Preparation method of collagen-based microspheres and application of collagen-based microspheres in preparation of subcutaneous tissue filling material

Abstract

The invention provides a preparation method of collagen-based microspheres and application of the collagen-based microspheres in preparation of a subcutaneous tissue filling material, and relates to the technical field of medical treatment and medical cosmetology. According to verification of a large number of experiments, the collagen-based microspheres are prepared through a two-step cross-linking method and adjustment of polarity of a solvent used in the second-step chemical cross-linking process; the mass swelling ratio and the effective crosslinking density of the collagen-based microspheres can be regulated and controlled; different from a traditional filling material which only depends on physical support, the collagen-based microspheres with specific mass swelling ratio and crosslinking density have good injectability and mechanical support performance and can realize subcutaneous cell recruitment and promote cell proliferation and extracellular matrix secretion, so that a long-acting and stable subcutaneous tissue filling effect is realized. In addition, the collagen-based microspheres have good biocompatibility and degradability, and conform to the idea of cell-free tissue engineering.

Description

Technical Field

[0001] This invention relates to the fields of medical and cosmetic medicine, and in particular to a method for preparing collagen-based microspheres and their application in the preparation of subcutaneous tissue filling materials. Background Technology

[0002] Subcutaneous tissue defects and extracellular matrix loss caused by aging, surgery, and trauma are common clinical conditions. Currently widely used subcutaneous tissue filler materials include collagen hydrogels, hyaluronic acid hydrogels, polylactic acid microspheres, and polycaprolactone microspheres.

[0003] Collagen hydrogels and hyaluronic acid hydrogels have good injectability, allowing for in-situ cross-linking and shaping after subcutaneous injection. Their dense gel structure can fill and support subcutaneous tissue, providing immediate results. However, the dense gel structure hinders the migration of autologous cells and prevents the formation of new tissue within the gel. Degradation of the hydrogel leads to a reduction or even loss of its filling effect. Therefore, the duration of the tissue-filling effect of hydrogels is limited.

[0004] Currently, commercially available polylactic acid (PLA) microspheres and polycaprolactone (PVC) microspheres can not only fill defects but also promote the formation of extracellular matrix components such as collagen and polysaccharides through the body's foreign body reaction. However, the extracellular matrix formed through the foreign body reaction process is very limited. Therefore, as polyester microsphere materials degrade, the support effect on subcutaneous tissue or skin will significantly decrease. In addition, the strong hydrophobicity of polyester microsphere materials and their acidic degradation products can easily lead to adverse reactions such as allergies, nodules, and non-bacterial inflammation in the body.

[0005] Cellular tissue engineering aims to recruit cells and promote in situ tissue formation using bioactive scaffolds, showing great promise for clinical applications. However, research on optimizing the properties of collagen-based microspheres to promote subcutaneous tissue regeneration and tissue filling is still scarce. Existing technologies lack reports on how to use innovative preparation methods to regulate the crosslinking density and composition of collagen-based microspheres to improve their stability in subcutaneous tissue and enhance their promoting effect on subcutaneous tissue formation.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] One of the objectives of this invention is to provide a method for preparing collagen-based microspheres and their application in the preparation of subcutaneous tissue filling materials, so as to at least solve one of the technical problems existing in the prior art.

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

[0009] This invention provides a method for preparing collagen-based microspheres and their application in the preparation of subcutaneous tissue filling materials.

[0010] Furthermore, the collagen-based microspheres also contain protein components, polysaccharide components, or inorganic components.

[0011] Furthermore, the protein component includes at least one of polypeptides, albumin, gelatin, or laminin;

[0012] Preferably, the polysaccharide component includes at least one of hyaluronic acid, chondroitin sulfate, chitosan, carboxymethyl chitosan, or carboxymethyl cellulose;

[0013] Preferably, the inorganic component includes at least one of hydroxyapatite, calcium carbonate, calcium oxide, magnesium oxide, and zinc oxide.

[0014] Furthermore, the collagen-based microspheres have a mass swelling ratio of 1.7–16 and / or an effective crosslinking density of 120–5400 mol / m³. 3 .

[0015] Furthermore, the collagen-based microspheres are prepared by the following method:

[0016] 1) Low-crosslinked collagen-based microsphere precursors were prepared by water-in-oil emulsion crosslinking method;

[0017] 2) The collagen-based microsphere precursor is subjected to heterogeneous chemical cross-linking to obtain the collagen-based microspheres.

[0018] Furthermore, step 1) includes:

[0019] After mixing the raw material solution and the mixed oil solution, the first crosslinking agent is added under stirring. After stirring for 30 minutes, the mixture is allowed to stand to obtain a precipitate. The precipitate is washed with an amphiphilic solvent and then soaked in ethanol. Microspheres with a particle size distribution of 20-200 micrometers are selected to obtain the collagen-based microsphere precursor.

[0020] Preferably, 0.5g of collagen is dissolved in 20mL of acidic solution to obtain the raw material solution;

[0021] Preferably, the acidic solution comprises a 0.01M dilute hydrochloric acid solution;

[0022] Preferably, the mixed oil solution comprises edible oil and paraffin oil in a mass ratio of 1:3;

[0023] Preferably, the volume ratio of the raw material solution to the mixed oil solution is 1:6;

[0024] Preferably, the first crosslinking agent comprises a PBS solution of EDC;

[0025] Preferably, the volume ratio of the raw material solution to the first crosslinking agent is 4:1;

[0026] Preferably, the amphiphilic solvent includes 1,4-dioxane, acetone, or tetrahydrofuran.

