example 1, Comparative Example 1 had lower propulsion and viscoelasticity, demonstrating that low-temperature cyclic freezing can promote the physical cross-linking of sodium hyaluronate solution.

[0157] Comparative Example 2, which did not contain hydroxyapatite microspheres, showed higher viscoelasticity compared to Comparative Example 1. This demonstrates that physical cross-linking of hyaluronic acid was achieved under these freezing cycle conditions. Compared to Example 1, the viscoelasticity of cross-linked HA was lower, which was due to the lack of hydroxyapatite, making it impossible to achieve secondary physical cross-linking of HA and hydroxyapatite.

[0158] In Comparative Example 3, the elasticity of the poly-L-lactic acid microspheres was lower than that of the hydroxyapatite microspheres. This is because, in addition to the physically cross-linked HA, the calcium and magnesium ions on the surface of hydroxyapatite can form ionic cross-links with hyaluronic acid, further enhancing the cross-linking strength of the gel system.

[0159] Comparative Example 4 uses hyaluronic acid with a molecular weight of 200,000. Its viscoelasticity is significantly lower than that of Example 1, while its viscoelasticity and propulsion force data are comparable to Comparative Example 1. This is because the lower molecular weight of the hyaluronic acid results in poorer molecular chain entanglement and weaker cross-linking of the molecular chains during the freeze-thaw cycle, making physical cross-linking between hyaluronic acid molecules virtually impossible. Therefore, lower molecular weights are not suitable for freeze-thaw physical cross-linking conditions.

[0160] Comparative Example 5, which involved a freeze-thaw cycle with a blast temperature of -20°C, showed that the elasticity data was significantly lower than that of Example 1, indicating that the freezing temperature had a greater impact on crosslinking.

[0161] Experiment Example 2

[0162] This experimental example uses the tissue repair and filling composite gel prepared in Example 1 as an example to analyze the microstructure of the tissue repair and filling composite gel of this application. The specific method is as follows:

[0163] The tissue repair and filling composite gel samples prepared in Example 1 and Comparative Example 1 were frozen at -20 °C for 12 hours, then freeze-dried for 24 hours. Small pieces of the samples were obtained by liquid nitrogen fracturing and then sputter-coated with gold. The cross-section of the hydrogel was scanned. SEM images of the physically cross-linked hyaluronic acid showed an interconnected three-dimensional network porous structure.

[0164] Figure 1 This is a SEM image of the tissue repair and filling composite gel prepared in Example 1 of the present invention.

[0165] Figure 2 This is a SEM image of the tissue repair and filling composite gel prepared in Comparative Example 1 of this invention.

[0166] Depend on Figure 1 , Figure 2It can be seen that the cross-sectional pore structure of the physically cross-linked hyaluronic acid hydrogel in Comparative Example 1 is more porous, while the microspheres formed after the introduction of hydroxyapatite in the gel system of Example 1 of this application are uniformly distributed inside the gel. SEM electron microscopy images show the porous structure of the freeze-thawed HA of the present invention and the uniform distribution of microspheres.

[0167] Experimental Example 3

[0168] This experimental example uses the tissue repair and filling composite gel prepared in Example 1 as an example to analyze the cell activity of the tissue repair and filling composite gel of this application. The specific method is as follows:

[0169] Cell viability assay: The biocompatibility of hydrogels plays a crucial role in maintaining cell viability, proliferation and differentiation in tissue engineering. The effect of the gel filler system prepared in this invention on the viability of rat-derived mesenchymal cells was determined by dual dyeing with Calcein-AM and PI.

[0170] This experiment included a saline control group (blank), a comparative example group (physical cross-linking group), and an example group (physical cross-linked hydrogel composite hydroxyapatite system group), with 6 parallel wells in each group. Extracts of different gel materials were added to the wells of the cell-seeded plates. After 24 hours of incubation, Calcein-AM and PI dual dyes were added, and after 30 minutes of incubation, cell viability and death staining were detected under a fluorescence microscope at 490 nm and 545 nm excitation. Live cells appeared green, and dead cells appeared red. The experimental results are as follows: Figure 3 As shown.

