Injection filling material and preparation and application thereof

Abstract

The application relates to the field of biomedical cosmetology, and provides an injection filling material and a preparation and application thereof. The preparation method of the injection filling material is that slurry formed by decellularized allogenic dermal microparticles with D50 of 100-155 mu m is mixed with an aqueous solution of sodium hyaluronate with a concentration of 1-1.5%, and then irradiation is carried out under the condition of 5-25 kGy for 10-30 min; the slurry is obtained by jointly crushing decellularized allogenic dermis and purified water; the injection filling material of the application has a wide application range, the injection filling material of the application not only makes up for the defect that the degradation time of pure sodium hyaluronate is short, but also makes up for the defect that the decellularized allogenic dermal filling material is prone to delamination by utilizing the chemical bonding reaction of sodium hyaluronate and dermal microparticles after mixing and irradiation, so that the stability of the system is increased, and the injection filling material is more suitable for medical and cosmetic filling materials.

Description

Technical Field

[0001] This invention relates to the field of biomedical aesthetics technology, specifically to an injectable filler material, its preparation and application, and more particularly to a filler material composed of sodium hyaluronate and decellularized allogeneic dermis, its preparation and application. Background Technology

[0002] Acellular allogeneic dermis is a natural human-derived extracellular matrix material with a natural three-dimensional collagen framework. It is free of immunogenic substances and exhibits excellent biocompatibility, finding wide application in dermal injury, oral mucosal repair, breast augmentation, and hernia repair. Without cross-linking, it can remain in vivo for at least one year, making it an excellent natural support material in the field of cosmetic fillers.

[0003] However, research into expanding the applications of acellular allogeneic dermis revealed that when the acellular allogeneic dermis was pulverized into particles and mixed with water, the solution separated into layers, rendering it unusable. This separation was even more pronounced after irradiation sterilization. This is likely because the water-based solution lacks viscosity and the mixture with the dermis is purely physical, leading to gradual separation due to differences in their densities. In the laboratory, the slurry formed from acellular allogeneic dermis particles exhibited separation after standing for 72 hours, resulting in uneven injection when used as a filler material in cosmetic procedures.

[0004] To improve the layering phenomenon, researchers have attempted to add substances such as hyaluronic acid, collagen, and gelatin, utilizing their unique viscosity to provide a suspending effect. However, in practical applications, they have found that even with the addition of suspending agents, the layering phenomenon still occurs under motion or irradiation conditions. Furthermore, transportation and irradiation sterilization processes are unavoidable for filler materials in the medical aesthetics field.

[0005] Therefore, this invention is proposed. Summary of the Invention

[0006] To address the issue of layering, suspending agents are currently the mainstream solution. Among the existing suspending agents, hyaluronic acid, as a base material, has many applications in the medical field, especially in injectable fillers for cosmetic surgery and wrinkle removal. It has good biocompatibility and safety, and is widely used in various fields. However, it also has certain problems. For example, natural sodium hyaluronate generally degrades quickly after being injected subcutaneously, and cannot maintain its effects for a long time, leading to the need for frequent re-injections. Therefore, cross-linking or modification methods are generally used to extend the degradation time of sodium hyaluronate. For example, cross-linked injectable sodium hyaluronate aqueous solutions or gels have been developed, which can generally maintain support for 6 to 18 months. However, this increases the granular feel of the sodium hyaluronate, and the introduction of cross-linking agents undoubtedly increases safety risks.

[0007] In other words, both decellularized allogeneic dermal microparticles and natural sodium hyaluronate have stability issues when used as filler materials.

[0008] In the experiment, it was unexpectedly discovered that by mixing a slurry formed from decellularized allogeneic dermal microparticles with a D50 of 100μm~155μm with an aqueous solution of sodium hyaluronate at a concentration of 1~1.5%, and then irradiating it at 5~25kGy for 10~30min, the stability of the resulting injectable filler material can be significantly improved. Specifically, it does not stratify after being stored at room temperature and 0~8℃ for more than 24 weeks. Especially at 15kGy, after irradiation, the injectable filler material can still maintain a stable state without stratification under high-speed centrifugation (3000r / min). Moreover, the degradation rate of the injectable filler material in vivo can reach less than 10% within 1 week and less than 50% within 48 weeks.