[0027] Furthermore, step 2) includes:

[0028] After the collagen-based microsphere precursor is soaked in a solvent to swell or shrink, it is placed in a second crosslinking agent solution prepared with the corresponding solvent for heterogeneous crosslinking. After soaking and washing with ethanol and drying, the collagen-based microspheres are obtained.

[0029] Preferably, the solvent includes at least one of dichloromethane, ethanol, and PBS;

[0030] Preferably, the second crosslinking agent comprises an EDC solution, wherein the solvent of the EDC solution comprises at least one of dichloromethane, ethanol, and PBS;

[0031] Preferably, the concentration of the second crosslinking agent is 0.3% to 5% w / v;

[0032] Preferably, the soaking time in the solvent for swelling or shrinkage is 10 to 24 hours.

[0033] Furthermore, the raw material solution also includes a mixed solution of collagen and chitosan; the collagen-chitosan mixed solution is prepared by the following method:

[0034] Dissolve 0.5g of collagen and 0.3g of carboxymethyl chitosan in 20mL of acidic solution to obtain the raw material solution;

[0035] Preferably, the acidic solution comprises a 0.01M dilute hydrochloric acid solution.

[0036] Furthermore, the raw material solution also includes a mixed solution of collagen and bovine serum albumin; the collagen-bovine serum albumin mixed solution is prepared by the following method:

[0037] Dissolve 0.5g of collagen and 0.3g of bovine serum albumin in 20mL of acidic solution to obtain the raw material solution;

[0038] Preferably, the acidic solution comprises a 0.01M dilute hydrochloric acid solution.

[0039] Furthermore, the raw material solution also includes a mixed solution of collagen and hyaluronic acid; the collagen-hyaluronic acid mixed solution is prepared by the following method:

[0040] Dissolve 0.5g of collagen and 0.3g of hyaluronic acid in 20mL of acidic solution to obtain the raw material solution;

[0041] Preferably, the acidic solution comprises a 0.01M dilute hydrochloric acid solution.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] Through extensive experimental exploration and verification, the inventors of this invention discovered that (1) by using a two-step cross-linking method and adjusting the polarity of the solvent used in the second chemical cross-linking process, the swelling rate and effective cross-linking density of collagen-based microspheres can be controlled; (2) unlike traditional filler materials that rely solely on physical support, collagen-based microspheres with specific swelling rates and cross-linking densities not only possess good injectability and mechanical support, but also enable cell recruitment, cell proliferation, and extracellular matrix secretion under the skin, thereby achieving a long-lasting and stable subcutaneous tissue filling effect; (3) by adjusting the components, the performance of the microspheres can be precisely controlled, thereby optimizing the therapeutic effect. In addition, collagen-based microspheres have good biocompatibility and biodegradability, which aligns with the concept of cell-free tissue engineering. In summary, collagen-based microspheres with specific swelling rates, effective cross-linking densities, and components have significant potential to outperform existing products in areas such as anti-aging and wrinkle removal, wound repair, and postoperative defect filling, and are well-positioned to become a new generation of high-performance subcutaneous tissue filler materials. Attached Figure Description

[0044] 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.

[0045] Figure 1 The in vitro degradation results of collagen-based microspheres provided in the experimental examples of this invention are shown in the figure.

[0046] Figure 2A This is a diagram showing the results of live / dead cell staining in an experimental example of the present invention.

[0047] Figure 2B This is a diagram showing the cell proliferation results provided in the experimental examples of this invention.

[0048] Figure 3 The images show the appearance, morphological characterization, and feasibility results of subcutaneous injection filling of collagen-based microspheres-1 provided in the experimental examples of this invention; where A is the appearance of collagen-based microspheres-1; B is an optical micrograph; C is the particle size distribution; D is a diagram of the subcutaneous injection process in mice; and E is a photograph of the subcutaneous microspheres 24 hours after subcutaneous injection, where the microspheres are within the dashed circle.

[0049] Figure 4The experimental examples of this invention show the effects of collagen-based microspheres and PLLA microspheres with different crosslinking densities on the formation of subcutaneous extracellular matrix; where A is the hematoxylin-eosin (H&E) staining result of subcutaneous regenerated tissue; and B is the extracellular matrix density result. Detailed Implementation

[0050] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0051] Generally, the nomenclature and techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, together with those described herein, are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well-known in the art and described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature, laboratory procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, together with those described herein, are those well-known and commonly used in the art.

[0052] 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.

[0053] According to one aspect of the present invention, a method for preparing collagen-based microspheres and their application in the preparation of subcutaneous tissue filling materials are provided.

[0054] Through extensive experimental exploration and verification, the inventors of this invention discovered that (1) by using a two-step cross-linking method and adjusting the polarity of the solvent used in the second chemical cross-linking process, the swelling rate and effective cross-linking density of collagen-based microspheres can be controlled; (2) unlike traditional filler materials that rely solely on physical support, collagen-based microspheres with specific swelling rates and cross-linking densities not only possess good injectability and mechanical support, but also enable cell recruitment, cell proliferation, and extracellular matrix secretion under the skin, thereby achieving a long-lasting and stable subcutaneous tissue filling effect; (3) by adjusting the components, the performance of the microspheres can be precisely controlled, thereby optimizing the therapeutic effect. In addition, collagen-based microspheres have good biocompatibility and biodegradability, which aligns with the concept of cell-free tissue engineering.