[0171] Figure 3 This is a cell activity analysis diagram of the tissue repair and filling composite gel prepared in Example 1 and Comparative Example 1 of the present invention.

[0172] Depend on Figure 3 It can be seen that, compared with the blank group, the cells in Example 1 and Comparative Example 1 maintained high activity, indicating that the physically cross-linked hydrogel composite hydroxyapatite system has no obvious toxicity to cells and has high biocompatibility.

[0173] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A tissue repair filling composite gel, characterized by, The tissue repair filling composite gel is mainly prepared by freeze-thaw physical crosslinking of hyaluronic acid and biodegradable microspheres. The hyaluronic acid has a molecular weight of 80-3 million, and the biodegradable microspheres are hydroxyapatite microspheres. The freeze-thaw physical crosslinking includes 2-8 cycles of freezing-thawing, each cycle including rapid freezing at-80℃ to-60℃ for 1-6 hours and then thawing at 15-30℃. The preparation method of the tissue repair filling composite gel comprises: (A) dissolving hyaluronic acid in a buffer solution, then adding biodegradable microspheres and mixing to obtain a mixed solution; (B) freeze-thaw physical crosslinking of the mixed solution to form an intermediate gel; (C) cutting and homogenizing the intermediate gel, then adding mobile phase hyaluronic acid and excipients and mixing to obtain the tissue repair filling composite gel.

2. The tissue repair filling composite gel according to claim 1, wherein, The hydroxyapatite microspheres have a particle size of 10-100 μm.

3. The tissue repair filling composite gel according to claim 1, wherein, The hyaluronic acid has a molecular weight of 2 million.

4. A method for preparing the tissue repair filling composite gel according to any one of claims 1 to 3, characterized in that, The preparation method of the tissue repair filling composite gel comprises: (A) dissolving hyaluronic acid in a buffer solution, then adding biodegradable microspheres and mixing to obtain a mixed solution; (B) freeze-thaw physical crosslinking of the mixed solution to form an intermediate gel; (C) cutting and homogenizing the intermediate gel, then adding mobile phase hyaluronic acid and excipients and mixing to obtain the tissue repair filling composite gel.

5. The method of claim 4, wherein the tissue repair filler composite gel is prepared by the steps of: In the mixed solution of step (A), the content of hyaluronic acid is 1.5-5.0%; And / or, in the mixed solution of step (A), the content of biodegradable microspheres is 10-20%, and the biodegradable microspheres are hydroxyapatite microspheres; And / or, the buffer solution in step (A) comprises one of phosphate buffer, tris, tris hydrochloride or 4-hydroxyethylpiperazine ethanesulfonic acid.

6. The method of claim 4, wherein the tissue repair filler composite gel is prepared by the steps of: After homogenization, the mass ratio of the intermediate gel to mobile phase hyaluronic acid in step (C) is 4-9:1-9.

7. The method of claim 4, wherein the tissue repair filler composite gel is prepared by the steps of: The excipients in step (C) include local anesthetics and / or nutrients; And / or, the local anesthetics are lidocaine or pharmaceutically acceptable salts thereof; And / or, the proportion of the local anesthetics in the tissue repair filling composite gel is 3%; And / or, the nutrients include at least one of amino acids and vitamins; And / or, the proportion of the nutrients in the tissue repair filling composite gel is 0.005-0.8%.

8. The method of claim 4, wherein the tissue repair filler composite gel is prepared by the steps of: The preparation method further comprises: (D) a step of moist heat sterilization of the tissue repair filling composite gel; And / or, the F0 of the moist heat sterilization is 8.

9. Use of the tissue repair filling composite gel of any one of claims 1-3 in the preparation of medical and aesthetic plastic filling products.

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