Monodisperse composite microsphere for tissue filling and regeneration, composite microsphere hydrogel as well as preparation method and application of composite microsphere hydrogel

Abstract

The invention belongs to the field of soft tissue repair, and particularly relates to injectable monodisperse composite microspheres for tissue filling and regeneration, composite microsphere hydrogel as well as a preparation method and application of the composite microsphere hydrogel. The invention aims to provide a composite microsphere capable of simultaneously meeting instant filling and long-term effectiveness, the composite microsphere is prepared by shearing an internal phase solution, an intermediate phase solution and an external phase solution into an emulsion and then curing by adopting a microfluidic technology, a core material of the composite microsphere is poly-L-lactic acid and the like, a shell layer is hyaluronic acid, and the composite microsphere is prepared by adopting a micro-fluidic technology. The obtained composite microspheres are uniform in morphology, size and height, and the sizes of the core layer and the shell layer are controllable. The shell layer is made of hyaluronic acid, tissue occupation can be achieved, instant filling is met, the core material in the composite microsphere is gradually released, growth of subcutaneous collagenous fibers is stimulated, the filled part is gradually replaced by dermal fibers and collagen, the degradation period of the material of the internal microsphere is generally 9-12 months, the effect is more natural, and no false face feeling exists; and the inflammatory response is smaller.

Description

Technical Field

[0001] The present invention belongs to the field of soft tissue repair, and particularly relates to an injectable monodisperse composite microsphere for tissue filling and regeneration, a composite microsphere hydrogel, and a preparation method and application thereof. Background Art

[0002] Skin aging is a complex evolution process, which is divided into endogenous aging and exogenous aging, and is mainly manifested as skin relaxation, decreased elasticity, dryness, increased wrinkles, decreased immune regulation ability, etc. Among them, endogenous factors mainly include time, genetic factors and hormones, while exogenous aging factors include ultraviolet radiation, environmental pollution, living habits, etc. Healthy skin is supported by dense collagen and pulled by elastin, so the skin is plump and elastic. However, the collagen in aging skin is thin and loose, and the ability to repair elastin is also weakened, resulting in decreased elasticity and increased wrinkles. In addition, the renewal and metabolism rate of skin cells also gradually slows down with age, leading to pigment accumulation, dull skin and the appearance of spots.

[0003] With the increasing understanding of the aging process and the growing desire for minimally invasive treatments, injectable fillers have great potential in correcting and restoring facial wrinkles / folds and shaping facial contours. Materials such as injectable fillers are injected into the skin layer or subcutaneous tissue to restore lost volume, smooth lines, soften wrinkles, and enhance facial contours. Filling and shaping soft tissue contours through injectable cosmetic techniques is one of the effective treatment methods for filling depressions, changing contours, and eliminating wrinkles. Most soft tissue filling materials are injected into the skin tissue of wrinkles and depressions, and the volume of the skin tissue is increased by the volume of the material itself to improve skin wrinkles or depressions. Currently, the soft tissue filling materials commonly selected in clinical applications can be roughly divided into three categories according to their sources: (1) Natural biological materials: collagen, hyaluronic acid (HA), etc.; (2) Autologous transplantation materials: autogenous fat granules, autoloius fibroblast cells, etc.; (3) Synthetic materials: acrylic acid (AA), polyacrylamide hydrogel (PAAG), poly-methylmethlacrylate (PMMA), poly-acrylic acidcationic polymer, poly-L-lactic acid (or poly-L-lactide, PLLA), etc. With the increasing demand for facial rejuvenation and the gradual maturity of injectable filling techniques, new products developed based on the above three types of injection materials are emerging continuously.