[0055] In some preferred embodiments, the collagen-based microspheres further contain protein components, polysaccharide components, or inorganic components. Typical protein components may be at least one of polypeptides, albumin, gelatin, or laminin; typical polysaccharide components may be at least one of hyaluronic acid, chondroitin sulfate, chitosan, carboxymethyl chitosan, or carboxymethyl cellulose; typical inorganic components include at least one of hydroxyapatite, calcium carbonate, calcium oxide, magnesium oxide, and zinc oxide.

[0056] Introducing specific proteins, polysaccharides, or inorganic substances into collagen-based microspheres can not only regulate their cross-linking structure and degradation behavior but also modulate their ability to promote subcutaneous tissue regeneration. This is one of the key strategies for achieving long-lasting, safe, and functional soft tissue filling. For example, introducing chitosan into collagen-based microspheres can effectively regulate their cross-linking structure and swelling behavior, and also endow the material with antibacterial properties, immunomodulatory capabilities, and a moderate tissue regeneration promoting effect. Introducing bovine serum albumin into collagen-based microspheres can enhance the cross-linking density of the microspheres, reduce swelling, and improve structural stability through physical regulation. Introducing hyaluronic acid into collagen-based microspheres further enhances the active tissue regeneration induction capacity of the collagen-based microspheres, significantly improving the clinical value of the material.

[0057] In some preferred embodiments, the mass swelling ratio of the collagen-based microspheres is 1.7 to 16, for example, but not limited to 1.7, 3, 5, 8, 10, 12, 14, or 16; and / or, the effective crosslinking density is 120 to 5400 mol / m³. 3 For example, it can be, but is not limited to, 120 mol / m 3 500mol / m 3 1000mol / m 3 2000mol / m 3 3000mol / m 3 4000mol / m 3 5000mol / m 3Or 5400mol / m 3 .

[0058] By regulating the swelling rate and effective cross-linking density of collagen-based microspheres, cell proliferation and extracellular matrix secretion can be further promoted, thereby achieving a long-lasting and stable subcutaneous tissue filling effect.

[0059] In some preferred embodiments, the collagen-based microspheres are prepared by the following method:

[0060] 1) Low-crosslinked collagen-based microsphere precursors were prepared by water-in-oil emulsion crosslinking method;

[0061] 2) The collagen-based microsphere precursor is subjected to heterogeneous chemical cross-linking to obtain the collagen-based microspheres.

[0062] The preparation method provided by this invention is simple in process, easy to operate, and improves batch consistency and repeatability.

[0063] In some preferred embodiments, step 1) includes:

[0064] After mixing the raw material solution and the mixed oil solution, the first crosslinking agent is added under stirring. After stirring for 30 minutes, the mixture is allowed to stand to obtain a precipitate. The precipitate is washed with an amphiphilic solvent and then soaked in ethanol. Microspheres with a particle size distribution of 20-200 micrometers are selected to obtain the collagen-based microsphere precursor.

[0065] The above steps can safely and efficiently prepare collagen-based microsphere precursors, providing a solid foundation for subsequent precise adjustment of crosslinking density, swelling behavior, and biological properties through the "second-step crosslinking".

[0066] Specifically, step 1) includes:

[0067] Weigh 0.5g of collagen and dissolve it in 20mL of 0.01M dilute hydrochloric acid solution. Add 120mL of mixed oil (edible oil: paraffin oil = 1:3) solution to a 250mL beaker and stir at a constant speed (800rpm) for 30min to ensure thorough mixing of the two oils. Slowly pour the completely dissolved collagen solution into the mixed oil and continue stirring at a constant speed for 30min. While stirring, add 5mL of 1% (g / mL) EDC PBS solution and continue stirring for 30min. After standing for 10min, remove oil stains from the microspheres by soaking them successively with 1,4-dioxane and acetone. Soak the washed microspheres in anhydrous ethanol, wash three times, and sieve to select collagen-based microsphere precursors with a particle size of 20-200μm. Store in a refrigerator at 4°C for later use.

[0068] In some preferred embodiments, step 2) includes:

[0069] The collagen-based microsphere precursor is soaked in a solvent to swell or shrink, then placed in a second crosslinking agent solution prepared with the corresponding solvent for heterogeneous crosslinking. After soaking and washing with ethanol and drying, the collagen-based microspheres are obtained.

[0070] Step 2) Based on the formed collagen-based microsphere precursor, the crosslinking density and network structure of the microspheres are finely adjusted by precisely controlling the solvent environment and crosslinking conditions.

[0071] In some preferred embodiments, the solvent includes at least one of dichloromethane, ethanol, and PBS.

[0072] In some preferred embodiments, the second crosslinking agent comprises an EDC solution, wherein the solvent of the EDC solution comprises at least one of dichloromethane, ethanol, and PBS.

[0073] In some preferred embodiments, the concentration of the second crosslinking agent is 0.3% to 5% w / v, for example, but not limited to 0.3% w / v, 1% w / v, 2% w / v, 3% w / v, 4% w / v or 5% w / v.

[0074] In some preferred embodiments, the soaking time in the solvent for swelling or shrinkage is 10 to 24 hours, preferably 12 hours.

[0075] Specifically, step 2) includes:

[0076] Collagen-based microsphere precursors were soaked in different solvents for 12 hours, then placed in cross-linking solutions prepared with the corresponding solvents, and cross-linked by shaking while lying flat. After being soaked and washed three times with ethanol, dried and sieved, a series of collagen-based microspheres with different cross-linking densities were obtained and stored in a refrigerator at 4°C for later use.

