A gel filler capable of promoting collagen regeneration, and a preparation method and application thereof

Abstract

This invention discloses a gel filler that promotes collagen regeneration, its preparation method, and its application. The gel filler comprises polycaprolactone microspheres, cross-linked sodium hyaluronate, polynucleotides, water-soluble amino acids, and a phosphate buffer solution. The gel filler of this invention exhibits good biocompatibility, excellent elasticity and mechanical strength, and good shape retention. Furthermore, the use of modified sodium hyaluronate mixed with a microsphere suspension ensures smooth application. In this gel filler, the microspheres are uniformly dispersed and do not accumulate or precipitate, effectively reducing the probability of granuloma formation. The addition of polynucleotides also has a certain effect on post-injection facial redness and swelling.

Description

Technical Field

[0001] This invention belongs to the fields of medicine and cosmetic filler technology, and in particular relates to a gel filler that can promote collagen regeneration, its preparation method and application. Background Technology

[0002] The skin, the largest organ in the human body, is the first line of defense against external environmental factors, playing a vital role in repelling pathogens and preventing mechanical, chemical, and physical damage. Key characteristics of skin aging include wrinkle formation, sagging skin, and the appearance of blemishes. Collagen, an abundant structural protein in the dermal matrix, plays a crucial role in maintaining skin integrity. Rapid collagen degradation is a significant cause of skin aging.

[0003] Soft tissue injections of regenerative materials can not only quickly improve facial wrinkles but also promote collagen regeneration and improve skin aging. Currently, commonly used regenerative fillers in clinical practice include collagen, polylactic acid, hyaluronic acid, hydroxyapatite, and polycaprolactone. Among them, polycaprolactone (PCL) is a completely biodegradable synthetic polymer with broad application prospects. In recent years, PCL has attracted much attention due to its excellent biocompatibility, biodegradability, low toxicity, plasticity, controllable degradation rate, and good bioactivity. These properties make PCL one of the ideal materials in the cosmetic and medical fields. Moreover, PCL can stimulate the regeneration of the body's own collagen, resulting in a naturally firmer skin.

[0004] Existing technologies disclose filler compositions comprising polycaprolactone microspheres, polynucleotides, suspension stabilizers, and buffer solutions, in which polycaprolactone is dispersed among the other substances. However, direct mixing cannot guarantee the uniformity of the polycaprolactone phenolic acid, easily leading to inflammation. The reason for this may be that existing polycaprolactone fillers are irregularly gel-like, granular, or spherical, often inducing an inflammatory response within a few months, stimulating the body to produce collagen. Prolonged inflammation can lead to inflammatory aging, granulomas, and excessive scarring. The immediate filling effect of these fillers is generally due to the physical support of the matrix material. The matrix material is absorbed quickly, while subsequent new collagen production is slow, resulting in a window period without filling effect, affecting aesthetics. The long-term effect of filling mainly depends on the subsequent production of new collagen. An imbalance in the ratio of type I and type III collagen during collagen deposition can lead to the formation of keloids. If the newly generated collagen is only dense and firm type I or has an excessively high proportion of easily lost type III collagen, it can lead to facial stiffness, keloids, and wrinkles after the loss of type III collagen. Summary of the Invention

[0005] In view of the above-mentioned problems existing in the prior art, the purpose of this invention is to provide a gel filler that can promote collagen regeneration, its preparation method, and its application. The technical solution provided by this invention is as follows:

[0006] In a first aspect, the present invention provides a gel filler that can promote collagen regeneration, comprising the following components in weight percentage: 0.05%-4% polycaprolactone microspheres, 0.8%-2.5% cross-linked sodium hyaluronate, 0.05%-1.0% polynucleotides, 0.0001%-0.02% water-soluble amino acids, and the remainder being a phosphate buffer solution.

[0007] Polynucleotides (PNs) are high-molecular-weight compounds composed of nucleotides linked by 3',5'-phosphodiester bonds, exhibiting a chain-like structure. Their large molecular weight typically results in a three-dimensional scaffold structure, which gives them unique physical and chemical properties in both in vivo and in vitro applications. The double helix and three-dimensional support structure of PNs allows cells to attach to and grow within the skin's internal environment, achieving an effective anti-aging effect. Furthermore, the 3D porous structure of PNs facilitates the movement of nutrients and moisture. In addition, PNs can reverse the skin aging process in a short period; their unique scaffold function provides sufficient space for extracellular matrix regeneration, activates endogenous secretion, and exerts the anti-inflammatory and soothing effects of polydeoxyribonucleotides (PDRNs).