[0009] Furthermore, for the selected material composed of an aqueous solution of sodium hyaluronate and decellularized allogeneic dermis, the degradation time of the sodium hyaluronate aqueous solution in vivo is extended without the use of a cross-linking agent. When the material of the present invention, which is composed of an aqueous solution of sodium hyaluronate and decellularized allogeneic dermis, is used as an injectable filler, both materials are naturally derived and have good biocompatibility. This not only makes up for the short degradation time of sodium hyaluronate alone, but also uses sodium hyaluronate to compensate for the disadvantage of decellularized allogeneic dermis filler material being prone to delamination when placed alone.

[0010] Specifically, the present invention provides a method for preparing an injectable filler material, which involves mixing a slurry formed from decellularized allogeneic dermal microparticles with a D50 of 100μm~155μm with an aqueous solution of sodium hyaluronate at a concentration of 1~1.5%, and then irradiating the mixture at a temperature of 5~25kGy for 10~30min; the slurry is obtained by pulverizing decellularized allogeneic dermal microparticles and purified water together.

[0011] The aforementioned D50 refers to the particle size of the decellularized allogeneic dermal microparticles in the slurry formed by the decellularized allogeneic dermal microparticles.

[0012] The experiment found that the stability of the injection filler was significantly improved when the irradiation dose was 15 kGy; moreover, the concentration of the sodium hyaluronate aqueous solution affected the stability of the injection filler, and the preferred concentration was 1% to further improve the stability of the injection filler.

[0013] This invention improves the sterilizability and stability of materials during transportation by combining sodium hyaluronate and decellularized allogeneic dermal microparticles under specific conditions. Furthermore, the method provided by this invention can further control the degradation time by adjusting the pulverization time of the decellularized allogeneic dermal microparticles and controlling the particle size; preferably, stirring at a speed of 1000-3000 r / min for 10-30 min is used.

[0014] According to the preparation method of the injectable filler material provided by the present invention, the preparation process of the slurry is as follows: healthy donor skin is chemically treated to remove cells and impurities, retaining the natural three-dimensional collagen network structure of extracellular matrix (ECM), residual reagents are washed off, and the washed decellularized allogeneic dermal extracellular matrix is ​​mixed with purified water and then pulverized.

[0015] According to the method for preparing injection filler material provided by the present invention, the specific preparation steps of the slurry are as follows:

[0016] (1) The donor skin is treated with sodium hydroxide solution of 1-10% mass concentration to remove cells, and after washing 4-10 times, decellularized allogeneic dermis is obtained;

[0017] (2) Weigh out decellularized allogeneic dermis, add purified water and pulverize it; when pulverizing, the ratio of decellularized allogeneic dermis to purified water is 1:5~1:20; the pulverization time is 1~30min.

[0018] According to the preparation method of the injection filler material provided by the present invention, the mass ratio of the slurry to the aqueous solution of sodium hyaluronate during mixing is 1:1 to 1:10.

[0019] According to the preparation method of the injection filler material provided by the present invention, the mass ratio of the slurry to the aqueous solution of sodium hyaluronate during mixing is 1:1.

[0020] According to the preparation method of the injection filler material provided by the present invention, the molecular weight of sodium hyaluronate in the aqueous solution of sodium hyaluronate is above 1000kd.

[0021] This concentration of sodium hyaluronate aqueous solution is non-crosslinked, stirs evenly without bubbles, and is clear and transparent.

[0022] The preparation process of the sodium hyaluronate aqueous solution is as follows: weigh sodium hyaluronate and add purified water, stir until clear and free of bubbles.

[0023] According to the preparation method of the injection filling material provided by the present invention, the irradiation is carried out by filling the mixed mixture with a pre-filled syringe in a clean room of Class 10,000 or above, and then sealing it in a blister box.

[0024] The present invention also provides an injection filler material prepared by the method described above.

[0025] This invention optimizes the storage stability of filler materials by optimizing the selection of raw materials and controlling irradiation conditions, so that sodium hyaluronate in the filler material reaches its optimal state during irradiation, making the injectable filler material uniform and stable during multiple sterilization and transportation storage processes.

[0026] The present invention also provides the application of the injectable filler material as described above, which is to use the injectable filler material as a medical aesthetic filler material; the filler material is used by injection.

[0027] The injectable filler material provided by this invention also achieves uniform stability during multiple sterilization and transportation storage processes, while making the degradation time of the injectable filler material more suitable for use as an injectable filler material.

[0028] This invention provides an injectable filler material based on decellularized allogeneic dermis, its preparation, and its application. By controlling the particle size of the decellularized allogeneic dermis microparticles and compounding them with an aqueous solution of sodium hyaluronate at a specific concentration, under specific irradiation conditions, the injectable filler material of this invention can remain stable and non-stratified after irradiation and high-speed centrifugation, making it suitable for use as an injectable filler material. Furthermore, the degradation time of the injectable filler material is also adjusted.