[0077] In some preferred embodiments, the raw material solution further includes a mixed solution of collagen and chitosan; the collagen-chitosan mixed solution is prepared by the following method:

[0078] Dissolve 0.5g of collagen and 0.3g of carboxymethyl chitosan in 20mL of acidic solution to obtain the raw material solution.

[0079] In some preferred embodiments, the raw material solution further includes a mixed solution of collagen and bovine serum albumin; the collagen-bovine serum albumin mixed solution is prepared by the following method:

[0080] Dissolve 0.5g of collagen and 0.3g of bovine serum albumin in 20mL of acidic solution to obtain the raw material solution.

[0081] In some preferred embodiments, the raw material solution further includes a mixed solution of collagen and hyaluronic acid; the collagen-hyaluronic acid mixed solution is prepared by the following method:

[0082] Dissolve 0.5g of collagen and 0.3g of hyaluronic acid in 20mL of acidic solution to obtain the raw material solution.

[0083] It is understood that the above acidic solution includes a 0.01M dilute hydrochloric acid solution.

[0084] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0085] Example 1

[0086] This embodiment provides a collagen-based microsphere (collagen microsphere-1), which is prepared by the following method:

[0087] Step 1: Weigh 0.5g of collagen and dissolve it in 20mL of 0.01M dilute hydrochloric acid solution. Add 120mL of mixed oil (edible oil: paraffin oil = 1:3) solution to a 250mL beaker and stir at a constant speed (800rpm) for 30min to ensure thorough mixing of the two oils. Slowly pour the completely dissolved collagen solution into the mixed oil and continue stirring at a constant speed for 30min. While stirring, add 5mL of 2% (g / mL) EDC PBS solution and continue stirring for 30min. After standing for 10min, soak the microspheres successively with 1,4-dioxane and acetone to remove oil stains. Soak the washed microspheres in anhydrous ethanol, wash three times, and sieve to select collagen-based microsphere precursors with a particle size of 20-200μm. Store in a 4°C refrigerator for later use.

[0088] Step 2: Soak the collagen-based microsphere precursor in different solvents for 12 hours, then place it in an EDC dichloromethane solution (5%, w / v), shake it horizontally for crosslinking, soak and wash it three times with ethanol, dry it and sieve it to obtain collagen-based microspheres, and store it in a refrigerator at 4°C for later use.

[0089] Example 2

[0090] This embodiment provides a collagen-based microsphere (collagen microsphere-2), which differs from Example 1 in that: dichloromethane is replaced with a dichloromethane / ethanol mixed solvent with a volume ratio of 1:1; and the solvent of the EDC dichloromethane solution is replaced with a dichloromethane / ethanol mixed solvent with a volume ratio of 1:1.

[0091] Example 3

[0092] This embodiment provides a collagen-based microsphere (collagen microsphere-3), which differs from Example 1 in that: dichloromethane is replaced with ethanol; and the solvent of the EDC dichloromethane solution is replaced with ethanol.

[0093] Example 4

[0094] This embodiment provides a collagen-based microsphere (collagen microsphere-4), which differs from Example 1 in that: dichloromethane is replaced with a 1:1 volume ratio of ethanol / PBS mixed solvent; and the solvent of the EDC dichloromethane solution is replaced with a 1:1 volume ratio of ethanol / PBS mixed solvent.

[0095] Example 5

[0096] This embodiment provides a collagen-based microsphere (collagen microsphere-5), which differs from Example 1 in that: dichloromethane is replaced with a 1:1 volume ratio ethanol / PBS mixed solvent; and the solvent of the EDC dichloromethane solution is replaced with a 1:1 volume ratio ethanol / PBS mixed solvent. The concentration of EDC is 2% w / v.

[0097] Example 6

[0098] This embodiment provides a collagen-based microsphere (collagen microsphere-6), which differs from Example 1 in that: dichloromethane is replaced with PBS; and the solvent of the EDC dichloromethane solution is replaced with PBS. The concentration of EDC is 2% w / v.

[0099] Example 7

[0100] This embodiment provides a collagen-based microsphere (collagen microsphere-7), which differs from Example 1 in that: dichloromethane is replaced with PBS; and the solvent of the EDC dichloromethane solution is replaced with PBS. The concentration of EDC is 1% w / v.

[0101] Example 8

[0102] This embodiment provides a collagen-based microsphere (collagen microsphere-8), which differs from Example 1 in that: dichloromethane is replaced with PBS; and the solvent of the EDC dichloromethane solution is replaced with PBS. The concentration of EDC is 0.3% w / v.

[0103] Example 9

[0104] This embodiment provides a collagen-based microsphere (collagen microsphere-9), which differs from Example 1 in that: dichloromethane is replaced with PBS; and the solvent of the EDC dichloromethane solution is replaced with PBS. The concentration of EDC is 0.2% w / v.

[0105] Example 10

[0106] This embodiment provides a collagen-based microsphere (collagen microsphere-10), which differs from Example 1 in that the EDC dichloromethane solution is replaced with PBS. The concentration of EDC is 0% w / v.

[0107] Example 11

[0108] This embodiment provides a collagen-based microsphere (collagen microsphere-11), which differs from Embodiment 1 in that:

[0109] In the first step, the concentration of the PBS solution in the EDC is replaced with 1%;

[0110] In the second step, the EDC dichloromethane solution is replaced with PBS. The concentration of EDC is 0% w / v.