[0008] Preferably, the gel filler that promotes collagen regeneration comprises, by mass percentage, 0.5%-1.5% of the polycaprolactone microspheres, 1.2%-2% of the cross-linked sodium hyaluronate, 0.1%-0.5% of the polynucleotides, 0.0002%-0.008% of the water-soluble amino acids, with the remainder being phosphate buffer.

[0009] Preferably, the molecular weight of the cross-linked sodium hyaluronate is 1.2-2.5 million Da.

[0010] Preferably, the polycaprolactone microspheres in the gel filler have a particle size of 20-55 μm, more preferably 20-45 μm. Excessively large particle size or uneven distribution may trigger foreign body reactions (such as inflammation or granulomas), while excessively small particle size may lead to over-phagocytosis by immune cells (such as phagocytes), resulting in a local immune response. Therefore, controlling the microsphere size within the 20-55 μm range is suitable for injection. Furthermore, excessively large particle size increases product viscosity, potentially causing needle blockage or difficulty in injection, while uneven particle size distribution may cause local accumulation or uneven diffusion. Microspheres within the 20-55 μm range ensure uniform distribution of particles in the tissue after injection, avoiding clumping or unevenness.

[0011] Preferably, the gel filler further comprises lidocaine hydrochloride, wherein the mass percentage of lidocaine hydrochloride in the gel filler is 0.1%-0.5%.

[0012] Preferably, the cross-linked sodium hyaluronate is obtained through a cross-linking reaction of sodium hyaluronate. Specifically, sodium hyaluronate is added to a BDDE solution (cross-linking agent, 1,4-butanediol glycidyl ether), mixed thoroughly, and then NaOH solution is added. After complete dissolution, the mixture is heated to induce a cross-linking reaction, resulting in cross-linked sodium hyaluronate gel. The cross-linking technology gives the sodium hyaluronate support, enabling it to hold PCL and prevent PCL from settling. This ensures that PCL is uniformly dispersed in the solution.

[0013] Preferably, the water-soluble amino acid is selected from one or more combinations of glycine, alanine, leucine, proline, threonine, hydroxyproline, lysine hydrochloride, serine, phenylalanine, valine, tryptophan, and methionine.

[0014] Based on the technical solution of this application, inflammation is reduced, collagen production is efficiently stimulated, and the ratio of type I and type III collagen is reshaped, which can achieve a more uniform filling effect, give the newly formed collagen a ratio closer to the internal composition of normal skin, reduce the formation of foreign body granulomas and keloids, make the facial filling effect more lasting, reduce the number of injections, make the expression more natural, and prevent swelling, stiffness, and wrinkles.

[0015] Secondly, the present invention also provides a method for preparing a gel filler that can promote collagen regeneration, comprising the following steps:

[0016] S1. Sodium hyaluronate is added to a crosslinking agent to prepare a crosslinked sodium hyaluronate gel;

[0017] S2. Dissolve the polynucleotide (PN) completely in phosphate buffer, add polycaprolactone (PCL) microspheres and mix well to obtain PN-PCL suspension;

[0018] S3. Prepare excipient solution;

[0019] S4. After dialysis and swelling of the cross-linked sodium hyaluronate gel obtained in step S1, it is mixed with the PN-PCL suspension prepared in step S2 and the excipient solution prepared in step S3 to obtain the gel filler.

[0020] Preferably, in step S3, the excipient solution mainly includes water-soluble amino acids, lidocaine hydrochloride, and phosphate buffer. Preferably, in step S4, the cross-linked sodium hyaluronate from step S1 is soaked in phosphate buffer, dialyzed to remove the cross-linking agent, and the pH of the buffer is adjusted to neutral during the process. After the gel swells and dialysis is stopped, it is sieved and granulated. The dialyzed cross-linked sodium hyaluronate gel, excipient solution, and PN-PCL suspension are mixed evenly to obtain the gel filler.

[0021] Preferably, in step S4, the ratio of cross-linked sodium hyaluronate gel: excipient solution: PN-PCL suspension is 5:1:1 to 40:1:1.