[0004] Poly-L-lactic acid (PLLA) is a medical aesthetic product approved by the US FDA for repairing subcutaneous soft tissue collagen loss. It is a typical material for soft tissue filling materials prepared from biodegradable polymers, with good biocompatibility and biodegradability. The degradation period is 2 to 12 months, and the degradation period can also be changed according to different modifiers added. Poly-L-lactic acid (PLLA) can be slowly hydrolyzed into lactic acid over time. Lactic acid can cause foreign body giant cell reactions and stimulate the growth of subcutaneous collagen. Eventually, PLLA is enzymatically decomposed in the body to form carbon dioxide and water and is completely degraded, and the filled site is replaced by its own dermal fibers and collagen. However, the generation of collagen after PLLA filling is a long-term and slow process, and it does not take effect immediately after injection. Generally, it begins to take effect 4 to 6 weeks after injection. Moreover, the commercially available PLLA microspheres have poor monodispersity and are prone to adverse reactions such as nodules and granulomas. For example, taking poly-L-lactic acid (PLLA) as the main material of the baby face needle, traditional baby face needle products are freeze-dried powder products mixed with CMC, mannitol and PLLA microspheres. The use of such products requires a lot of experience from doctors in operations such as reconstitution, injection volume, injection site, injection plane, and injection times, and the operation has a high degree of difficulty. And because it is non-volume occupying filling, the injection site will return to the situation before injection within a few days after injection, and collagen gradually begins to be generated 4 to 6 weeks later, and the repair of tissues begins to take effect.

[0005] Hyaluronic acid (HA) is one of the components of the natural extracellular matrix, with good biocompatibility, biodegradability, and strong water retention ability. The micro-nano gels based on hyaluronic acid have the characteristics of adjustable size, easy modification, soft deformability, and can maintain the structural integrity. They can not only meet the application requirements of most bulk gels, but also have unique advantages due to their small size characteristics, so they are widely used in the field of biomedicine. However, the half-life of unmodified HA is about 12 hours and it decomposes rapidly under the action of hyaluronidase. Therefore, predecessors have changed the chemical properties of HA by cross-linking to design a more stable molecule with a longer filling time (about 6 months) in tissues. However, the in vivo filling time of cross-linked HA still cannot meet the requirements. In addition, commercially available products are all gel products, and filling is prone to side effects such as embolism.

[0006] The inventors of the present invention expect to develop a tissue repair filling material that can simultaneously meet immediate filling and long-term effectiveness, combine volume occupying filling and collagen regeneration, and achieve a natural filling effect on the basis of existing products, which can meet the tissue repair products that combine immediate and long-term effects. Summary of the Invention

[0007] The first technical problem to be solved by the present invention is to provide a monodisperse composite microsphere for tissue filling and regeneration that can simultaneously meet immediate filling and long-term effectiveness.

[0008] The monodisperse composite microspheres for tissue filling, regeneration and repair of the present invention are prepared by using microfluidic technology to shear an internal phase solution, an intermediate phase solution, and an external phase solution into an emulsion and then solidifying it. The core material of the composite microspheres is any one or more of poly-L-lactic acid, polycaprolactone, and racemic poly-lactic acid, and the shell layer is hyaluronic acid:

[0009] The total amount of the internal phase solution is calculated as 100% w / v, and includes components with the following weight ratio relationship:

[0010] Any one or more of poly-L-lactic acid, polycaprolactone, and racemic poly-lactic acid 1-20% w / v

[0011] The balance of the solvent is the solvent;

[0012] The total amount of the intermediate phase solution is calculated as 100% w / v, and includes components with the following weight ratio relationship:

[0013]

[0014]

[0015] The internal phase solution in the above technical solution:

[0016] Poly-L-lactic acid (PLLA) has good biocompatibility and is mainly used in the medical fields such as surgical sutures, dentistry, ophthalmology, pharmaceutical controlled release systems, artificial skin, artificial blood vessels, fillers for bone and soft tissue defects, and bioabsorbable stents. In the present invention, it is used as a core material of composite microspheres to stimulate collagen regeneration in the middle and late stages of injection.

[0017] Polycaprolactone (PCL) is a biocompatible, biodegradable, and bioabsorbable polymer, which is widely used in surgical sutures, artificial blood vessels / skin, bone and soft tissue fillers, and tissue scaffolds. Similar to PLLA, the final degradation products of PCL are CO2 and H2O, which can be completely eliminated from the body. In the present invention, it can also be used as a core material of composite microspheres to stimulate collagen regeneration in the middle and late stages of injection.

[0018] Poly-racemic lactic acid (PDLLA) is also a biocompatible, biodegradable, biostimulatory, and persistent material, and is also a subcutaneous stimulatory filler. In the present invention, it can also be used as a core material of composite microspheres to stimulate collagen regeneration in the middle and late stages of injection.