[0111] Example 12

[0112] This embodiment provides a collagen-based microsphere (collagen microsphere-12), which differs from Embodiment 1 in that:

[0113] In the first step, the concentration of the PBS solution in the EDC is replaced with 5%;

[0114] In the second step, the EDC dichloromethane solution is replaced with PBS. The concentration of EDC is 0% w / v.

[0115] Example 13

[0116] This embodiment provides a collagen-chitosan microsphere (Col-CS microsphere), which differs from Embodiment 3 in that:

[0117] Weigh out 0.5g of collagen and dissolve it in 20mL of 0.01M dilute hydrochloric acid solution. Alternatively, weigh out 0.5g of collagen (Col) and 0.3g of carboxymethyl chitosan (CS) and dissolve them in 20mL of 0.01M dilute hydrochloric acid solution.

[0118] Example 14

[0119] This embodiment provides a collagen-bovine serum albumin microsphere (Col-BSA microsphere), which differs from Example 3 in that:

[0120] Weigh out 0.5g of collagen and dissolve it in 20mL of 0.01M dilute hydrochloric acid solution. Alternatively, weigh out 0.5g of collagen (Col) and 0.3g of bovine serum albumin (BSA) and dissolve them in 20mL of 0.01M dilute hydrochloric acid solution.

[0121] Example 15

[0122] This embodiment provides a collagen-hyaluronic acid microsphere (Col-HA microsphere), which differs from Embodiment 3 in that:

[0123] Weigh out 0.5g of collagen and dissolve it in 20mL of 0.01M dilute hydrochloric acid solution. Alternatively, weigh out 0.5g of collagen (Col) and 0.3g of hyaluronic acid (HA) and dissolve them in 20mL of 0.01M dilute hydrochloric acid solution.

[0124] Example 16

[0125] This embodiment provides a collagen-magnesium oxide microsphere (Col-MgO microsphere), which differs from Example 3 in that:

[0126] Weigh out 0.5g of collagen and dissolve it in 20mL of 0.01M dilute hydrochloric acid solution. Alternatively, weigh out 0.5g of collagen (Col) and 0.3g of nano magnesium oxide (MgO) and dissolve them in 20mL of 0.01M dilute hydrochloric acid solution.

[0127] Example 17

[0128] This embodiment provides a collagen-based microsphere (collagen microsphere-13), which differs from Embodiment 1 in that:

[0129] In the second step, the concentration of EDC is 10% w / v.

[0130] Comparative Example 1

[0131] This comparative example provides PLLA microspheres, which are prepared by the following method:

[0132] Dissolve 0.64 g of polylactic acid (PLLA, 100 kDa–150 kDa) in 8 mL of dichloromethane, then add the solution dropwise to a 200 mL beaker containing 100 mL of PVA solution (0.3%, w / v). Stir at 700 rpm for 4 hours at room temperature to allow the dichloromethane to fully evaporate, then stop stirring. Let the beaker stand, discard the supernatant, wash twice with water, and use a sieve to separate PLLA microspheres with a particle size of 20–100 μm. Store the microspheres at 4°C for later use.

[0133] Comparative Example 2

[0134] This comparative example provides a glutaraldehyde-crosslinked collagen-based microsphere_1 (collagen microsphere_glutaraldehyde-1), which differs from Example 1 in that:

[0135] In the first step, EDC is replaced with glutaraldehyde, and the concentration of glutaraldehyde in the PBS solution is 0.2%.

[0136] In the second step, EDC in the dichloromethane solution is replaced with glutaraldehyde. The concentration of glutaraldehyde is 2% w / v.

[0137] Comparative Example 3

[0138] This comparative example provides a collagen-based microsphere crosslinked with glutaraldehyde, No. 2 (collagen microspheres_glutaraldehyde-2), which differs from Comparative Example 2 in that dichloromethane is replaced with ethanol.

[0139] Comparative Example 4

[0140] This comparative example provides a collagen-based microsphere crosslinked with glutaraldehyde, No. 3 (collagen microspheres_glutaraldehyde-3), which differs from Comparative Example 2 in that dichloromethane is replaced with a 1:1 volume ratio of ethanol / PBS mixed solvent.

[0141] Comparative Example 5

[0142] This comparative example provides a glutaraldehyde-crosslinked collagen-based microsphere_4 (collagen microsphere_glutaraldehyde-4), which differs from Comparative Example 2 in that dichloromethane is replaced with PBS.

[0143] Experimental Example 1: Calculation of Effective Crosslinking Density of Microspheres

[0144] The collagen microspheres provided in Examples 1-17 and Comparative Examples 2-5 were placed in pure water and allowed to swell overnight at room temperature until equilibrium was reached. The microspheres were then weighed after gently absorbing surface moisture with filter paper to obtain their wet weight (W). w Dry in an oven for 12–24 hours, weighing multiple times during this period until the weight no longer changes, to obtain the dry weight (W). d Based on the equilibrium swelling ratio (Q) of mass m The calculation formula for ) is as follows:

[0145]

[0146] The equilibrium swelling ratio (Qv) based on volume is calculated as follows:

[0147]

[0148] Where ρ p This is the density of collagen (1.31 g / mL); ρ s (1.0 g / mL) is the density of water. The number-average molecular weight between crosslinking points (M) c The calculation formula for ) is as follows:

[0149]

[0150] Where υ is the specific volume of collagen, V1 is the molar volume of water (18 mL / mol), and χ is the collagen-water interaction constant (χ = 0.161).