[0022] Preferably, the solvent used in steps S2, S3 and S4 is a phosphate buffer solution; the pH of the buffer solution is 6.7 to 8.0; and the osmotic pressure of the buffer solution is 270 to 320 mOsm / L.

[0023] Thirdly, the present invention also provides the application of the gel filler that promotes collagen regeneration in the preparation of medical aesthetic materials, including injectable fillers.

[0024] Fourthly, the present invention also provides the application of the filler composition as an injection filler, the filler composition comprising the following components in weight percentages: 0.05%-4% polycaprolactone microspheres, 0.8%-2.5% cross-linked sodium hyaluronate, 0.05%-1.0% polynucleotides, 0.0001%-0.02% water-soluble amino acids, and phosphate buffer solution.

[0025] Fifthly, the present invention also provides a pre-filled syringe comprising the gel filler described herein that promotes collagen regeneration.

[0026] Compared with the prior art, the gel filler that promotes collagen regeneration and its preparation method provided by the present invention have the following advantages:

[0027] 1. The gel filler of the present invention has good biocompatibility, good elasticity and mechanical strength, and good shape retention; at the same time, it is made by mixing modified sodium hyaluronate with microsphere suspension, which makes it easy to push during use.

[0028] 2. In the gel filler of the present invention, the microspheres are evenly dispersed and will not accumulate or precipitate, effectively reducing the probability of granuloma formation. At the same time, the addition of polynucleotides has a certain effect on redness and swelling of the face after injection.

[0029] 3. In the gel filler of the present invention, the effects of different component ratios on the production of type I and type III collagen in human skin were studied, and a ratio that can generate higher collagen content and a ratio of type I and type III collagen that is closer to the collagen ratio in normal skin was proposed, resulting in better filling effect and more natural facial expressions.

[0030] 4. In the gel filler of the present invention, the added nutrients are formulated according to the skin aging mechanism, aiming to optimize fibroblast function: create a good microenvironment to ensure optimal operation; provide sufficient substrates to promote biosynthesis; and resist oxidative stress damage.

[0031] 5. The gel filler of this invention has a dual filling effect: an immediate effect and a lasting effect. Cross-linked sodium hyaluronate provides an immediate filling effect upon injection. As the cross-linked sodium hyaluronate decomposes in the body, the polycaprolactone microspheres stimulate surrounding tissues to produce collagen, resulting in a lasting effect, avoiding a gap in the filling effect and maintaining the aesthetic appearance after filling. Attached Figure Description

[0032] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0033] Figure 1 The present invention provides a flowchart of a method for preparing a gel filler that can promote collagen regeneration.

[0034] Figure 2 The results show the particle size of PCL in the gel filler of Example 1 in Experimental Example 5. Detailed Implementation

[0035] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0036] Abbreviation meaning:

[0037] PN stands for polynucleotide;

[0038] PCL stands for polycaprolactone;

[0039] BDDE is a crosslinking agent, 1,4-butanediol glycidyl ether.

[0040] Example 1

[0041] A gel filler that promotes collagen regeneration includes a modified sodium hyaluronate gel and a PN-PCL suspension uniformly distributed in the gel. The modified sodium hyaluronate gel includes cross-linked sodium hyaluronate, lidocaine hydrochloride, and water-soluble amino acids. Taking 100 mL of the gel filler as an example, the content of each component is as follows: cross-linked sodium hyaluronate 15 mg / mL; lidocaine hydrochloride 3 mg / mL; PN 10 mg / mL; PCL 10 mg / mL; and water-soluble amino acids totaling 0.12 mg / mL: wherein the water-soluble amino acids include alanine 0.015 mg / mL, leucine 0.015 mg / mL, glycine 0.02 mg / mL, proline 0.045 mg / mL, and lysine hydrochloride 0.025 mg / mL.

[0042] The preparation method of a gel filler that can promote collagen regeneration is as follows (taking 100mL as an example): The process is as follows Figure 1 As shown, it includes the following steps:

[0043] S1. Weigh out sodium hyaluronate with a molecular weight of 120 wDa and add it to the BDDE solution (the mass ratio of BDDE to sodium hyaluronate is 1:50). After mixing evenly, add 1.2% NaOH solution (the mass percentage concentration of BDDE in the alkaline solution is 0.6%). After complete dissolution, perform a cross-linking reaction in a 40℃ water bath for 24 hours to obtain cross-linked sodium hyaluronate gel. Under the conditions of sodium hyaluronate, molecular weight, and BDDE ratio provided by this invention, suitable gel hardness and elasticity can be obtained.