[0029] This invention provides an injectable filler material obtained by mixing an aqueous solution of sodium hyaluronate with a slurry formed from decellularized allogeneic dermal microparticles. This solution addresses the issue of stratification of decellularized allogeneic dermal microparticles during application and yields a natural filler material that provides stable filling over a long period without the use of any cross-linking agents. Furthermore, because dermis possesses the same elasticity and extensibility as human skin, it avoids the grainy feel associated with cross-linked injectable sodium hyaluronate solutions or gel-like materials. In summary, this invention utilizes the stabilizing effect of sodium hyaluronate and the long degradation time of decellularized allogeneic dermal microparticles. By uniformly mixing a certain concentration of uncross-linked sodium hyaluronate aqueous solution with decellularized allogeneic dermal microparticles, the resulting composite filler material fully leverages the advantages of both materials, achieving stable filling of a natural material over a long period without the use of any cross-linking agents. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0031] Figure 1 This is a particle size distribution diagram of the slurry formed from decellularized allogeneic dermal microparticles obtained in Example 1 of the present invention;

[0032] Figure 2 This is a particle size distribution diagram of the slurry formed from decellularized allogeneic dermal microparticles obtained in Example 2 of the present invention.

[0033] Figure 3 These are infrared spectra of the injected filler material before and after irradiation. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0035] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0036] Example 1

[0037] A method for preparing an injection filler material, comprising the following specific steps:

[0038] (1) Raw material preparation:

[0039] Preparation of a slurry from decellularized allogeneic dermal microparticles: First, healthy donor skin from a legitimate tissue bank was treated with 5% sodium hydroxide solution to remove cells for 16-24 hours at room temperature. After ultrasonic cleaning four times, decellularized allogeneic dermal microparticles were obtained, each time for 10 minutes. Then, a certain amount of decellularized allogeneic dermal microparticles was weighed, purified water was added, and the material-to-liquid ratio was 1:5. The microparticles were then pulverized (using a fixed-cell wall-breaking pulverizer) for a total time of 5 minutes (with a 3-minute pause every 1 minute to prevent local overheating during the pulverization process) to obtain the slurry.

[0040] Preparation of sodium hyaluronate aqueous solution: Weigh sodium hyaluronate with a molecular weight of 1000kd or higher (supplied by Bloomage Biotechnology Co., Ltd.) and add it to purified water to a concentration of 1%. Stir well until clear and free of bubbles to obtain an aqueous solution of sodium hyaluronate.

[0041] (2) Mix the slurry formed by decellularized allogeneic dermal microparticles with an aqueous solution of sodium hyaluronate at a mass ratio of 1:1, and continue stirring for 10 minutes at a stirring speed of 2000 r / min.

[0042] (3) The mixture in step (2) is filled in a clean room of Class 10,000 or above using a pre-filled syringe, sealed in a blister box and then irradiated with a dose of 5 kGy for 30 min to obtain the injection filling material.

[0043] Example 2

[0044] A method for preparing an injection filler material, the specific steps of which are basically the same as those in Example 1, except that: in step (1), during the preparation of the slurry formed by decellularized allogeneic dermal microparticles, the total crushing time is adjusted to 15 min to obtain an injection filler material based on decellularized allogeneic dermal.

[0045] The particle size distribution of the slurry formed from decellularized allogeneic dermal microparticles prepared in Examples 1 and 2 was analyzed. The results showed that the particle size D50 after pulverization for 5 minutes (Example 1) was 153.6 μm (see...). Figure 1 The particle size after pulverizing for 15 minutes (Example 2) was 101.9 μm (see Example 2). Figure 2 This shows that as the pulverization time increases, the particle size of the decellularized allogeneic dermal microparticles in the resulting slurry decreases, and the particle size can be flexibly controlled by the pulverization time.

[0046] Example 3

[0047] A method for preparing an injection filler material, the specific steps of which are basically the same as those in Example 2, except that: in step (3), the irradiation dose is adjusted to 15 kGy to obtain the injection filler material.

[0048] Example 4

[0049] A method for preparing an injection filler material, the specific steps of which are basically the same as those in Example 2, the only difference being that: in step (3), the irradiation dose is adjusted to 25 kGy to obtain the injection filler material.

[0050] Comparative Example 1

[0051] A method for preparing a material based on decellularized allogeneic dermis is basically the same as that in Example 2, except that: an aqueous solution of sodium hyaluronate is not prepared and step (2) is not included, thus obtaining an injection filler material.