[0151] Effective cross-linking density (v) of collagen-based microspheres e The calculation formula for ) is as follows:

[0152]

[0153] The results are shown in Table 1 below:

[0154] Table 1. Mass swelling ratio and effective crosslinking density of collagen microspheres

[0155] serial number <![CDATA[Quality swelling ratio (Q m )]]> <![CDATA[Effective crosslink density (v e )]]> Example 1 (Collagen Microspheres-1) 1.88±0.13 5329.18±727.06 Example 2 (Collagen Microspheres - No. 2) 2.50±0.22 3121.96±463.97 Example 3 (Collagen Microspheres - No. 3) 3.55±0.38 1662.94±81.04 Example 4 (Collagen Microspheres - No. 4) 4.83±0.46 963.91±162.65 Example 5 (Collagen Microspheres - No. 5) 5.85±0.73 696.61±148.92 Example 6 (Collagen Microspheres - No. 6) 8.95±0.40 326.59±24.44 Example 7 (Collagen Microspheres - No. 7) 10.53±0.97 250.71±42.55 Example 8 (Collagen Microspheres - No. 8) 13.55±0.73 161.39±14.95 Example 9 (Collagen Microspheres - No. 9) 17.23±0.49 107.11±5.21 Example 10 (Collagen Microspheres - No. 10) 18.03±0.39 99.14±3.49 Example 11 (Collagen Microspheres-11) 19.68±1.20 85.97±8.35 Example 12 (Collagen Microspheres - No. 12) 10.62±0.66 244.43±26.08 Example 17 (Collagen Microspheres - No. 13) 1.45±0.13 8866.93±1586.07 Example 13 (Col-CS microspheres) 4.15±0.35 NA Example 14 (Col-BSA microspheres) 3.23±0.29 NA Example 15 (Col-HA microspheres) 4.45±0.21 NA Example 16 (Col-MgO microspheres) 3.40±0.43 NA Comparative Example 2 (Collagen Microspheres_Glutaraldehyde-1) 2.25±0.26 3859.78±922.24 Comparative Example 3 (Collagen Microspheres_Glutaraldehyde-2) 4.98±0.36 907.38±120.37 Comparative Example 4 (Collagen Microspheres_Glutaraldehyde-3) 5.45±0.50 777.66±121.25 Comparative Example 5 (Collagen Microspheres_Glutaraldehyde-4) 7.33±0.61 464.83±66.78

[0156] Table 1 shows that the mass swelling ratio (Q) of collagen-based microspheres (collagen microspheres-1 to collagen microspheres-9 and collagen microspheres-13) prepared by the "two-step crosslinking method" is... m ) and effective crosslinking density (v e The swelling ratio of collagen-based microspheres is significantly affected by the "solvent type" and "EDC concentration" used in the second crosslinking process. For example, collagen-based microspheres-13 and-1 prepared using a non-polar organic solvent (dichloromethane) exhibit lower swelling ratios and higher crosslinking densities. With the addition of a polar organic solvent (ethanol), the swelling ratio of the resulting collagen-based microspheres increases, while their crosslinking density decreases significantly. Compared to ethanol solvent, a mixture of ethanol and PBS further increases the swelling ratio of collagen-based microspheres, accompanied by a decrease in their crosslinking density. Collagen-based microspheres prepared using a polar PBS solvent exhibit the highest swelling ratio and the lowest effective crosslinking density. Therefore, 1) at the same EDC concentration, reducing the polarity of the solvent can decrease the swelling ratio of collagen-based microspheres and increase their crosslinking density; 2) with the same solvent, increasing the EDC concentration also decreases the swelling ratio of collagen-based microspheres and increases their crosslinking density.

[0157] Furthermore, the inventors also discovered that: 1) In collagen-based microspheres (collagen microspheres-10 to collagen microspheres-12) obtained solely through the first-step crosslinking method, increasing the EDC concentration decreases their swelling rate and is accompanied by an increase in crosslinking density. However, even when the EDC concentration is increased to 5% (w / v), the crosslinking density of collagen microsphere-12 is much lower than that of collagen microspheres (collagen microspheres-1 to collagen microspheres-5) obtained using the "two-step crosslinking method" and "organic solvents"; collagen microsphere-12 has comparable swelling rates and effective crosslinking densities to collagen microsphere-7 prepared using the "two-step crosslinking method" based on PBS solvent and a lower EDC concentration (1%, w / v). 2) Combining other raw materials (such as carboxymethyl chitosan, bovine serum albumin, hyaluronic acid, and magnesium oxide) with collagen can regulate the swelling rate of collagen-based microspheres; for example, the combination of carboxymethyl chitosan and hyaluronic acid will increase the swelling rate of collagen-based microspheres, while the introduction of bovine serum albumin and magnesium oxide will slightly decrease the swelling rate of collagen-based microspheres.

[0158] In summary, this invention can effectively regulate the swelling rate and effective cross-linking density of collagen microspheres by adjusting the solvent type, EDC cross-linking agent concentration, or compounding with other raw materials (such as polysaccharides, proteins, and inorganic substances) in the "two-step cross-linking method".

[0159] Furthermore, the inventors used glutaraldehyde instead of EDC as a crosslinking agent to prepare four types of collagen-based microspheres. Experiments revealed that the increased polarity of the solvent used in the second crosslinking step led to an increase in the swelling ratio of the collagen-based microspheres and a decrease in the effective crosslinking density. This finding is consistent with the characteristics of EDC-based collagen-based microspheres, demonstrating the universality of this "two-step crosslinking method."