[0044] S2. Add PN to 100 mL of phosphate buffer (pH 7.0, osmotic pressure 280 mOsm / L, the following steps are the same), stir until there are no flocculent substances, then add PCL and stir evenly to obtain PN-PCL suspension (PN and PCL concentrations are calculated according to the mixing ratio in step S4).

[0045] S3. Add lidocaine hydrochloride, alanine, leucine, glycine, proline, and lysine hydrochloride to 500 mL of phosphate buffer and dissolve completely until the solution is clear and transparent. Then adjust the solution to neutral with 1 mol / L NaOH to obtain the excipient solution (the ratio of lidocaine hydrochloride, alanine, leucine, glycine, proline, and lysine hydrochloride is calculated according to the mixing ratio in step S4).

[0046] S4. Soak the cross-linked sodium hyaluronate from step S1 in phosphate buffer at a ratio of 1g:100mL. Dialyze the solution six times, changing the buffer solution each time. Adjust the pH of the buffer to neutral every 30 minutes. Stop dialysis when the gel swells to 80g. Granulate the gel by sieving through a 100-mesh stainless steel sieve. Mix 8 portions of the dialyzed cross-linked sodium hyaluronate gel, 1 portion of excipient solution, and 1 portion of PN-PCL suspension evenly to obtain the gel filler. The gel filler contains: 15mg / mL cross-linked sodium hyaluronate; 3mg / mL lidocaine hydrochloride; 10mg / mL PN; 10mg / mL PCL; and a total of 0.12mg / mL water-soluble amino acids: alanine 0.015mg / mL, leucine 0.015mg / mL, glycine 0.02mg / mL, proline 0.045mg / mL, and lysine hydrochloride 0.025mg / mL.

[0047] Example 2

[0048] A gel filler that promotes collagen regeneration includes a modified sodium hyaluronate gel and a PN-PCL suspension uniformly distributed in the gel. The modified sodium hyaluronate gel includes cross-linked sodium hyaluronate, lidocaine hydrochloride, and water-soluble amino acids. Taking 100 mL of the gel filler as an example, the content of each component is as follows: cross-linked sodium hyaluronate 10 mg / mL; lidocaine hydrochloride 3 mg / mL; PN 5 mg / mL; PCL 10 mg / mL; and water-soluble amino acids totaling 0.20 mg / mL: alanine 0.025 mg / mL, leucine 0.035 mg / mL, glycine 0.04 mg / mL, proline 0.065 mg / mL, and lysine hydrochloride 0.035 mg / mL.

[0049] The preparation method of a gel filler that can promote collagen regeneration is as follows (taking 100mL as an example):

[0050] S1. Weigh out sodium hyaluronate with a molecular weight of 120 wDa and add it to the BDDE solution (the mass ratio of BDDE to sodium hyaluronate is 1:25). After mixing evenly, add 1.5% NaOH solution (the mass percentage concentration of BDDE in the alkaline solution is 0.3%). After complete dissolution, perform a cross-linking reaction in a 40℃ water bath for 24 hours to obtain cross-linked sodium hyaluronate gel.

[0051] S2. Add PN to 100 mL of phosphate buffer (pH 7.0, osmotic pressure 280 mOsm / L, the following steps are the same), stir until there are no flocculent substances, then add PCL and stir evenly to obtain PN-PCL suspension (PN and PCL concentrations are calculated according to the mixing ratio in step S4).

[0052] S3. Add lidocaine hydrochloride, alanine, leucine, glycine, proline, and lysine hydrochloride to 500 mL of phosphate buffer and dissolve completely until the solution is clear and transparent. Then adjust the solution to neutral with 1 mol / L NaOH to obtain the excipient solution (the ratio of lidocaine hydrochloride, alanine, leucine, glycine, proline, and lysine hydrochloride is calculated according to the mixing ratio in step S4).