[0052] Comparative Example 2

[0053] A method for preparing an injection filler material is basically the same as that in Example 2, except that: in step (3), no irradiation is performed, and the product after being packaged in a blister box is directly used as the injection filler material.

[0054] The slurry formed from decellularized allogeneic dermal microparticles prepared in step (1) of Example 2-4, and the materials obtained in Comparative Example 1 and Comparative Example 2 were subjected to a stability test (ambient temperature 18-30℃, static setting) to observe whether the microparticles and the medium solution in the material separated into layers, as detailed in Table 1 below. Simultaneously, infrared spectroscopy was performed on the materials in Examples 2-4 and Comparative Example 2, as detailed in Table 1 below. Figure 3 .

[0055] Table 1

[0056]

[0057] As can be seen from Table 1, when purified water is used as the medium (the slurry formed by decellularized allogeneic dermal microparticles in Example 2 and Comparative Example 1), the stratification is easy before and after irradiation. When sodium hyaluronate aqueous solution is used as the medium (Comparative Example 2), the stratification is less likely to occur than when purified water is used, but stratification still occurs after long-term storage. In contrast, the sodium hyaluronate aqueous solution of the same concentration makes the injected filler material more uniform and stable under the action of irradiation, and it is less likely to stratify.

[0058] Furthermore, the stability of the materials obtained in Examples 2-4 was tested. The test conditions were as follows: the injection filling materials obtained in Examples 2-4 were centrifuged at high speed of 1000 r / min to see if the microparticles and the medium solution in the injection filling materials were separated into layers, as shown in Table 2 below.

[0059] Table 2

[0060]

[0061] The injection filling materials obtained in Examples 3 and 4 were centrifuged at different rates to see if the microparticles and the medium solution in the injection filling materials separated into layers, as shown in Table 3 below.

[0062] Table 3

[0063]

[0064] As can be seen from the table above, the injection filling material in Example 3 has better irradiation stability and centrifugal stability.

[0065] Testing of the injectable filler material in Example 3 revealed the formation of new functional groups, indicating a chemical bonding reaction between the free groups in sodium hyaluronate and some functional groups in the collagen matrix of the microparticle material during irradiation. Furthermore, increasing the irradiation dose promoted the bonding reaction to some extent, but excessively increasing the irradiation dose led to severe degradation of sodium hyaluronate, which affected its chemical bonding reaction with the collagen matrix to a certain degree.

[0066] Example 5

[0067] A method for preparing an injectable filler material, the specific steps of which are basically the same as those in Example 4, the only difference being that: when preparing the aqueous solution of sodium hyaluronate in step (1), the concentration of the aqueous solution of sodium hyaluronate is adjusted to 1.5% to obtain an injectable filler material based on decellularized allogeneic dermis.

[0068] Comparative Example 3

[0069] A method for preparing an injection filler material, the specific steps of which are basically the same as those in Example 4, the only difference being that: when preparing the aqueous solution of sodium hyaluronate in step (1), the concentration of the aqueous solution of sodium hyaluronate is adjusted to 0.5% to obtain the injection filler material.

[0070] The stability of the injection filler materials prepared from aqueous solutions of sodium hyaluronate of different concentrations in Examples 4, 5 and Comparative Example 3 was tested after 24 weeks to see if the microparticles in the material and the aqueous solution of sodium hyaluronate separated into layers, as shown in Table 4 below.

[0071] Table 4

[0072]

[0073] The results showed that, without irradiation, the solid and liquid phases would separate regardless of the HA concentration; under irradiation, when the initial medium concentration was 0.5%, the material was prone to separation after prolonged storage, indicating that the medium concentration should not be too low, but too high a concentration would also affect the flowability of the injection filler material, so it should not exceed 1.5%.

[0074] The specific process for applying injectable filler materials is as follows:

[0075] The injectable filling materials prepared in Example 1, the injectable filling materials prepared in Example 2, and the aqueous solution of sodium hyaluronate in Example 1 were injected subcutaneously into animals to observe intradermal degradation. The specific process was as follows: the hair on the back of each rabbit was removed, and three injection sites were marked on each side of the central axis. About 1 ml of different materials was injected into the three sites on the left side to form a raised wheal with a diameter of about 1 cm. During the injection filling, attention was paid to the consistency of the layers. Physiological saline was injected into the other side as a control. The results were observed at 1 week, 4 weeks, 8 weeks, 12 weeks, and 24 weeks.