[0160] Experiment Example 2 Degradation Experiment

[0161] Approximately 100 mg of the collagen-based microspheres provided in Examples 1, 5, 9 and 17 were added to tubes containing 5 mL of trypsin (0.15%, w / v) solution. The supernatant was removed after a predetermined time, and the tubes were washed three times with water, dried and weighed.

[0162] like Figure 1 As shown, under high trypsin levels in vitro, the degradation rate of collagen-based microspheres provided in Examples 1, 5, and 9 gradually increased with decreasing cross-linking density within 72 hours. Furthermore, after 96 hours of incubation, the collagen-based microspheres provided in Examples 5 and 9 achieved complete degradation, while the remaining rate of collagen microsphere-1 provided in Example 1 was less than 10%. However, the remaining rate of collagen microsphere-13 with high cross-linking density provided in Example 17 still exceeded 80% after 96 hours, indicating insufficient biodegradability and unsuitability for use as a subcutaneous soft tissue repair material.

[0163] Experiment Example 3: Cell Compatibility Test

[0164] Cell seeding:

[0165] 1g of collagen-based microspheres provided in Example 1 and Example 10 were resuspended in 1ml of cell suspension (containing 2×10⁻⁶ cells per 100ml). 6 (60 μL / well) of vascular smooth muscle cells were seeded into 24-well plates. After 30 minutes, 1 ml of complete culture medium was added to each well, and the plates were placed in a CO2 cell incubator at 37°C.

[0166] Live / dead cell staining:

[0167] After co-culturing collagen-based microspheres with cells for 1 or 7 days, calcein-AM (2 μM) and propidium iodide (PI, 4 μM) were added to the culture medium in each well. After incubation for 30 minutes, the cells were observed under a Nikon fluorescence microscope.

[0168] Cell proliferation detection:

[0169] The proliferation of cells on collagen-based microspheres was quantitatively detected using CCK-8 assay. At each predetermined time point, the cell culture medium in each well of a 24-well plate was replaced with 1.0 mL of CCK-8 working solution, and the plate was returned to a CO2 cell incubator for 30 min. The supernatant was then collected and its absorbance at 450 nm was measured.

[0170] The effects of collagen-based microspheres with different cross-linking densities on the activity of vascular smooth muscle cells were studied using in vitro cell culture methods. Figure 2A As shown, collagen-based microspheres with different cross-linking densities can all provide an adhesion interface for cells and promote cell survival; however, compared to collagen microsphere-10 with a lower cross-linking density, collagen microsphere-1 with a higher cross-linking density promotes cell proliferation more effectively. Figure 2B ).

[0171] Experimental Example 4: Subcutaneous Implantation Experiment

[0172] 100 μL of microspheres were injected subcutaneously into 10-week-old male Kunming mice (KM mice). The animals were euthanized at a predetermined time and samples were collected. Histological staining was used to assess cell recruitment and extracellular matrix formation.

[0173] Particle size and feasibility of subcutaneous injection of collagen-based microspheres

[0174] like Figure 3 As shown, the prepared collagen microspheres-1 are in powder form. Figure 3 A), almost all are in a single dispersed state ( Figure 3 B); In the dry state, its particle size ranges from 20 to 150 μm, mainly concentrated in the range of 40 to 100 μm. Figure 3 C); it can be dispersed in PBS and injected subcutaneously into nude mice. Figure 3 D); and one day later it aggregated in the subcutaneous tissue. Figure 3 E).

[0175] The effects of cross-linking status and composition of collagen microspheres on subcutaneous tissue formation

[0176] Collagen-based microspheres and PLLA microspheres (control group) with different crosslinking densities were subcutaneously injected for 21 days, followed by hematoxylin and eosin (H&E) staining. Figure 4 In section A), both collagen-based microspheres and PLLA microspheres promoted subcutaneous tissue formation to some extent. Quantitative analysis of the extracellular matrix in various regenerated tissues was performed using ImageJ (Table 2 and...). Figure 4 In the study (B), it was found that the extracellular matrix formed by the collagen microspheres-10 group was the sparsest, even lower than that of the PLLA microsphere group. Furthermore, this experiment showed that the microspheres in the collagen microspheres-10 group were significantly larger. Figure 4A) indicates that significant degradation has occurred. Furthermore, this experiment found no statistically significant difference in extracellular matrix density between the collagen microspheres-9 group and the PLLA microsphere group prepared using the "two-step crosslinking method"; the extracellular matrix density of the collagen microspheres-1 group was significantly higher than that of the other groups. For glutaraldehyde-crosslinked collagen-based microspheres, the extracellular matrix density decreased as the crosslinking density of these four types of collagen-based microspheres decreased. These results indicate that the crosslinking density of collagen-based microspheres affects their bioactivity regardless of the crosslinking agent used; increasing the crosslinking density of collagen-based microspheres through the "two-step crosslinking method" can significantly enhance their tissue regeneration properties.

[0177] Table 2. Effects of different collagen-based microspheres on extracellular matrix formation.