[0053] S4. Soak the cross-linked sodium hyaluronate from step S1 in phosphate buffer at a ratio of 1g:300mL. Dialyze the solution by changing the buffer six times, adjusting the pH of the buffer to neutral every 30 minutes. Stop dialysis when the gel swells to 70g, and then granulate it by sieving through a 100-mesh stainless steel sieve. Take 6 portions of the dialyzed cross-linked sodium hyaluronate gel, 1 portion of excipient solution, and 1 portion of PN-PCL suspension and mix them evenly to obtain the gel filler. The contents are: cross-linked sodium hyaluronate 10mg / mL; lidocaine hydrochloride 3mg / mL; PN 5mg / mL; PCL 10mg / mL; and a total of 0.20mg / mL of water-soluble amino acids: alanine 0.025mg / mL, leucine 0.035mg / mL, glycine 0.04mg / mL, proline 0.065mg / mL, and lysine hydrochloride 0.035mg / mL.

[0054] Example 3

[0055] A gel filler that promotes collagen regeneration includes a modified sodium hyaluronate gel and a PN-PCL suspension uniformly distributed in the gel. The modified sodium hyaluronate gel includes cross-linked sodium hyaluronate, lidocaine hydrochloride, and water-soluble amino acids. Taking 100 mL of the gel filler as an example, the content of each component is as follows: cross-linked sodium hyaluronate 20 mg / mL; lidocaine hydrochloride 3 mg / mL; PN 5 mg / mL; PCL 10 mg / mL; and water-soluble amino acids totaling 0.01 mg / mL: alanine 0.0025 mg / mL, leucine 0.001 mg / mL, glycine 0.001 mg / mL, proline 0.0035 mg / mL, and lysine hydrochloride 0.002 mg / mL.

[0056] The preparation method of a gel filler that can promote collagen regeneration is as follows (taking 100mL as an example):

[0057] S1. Weigh out sodium hyaluronate with a molecular weight of 120 wDa and add it to the BDDE solution (the mass ratio of BDDE to sodium hyaluronate is 1:75). After mixing evenly, add 0.6% NaOH solution (the mass percentage concentration of BDDE in the alkaline solution is 0.9%). After complete dissolution, perform a cross-linking reaction in a 40℃ water bath for 24 hours to obtain cross-linked sodium hyaluronate gel.

[0058] S2. Add PN to 100 mL of phosphate buffer (pH 7.0, osmotic pressure 280 mOsm / L, the following steps are the same), stir until no flocculent matter remains, then add PCL and stir until homogeneous to obtain the PN-PCL suspension. (The concentrations of PN and PCL are calculated based on the mixing ratio in step S4.)

[0059] S3. Dissolve lidocaine hydrochloride, alanine, leucine, glycine, proline, and lysine hydrochloride separately in 500 mL of phosphate buffer until the solution is clear and transparent. Then adjust the solution to neutral with 1 mol / L NaOH to obtain the excipient solution. (The proportions of lidocaine hydrochloride, alanine, leucine, glycine, proline, and lysine hydrochloride are calculated according to the mixing ratio in step S4.)

[0060] S4. Soak the cross-linked sodium hyaluronate from step S1 in phosphate buffer at a ratio of 1g:450mL. Dialyze the gel by changing the phosphate buffer six times to remove the cross-linking agent BDDE. Adjust the pH of the buffer to neutral every 30 minutes. After the gel swells to 90g, stop dialysis and granulate it through a 100-mesh stainless steel sieve. Take 18 portions of the dialyzed cross-linked sodium hyaluronate gel, 1 portion of excipient solution, and 1 portion of PN-PCL suspension and mix them evenly to obtain the gel filler. The cross-linked sodium hyaluronate is 20mg / mL; lidocaine hydrochloride is 3mg / mL; PN is 5mg / mL; PCL is 10mg / mL; and water-soluble amino acids are 0.01mg / mL in total: alanine 0.0025mg / mL, leucine 0.001mg / mL, glycine 0.001mg / mL, proline 0.0035mg / mL, and lysine hydrochloride 0.002mg / mL.

[0061] Examples 4-5

[0062] The difference between Examples 4-5 and Example 1 is that the contents of PN and PCL are different, while the other reaction conditions are the same as in Example 1.

[0063] Examples 6-8

[0064] The difference between Examples 6-8 and Example 1 is that the molecular weight and content of sodium hyaluronate are different, while the other reaction conditions are the same as in Example 1.

[0065] Examples 9-10

[0066] The difference between Examples 9-10 and Example 1 is that the contents of PN, PCL, and sodium hyaluronate are different, while the other reaction conditions are the same as in Example 1.