[0076] At each observation point, the injected wheal parameters were measured using vernier calipers: wheal length a i , width of the dermal papilla b i , Pemu height c i The parameters of the puddles were measured multiple times and the average value was taken; the volume V of the puddles was calculated. i and the dermal volume retention rate δ i Skin volume retention rate δi The higher the value, the lower the material absorption rate. The pituitary volume retention rate δ was recorded at different time points for different groups of materials. i Value. Where V i =2 / 3πa i b i c i (i=1,2,3…), δ i =(V i / V0)×100% (i=1,2,3…), where V0 is the volume of the wheal measured immediately after implantation. δ i The values ​​(in %) are shown in Table 5 below. (Note: This measurement and calculation method is based on the reference "Experimental Study on Autologous Hair Keratin as an Injectable Soft Tissue Filler Material")

[0077] Table 5

[0078]

[0079] The results showed that the injectable filler material in Example 1 had a longer degradation time than the injectable filler material in Example 2. This is because the slurry formed from decellularized allogeneic dermal microparticles with a shorter pulverization time in Example 1 had a larger particle size than the slurry formed from decellularized allogeneic dermal microparticles with a longer pulverization time in Example 2, resulting in a longer degradation time for the injectable filler material prepared from it. However, the aqueous solution of sodium hyaluronate began to degrade significantly after one month and was completely degraded after 2-3 months. The injectable filler material based on decellularized allogeneic dermal microparticles, obtained by combining the aqueous solution of sodium hyaluronate and the slurry formed from decellularized allogeneic dermal microparticles, degraded by only about 10% after one month and less than 50% after one year. It is expected that the injectable filler material of the present invention, when used as a facial filler material by injection, can last for more than one year, which is better than some cross-linked sodium hyaluronate products.

[0080] 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 of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing an injection filler material, characterized in that, A slurry formed from decellularized allogeneic dermal microparticles with a D50 of 100μm~155μm is mixed with an aqueous solution of sodium hyaluronate with a concentration of 1~1.5% and then irradiated at 15kGy for 10~30min to obtain an injection filler material. The slurry is obtained by co-pulverizing decellularized allogeneic dermal and purified water. During pulverization, the ratio of decellularized allogeneic dermal to purified water is 1:5~1:20, and the pulverization time is 1~30min. The mass ratio of the slurry to the aqueous solution of sodium hyaluronate is 1:1~1:

10. The molecular weight of sodium hyaluronate in the aqueous solution is above 1000kd. The injection filler material does not separate into layers under centrifugation at 3000r / min.

2. The method for preparing the injection filler material according to claim 1, characterized in that, The preparation process of the slurry is as follows: remove cells and impurities from the donor skin, retain the extracellular matrix with natural three-dimensional collagen network structure, wash away residual reagents, mix the washed decellularized allogeneic dermal extracellular matrix with purified water and then pulverize it.

3. The method for preparing the injection filler material according to claim 1, characterized in that, The specific preparation steps of the slurry are as follows: (1) The donor skin is treated with sodium hydroxide solution of 1-10% mass concentration to remove cells, and after washing 4-10 times, decellularized allogeneic dermis is obtained; (2) Weigh out decellularized allogeneic dermis, add purified water and pulverize it; when pulverizing, the ratio of decellularized allogeneic dermis to purified water is 1:5~1:20; the pulverization time is 1~30min.

4. The method for preparing the injection filler material according to claim 2, characterized in that, The specific preparation steps of the slurry are as follows: (1) The donor skin is treated with sodium hydroxide solution of 1-10% mass concentration to remove cells, and after washing 4-10 times, decellularized allogeneic dermis is obtained; (2) Weigh out decellularized allogeneic dermis, add purified water and pulverize it; when pulverizing, the ratio of decellularized allogeneic dermis to purified water is 1:5~1:20; the pulverization time is 1~30min.

5. The method for preparing the injection filler material according to claim 1, characterized in that, The mass ratio of the slurry to the aqueous solution of sodium hyaluronate during mixing is 1:

1.

6. The method for preparing the injection filler material according to any one of claims 1 to 5, characterized in that, The irradiation is performed by filling the mixed mixture with a pre-filled syringe in a cleanroom of Class 10,000 or higher, and then sealing it in a blister pack.

7. An injection filler material, characterized in that, It is prepared by the method of any one of claims 1 to 6.

8. An application of an injection filler material, characterized in that, The injectable filler material described in claim 7 is used as a filler material for medical aesthetic purposes.

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