[0178] serial number extracellular matrix density Collagen Microspheres-1 120.6±19.5 Collagen Microspheres - No. 3 93.1±16.2 Collagen Microspheres - No. 9 71.7±8.1 Collagen Microspheres-10 54.3±6.8 Collagen Microspheres-11 48.6±7.2 Collagen Microspheres-12 79.7±6.4 Col-CS microspheres 89.3±9.2 Col-BSA microspheres 79.4±7.9 Col-HA microspheres 103.2±11.9 Col-MgO microspheres 91.27±7.6 PLLA microspheres 65.1±7.4 Collagen microspheres_glutaraldehyde-1 103.4±10.8 Collagen microspheres_glutaraldehyde-2 88.1±8.6 Collagen microspheres_glutaraldehyde-3 85.5±9.1 Collagen microspheres_glutaraldehyde-4 77.9±5.7

[0179] To fully investigate the cross-linking status and component effects of collagen-based microspheres on subcutaneous tissue formation, the inventors further studied the subcutaneous tissue formation promotion capabilities of collagen microsphere-3 and its counterparts, Col-CS, Col-BSA, Col-HA, and Col-MgO microspheres with the same cross-linking parameters. They found that the addition of the HA component significantly promoted extracellular matrix secretion. Furthermore, the extracellular matrix density of collagen microsphere-12, prepared only through the first step of cross-linking with EDC aqueous solution (5%, w / v), was significantly lower than that of collagen microsphere-3.

[0180] 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. Application of collagen-based microspheres in the preparation of subcutaneous tissue filling materials.

2. The application according to claim 1, characterized in that, The collagen-based microspheres also contain protein components, polysaccharide components, or inorganic components.

3. The application according to claim 2, characterized in that, The protein component includes at least one of polypeptides, albumin, gelatin, or laminin. Preferably, the polysaccharide component includes at least one of hyaluronic acid, chondroitin sulfate, chitosan, carboxymethyl chitosan, or carboxymethyl cellulose; Preferably, the inorganic component includes at least one of hydroxyapatite, calcium carbonate, calcium oxide, magnesium oxide, and zinc oxide.

4. The application according to any one of claims 1-3, characterized in that, The collagen-based microspheres have a mass swelling ratio of 1.7–16 and / or an effective crosslinking density of 120–5400 mol / m³. 3 .

5. The method for preparing collagen-based microspheres in any one of claims 1-4, characterized in that, The collagen-based microspheres were prepared by the following two-step crosslinking method: 1) Low-crosslinked collagen-based microsphere precursors were prepared by water-in-oil emulsion crosslinking method; 2) The collagen-based microsphere precursor is subjected to heterogeneous chemical cross-linking to obtain the collagen-based microspheres.

6. The preparation method according to claim 5, characterized in that, Step 1) includes: After mixing the raw material solution and the mixed oil solution, the first crosslinking agent is added under stirring. After stirring for 30 minutes, the mixture is allowed to stand to obtain a precipitate. The precipitate is washed with an amphiphilic solvent and then soaked in ethanol. Microspheres with a particle size distribution of 20-200 micrometers are selected to obtain the collagen-based microsphere precursor. Preferably, 0.5g of collagen is dissolved in 20mL of acidic solution to obtain the raw material solution; Preferably, the acidic solution comprises a 0.01M dilute hydrochloric acid solution; Preferably, the mixed oil solution comprises edible oil and paraffin oil in a mass ratio of 1:3; Preferably, the volume ratio of the raw material solution to the mixed oil solution is 1:6; Preferably, the first crosslinking agent comprises a PBS solution of EDC; Preferably, the volume ratio of the raw material solution to the first crosslinking agent is 4:1; Preferably, the amphiphilic solvent includes 1,4-dioxane, acetone, or tetrahydrofuran.

7. The preparation method according to claim 5, characterized in that, Step 2) includes: After the collagen-based microsphere precursor is soaked in a solvent to swell or shrink, it is placed in a second crosslinking agent solution prepared with the corresponding solvent for heterogeneous crosslinking. After soaking and washing with ethanol and drying, the collagen-based microspheres are obtained. Preferably, the solvent includes at least one of dichloromethane, ethanol, and PBS; Preferably, the second crosslinking agent comprises an EDC solution, wherein the solvent of the EDC solution comprises at least one of dichloromethane, ethanol, and PBS; Preferably, the concentration of the second crosslinking agent is 0.3% to 5% w / v; Preferably, the soaking time in the solvent for swelling or shrinkage is 10 to 24 hours.

8. The preparation method according to claim 6, characterized in that, The raw material solution also includes a mixed solution of collagen and chitosan; the collagen-chitosan mixed solution is prepared by the following method: Dissolve 0.5g of collagen and 0.3g of carboxymethyl chitosan in 20mL of acidic solution to obtain the raw material solution; Preferably, the acidic solution comprises a 0.01M dilute hydrochloric acid solution.

9. The preparation method according to claim 6, characterized in that, The raw material solution also includes a mixed solution of collagen and bovine serum albumin; the collagen-bovine serum albumin mixed solution is prepared by the following method: Dissolve 0.5g of collagen and 0.3g of bovine serum albumin in 20mL of acidic solution to obtain the raw material solution; Preferably, the acidic solution comprises a 0.01M dilute hydrochloric acid solution.

10. The preparation method according to claim 6, characterized in that, The raw material solution also includes a mixed solution of collagen and hyaluronic acid; the collagen-hyaluronic acid mixed solution is prepared by the following method: Dissolve 0.5g of collagen and 0.3g of hyaluronic acid in 20mL of acidic solution to obtain the raw material solution; Preferably, the acidic solution comprises a 0.01M dilute hydrochloric acid solution.

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