[0067] Comparative Example 1

[0068] The difference between Comparative Example 1 and Example 1 is that cross-linked sodium hyaluronate is replaced with free sodium hyaluronate, while the other reaction conditions and steps are the same as in Example 1. Specifically, taking 100 mL of gel filler as an example, sodium hyaluronate with a molecular weight of 120 wDa is added to phosphate buffer and stirred until it becomes clear and transparent to obtain a free sodium hyaluronate solution.

[0069] Comparative Example 2

[0070] The difference between Comparative Example 2 and Example 1 is that the PN-PCL suspension was replaced with PCL microspheres. Specifically, after dialysis, the cross-linked sodium hyaluronate gel was mixed with the excipient solution and then directly added to the PCL microspheres.

[0071] Comparative Example 3

[0072] The difference between Comparative Example 3 and Example 1 is that PN and PCL were added to the gel separately. Specifically, PN was added to the excipient solution, then mixed evenly with the cross-linked sodium hyaluronate gel, and finally PCL microspheres were added and mixed evenly.

[0073] The specific implementation conditions and the reaction conditions of the comparative examples are shown in Table 1.

[0074] Table 1. Reaction conditions for each embodiment and comparative example.

[0075]

[0076] Experimental Example 1

[0077] The effect of different formulations on the extrusion force of the sample:

[0078] The pushing force of the examples and comparative examples was tested according to the method in "YY / T0962-2014 Cross-linked Sodium Hyaluronate Gel for Plastic Surgery". The pushing needle was 27G×1 / 2. The test results are shown in Table 2.

[0079] Table 2. Effects of different formulations on sample extrusion force

[0080]

[0081]

[0082] Table 2 shows that the extrusion force of the samples increases with the increase of sodium hyaluronate content, molecular weight, and PN addition. When the sodium hyaluronate concentration is 1% or 1.5%, the molecular weight is less than 120 wDa, and the PN addition is 0.5%, the extrusion force of the gel sample is less than 25 N, and the extrusion is smooth, suitable for doctors' injection operations. In addition, although the average extrusion force of Comparative Example 1 is less than 25 N, obvious problems occurred during the test, and visible needle blockage appeared. This is mainly because the sodium hyaluronate in the comparative example is in a free state and cannot support the PCL microspheres, and PCL is prone to aggregation. The extrusion forces of Comparative Examples 2 and 3 are also uneven, mainly because PCL was not mixed with PN and was directly added to the cross-linked sodium hyaluronate gel, resulting in uneven dispersion. The extrusion force of Examples 1 and 4 fluctuates little, mainly because the PCL in the samples is mixed with PN first, which allows for better dispersion in the cross-linked sodium hyaluronate gel.

[0083] Experimental Example 2

[0084] Effects of different formulations on total collagen and type I and type III remodeling

[0085] The following experiments were conducted using Examples 1, 4, 5, Comparative Example 1, and Comparative Example 2. Experimental methods: After subcutaneous injection of 100 μL of the material into the back of mice for 8 weeks, the skin was peeled back, and skin tissue was harvested from the corresponding locations and immediately fixed with tissue fixative. After paraffin embedding, sections were prepared for immunofluorescence staining of type I and type III collagen. The results were observed under a fluorescence confocal microscope, and the average fluorescence intensity was calculated using ImageJ. The experimental results are shown in Table 3.

[0086] Table 3. Effects of different formulations on total collagen and type I and type III remodeling.

[0087] Total collagen Collagen Type I: Type III Example 1 39.1425±1.87 3.6713:1 Example 4 15.1045±1.01 3.2056:1 Example 5 30.0501±0.97 3.5320:1 Comparative Example 1 21.6715±0.56 2.8964:1 Comparative Example 2 10.4601±0.23 0.7834:1

[0088] As shown in Table 3, Type I and Type III collagen are two important collagen proteins in human skin. However, under certain clinical conditions, imbalanced collagen synthesis can lead to scar tissue formation. Hypertrophic scar tissue is mainly characterized by an excess of Type III collagen, while keloids are caused by a disorder in the expression of both Type I and Type III collagen. The ratio of Type I to Type III collagen is shown in Table 3. Eight weeks after implantation, the collagen produced by stimulation around the material is mainly Type I collagen, with less Type III collagen. The type I collagen: type III collagen ratios in Comparative Examples 1 and 2 were 2.8964:1 and 0.7834:1, respectively, while the type I collagen: type III collagen ratios in Examples 1 and 5 were 3.6713:1 and 3.5320:1, respectively, close to 4:1, which is close to the collagen ratio in normal skin (Riita R, Mateleena P, Arja J (2002). Increased expression of collagen type and in human skin as a consequence of radiotherapy. Arch. Dermatol. Res. 294(4):178-184.), making the filling effect and facial expressions more natural. Compared to the comparative examples, the combination in Example 1 significantly stimulated collagen production in the skin, with the total collagen amount being 1.81 times and 3.74 times that of Comparative Examples 1 and 2, respectively. This significant increase in total collagen production allows for a more lasting filling effect. Simultaneously, the ratio of Type I collagen to Type III collagen was approximately 4:1, close to the ratio found in normal skin, resulting in better filling and more effective stimulation of collagen production. This also avoided post-injection facial redness and swelling caused by hypertrophic scar tissue.

[0089] Experimental Example 3

[0090] Effects of different formulations on the M1 / M2 ratio of macrophages

[0091] The following experiments were conducted using Examples 1, 4, 5, and Comparative Example 1. After subcutaneous injection of 100 μL into the back of ICR mice (weighing 20 ± 2 g) for 8 weeks, the skin was peeled back, and skin tissue was harvested from the corresponding locations and immediately fixed with tissue fixation solution. After paraffin embedding, sections were prepared for immunofluorescence staining of macrophage-related markers CD86, CD68, and CD207. The results were observed under a fluorescence confocal microscope, and the average fluorescence intensity was calculated using ImageJ. The experimental results are shown in Table 4.

[0092] Table 4. Effects of different formulation ratios on the M1 / M2 ratio of macrophages

[0093] Group M1 / M2 Example 1 0.7489±0.34 Example 4 3.1327±0.57 Example 5 1.9853±0.46 Comparative Example 1 7.5239±1.75 Comparative Example 3 4.6954±0.58

[0094] Table 4 shows that the amount of PCL added and its uniform dispersion significantly affect the M1 / M2 ratio. When the PCL addition is low, the M1 / M2 ratio is high, and the material is easily and rapidly phagocytosed by M1 macrophages, leading to difficulty in continuously stimulating subcutaneous collagen formation. When PCL is unevenly dispersed, the M1 / M2 ratio is high, attracting excessive M1 macrophages to fuse and form foreign body giant cells, causing inflammation. When the PCL addition is 1% or 1.5%, the M1 / M2 ratio is significantly reduced, and the sample promotes greater M2 polarization of macrophages, resulting in stronger collagen stimulation. Furthermore, the sustained M1 response around the biodegradable biomaterial is a cause of excessive scarring, while promoting the M2 response has been shown to be beneficial for material replacement by host tissue. Different proportions of microspheres can alter the M1 / M2 ratio of surrounding macrophages. When the PCL addition is 1%, the macrophages around the material are predominantly polarized as M2-type macrophages, which helps reduce excessive scarring from the implanted material and continuously stimulates subcutaneous collagen production.

[0095] Test Example 4

[0096] Effects of different prescription combinations on inflammation

[0097] The following experiments were conducted using Examples 1, 9, and Comparative Example 2. Experimental methods: After subcutaneous injection of 100 μL into the back of ICR mice (weighing 20 ± 2 g) for 8 weeks, the skin was peeled back, and skin tissue was harvested from the corresponding locations and immediately fixed with tissue fixation solution. After paraffin embedding, sections were stained with immunofluorescence for the inflammatory factors IL-1β and TNF-α. The tissues were observed and photographed under a fluorescence confocal microscope, and the average fluorescence intensity was calculated using ImageJ. The experimental results are shown in Table 5.

[0098] Table 5. Effects of different prescription combinations on inflammation.

[0099] Group IL-1β TNF-α Example 1 6.2876±0.44 4.7850±0.23 Example 9 4.2504±0.12 5.4901±0.34 Comparative Example 2 10.4513±0.70 20.5632±1.12

[0100] As shown in Table 5, M1 macrophages have strong phagocytic activity and can secrete a large number of acute inflammatory cytokines, such as IL-1β and TNF-α, thereby promoting acute inflammatory responses. Therefore, the continuous M1 response around the degradable biomaterial is the cause of excessive scar formation. After material implantation, Comparative Example 2 showed the highest level of inflammatory factors, while Example 9 showed the lowest level of inflammation. In Examples 1 and 9, the addition of PN significantly reduced the inflammation level compared to the microspheres and HA groups alone, thus reducing the risk of scarring at the filling site.

[0101] Experimental Example 5

[0102] PCL particle size detection in gel fillers

[0103] The PCL microspheres used in this invention have a size of 20-55 μm. On the one hand, this is to prove that the particle size of the microspheres will not be affected during the test. On the other hand, it is necessary to test the particle size of PCL in the finished product to ensure the safety of the filler.

[0104] Since all embodiments in this patent use the same type of PCL microspheres, Example 1 was selected for the following experiment. Experimental method: Using purified water as the dispersant, at a rotation speed of 1600 rpm / min, an appropriate amount of sample was taken, and excess sodium hyaluronate enzyme was added for enzymatic hydrolysis in a 42°C water bath until no transparent gel particles were found. The white powder was then poured into the sample cell until the opacity was above 2%, and the particle size was measured using a particle size analyzer.

[0105] Experimental results are as follows Figure 2 As shown, the measured particle size of PCL microspheres was 84% ​​in the range of 20 μm to 45 μm. The particle size corresponding to a cumulative particle size distribution of 10% was 32.154 μm, the cumulative particle size distribution of 50% was 42.676 μm, and the cumulative particle size distribution of 90% was 47.139 μm. It is evident that different embodiments do not affect the particle size of the PCL microspheres, and the safety is high at this particle size.

[0106] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A gel filler that promotes collagen regeneration, characterized in that, The composition includes the following components by weight percentage: polycaprolactone microspheres 0.05%-4%, cross-linked sodium hyaluronate 0.8%-2%, polynucleotides 0.05%-1.0%, water-soluble amino acids 0.0001%-0.02%, and the remainder being phosphate buffer solution; The polycaprolactone microspheres in the gel filler have a particle size of 20-55 μm; The extrusion force of the gel filler is less than 25N; Preparation method of gel filler that can promote collagen regeneration: S1. Sodium hyaluronate is added to a crosslinking agent to prepare a crosslinked sodium hyaluronate gel; S2. Dissolve the polynucleotide completely in phosphate buffer, add polycaprolactone microspheres and mix well to obtain PN-PCL suspension; S3. Prepare an excipient solution; the excipient solution includes water-soluble amino acids, lidocaine hydrochloride, and phosphate buffer; S4. After dialysis and swelling of the cross-linked sodium hyaluronate gel in step S1, it is mixed with the PN-PCL suspension prepared in step S2 and the excipient solution prepared in step S3 to obtain the gel filler. In step S4, the cross-linked sodium hyaluronate from step S1 is soaked in phosphate buffer and dialyzed to remove the cross-linking agent. During this process, the pH of the buffer is adjusted to neutral. After the gel swells and dialysis is stopped, it is sieved and granulated. The dialyzed cross-linked sodium hyaluronate gel, excipient solution, and PN-PCL suspension are mixed evenly to obtain the gel filler. In step S4, the ratio of cross-linked sodium hyaluronate gel: excipient solution: PN-PCL suspension is 5:1:1 to 40:1:

1.

2. The gel filler for promoting collagen regeneration according to claim 1, characterized in that, The polycaprolactone microspheres comprise 0.5%-1.5% by mass, the cross-linked sodium hyaluronate comprises 1.2%-2% by mass, the polynucleotide comprises 0.1%-0.5% by mass, the water-soluble amino acids comprise 0.0002%-0.008% by mass, and the remainder is phosphate buffer.

3. The gel filler for promoting collagen regeneration according to claim 1, characterized in that, The gel filler also contains lidocaine hydrochloride, and the mass percentage of lidocaine hydrochloride in the gel filler is 0.1%-0.5%.

4. The gel filler for promoting collagen regeneration according to claim 1, characterized in that, The water-soluble amino acid is selected from one or more combinations of glycine, alanine, leucine, proline, threonine, hydroxyproline, lysine hydrochloride, serine, phenylalanine, valine, tryptophan, and methionine.

5. The application of the gel filler that promotes collagen regeneration as described in any one of claims 1 to 4 in the preparation of medical aesthetic materials.

6. The application according to claim 5, characterized in that, The medical aesthetic materials include injectable fillers.

7. A pre-filled syringe comprising the gel filler according to any one of claims 1 to 4 that promotes collagen regeneration.

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