[0019] The molecular weight of the poly-L-lactic acid, polycaprolactone, and poly-racemic lactic acid is 40,000-400,000, and the preferred molecular weight is 70,000-140,000.

[0020] Among them, the solvent is at least one of dichloromethane and chloroform.

[0021] Further preferably, based on 100% w / v of the total amount of the internal phase solution, it includes components with the following weight ratio relationship:

[0022] Any one or more of poly-L-lactic acid, polycaprolactone, and racemic poly-lactic acid 1-10% w / v

[0023] The balance of the solvent is the solvent.

[0024] Most preferably, based on 100% w / v of the total amount of the internal phase solution, it includes components with the following weight ratio relationship:

[0025] Any one or more of poly-L-lactic acid, polycaprolactone, and racemic poly-lactic acid 2-8% w / v

[0026] The balance of the solvent is the solvent.

[0027] The above preferred conclusion is experimentally proven that when the mass-volume ratio of any one or more of poly-L-lactic acid, polycaprolactone, and racemic poly-lactic acid is lower than 2% w / v, the yield of the composite microspheres will be too low, and when it is higher than 8% w / v, the difficulty of preparing the composite microspheres by microfluidics will increase greatly.

[0028] The intermediate phase solution in the above technical solution:

[0029] The molecular weight of the hyaluronic acid is 400,000 - 3,000,000, and preferably the molecular weight is 1,000,000 - 2,000,000, which serves as the shell layer of the composite microspheres to achieve immediate occupancy filling during injection.

[0030] The butanediol diglycidyl ether (BDDE) is used as a cross-linking agent for hyaluronic acid in the present invention.

[0031] The polyethylene glycol (PEG) is used as a dispersant for the intermediate phase in the present invention, and its function is to prevent the composite emulsion from merging easily in the external phase solution before curing.

[0032] The volume ratio of the mixed solution of physiological saline and ultrapure water is 1:1 - 3.

[0033] Preferably, the volume ratio of the mixed solution of physiological saline and ultrapure water is 1:1.5

[0034] Further preferably, based on 100% w / v of the total amount of the intermediate phase solution, it includes components with the following weight ratio relationship:

[0035]

[0036] Most preferably, based on 100% w / v of the total amount of the intermediate phase solution, it includes components with the following weight ratio relationship:

[0037]

[0038] The external phase solution in the above technical solution:

[0039] The external phase solution comprises components with the following volume ratio relationship:

[0040] Heavy paraffin 60 - 100% v / v

[0041] Polyisobutylene succinimide (T - 154) 1 - 20% v / v

[0042] Butanediol diglycidyl ether (BDDE) 0 - 20% v / v;

[0043] Among them, the sum of the volume ratios of heavy paraffin, T - 154, and BDDE is 100% v / v.

[0044] Further preferably, the external phase solution comprises components with the following volume ratio relationship:

[0045] Heavy paraffin 80 - 100% v / v

[0046] Polyisobutylene succinimide (T - 154) 1 - 15% v / v

[0047] Butanediol diglycidyl ether (BDDE) 1 - 10% v / v

[0048] Among them, the sum of the volume ratios of heavy paraffin, T - 154, and BDDE is 100% v / v.

[0049] Most preferably, the external phase solution comprises components with the following volume ratio relationship:

[0050] Heavy paraffin 90 - 100% v / v

[0051] Polyisobutylene succinimide (T - 154) 1 - 10% v / v

[0052] Butanediol diglycidyl ether (BDDE) 1 - 5% v / v

[0053] Among them, the sum of the volume ratios of heavy paraffin, T - 154, and BDDE is 100% v / v.

[0054] The polyisobutylene succinimide (T - 154) is used as a viscosity regulator for the external phase solution in the present invention, so that the viscosity of the external phase matches that of the middle phase to successfully prepare composite microspheres, and at the same time, the stability of the composite emulsion can be improved.

[0055] The monodisperse composite microspheres for tissue filling, regeneration and repair of the present invention are prepared by microfluidic technology, which can control the monodispersity of the O / W / O composite emulsion and the size of the composite emulsion, ensuring that the morphology and size of the composite microspheres after curing are highly uniform and the sizes of the core layer and the shell layer are controllable. The prepared composite emulsion has a particle size of 300 - 1200 μm, the internal droplet size of the composite emulsion is 80 - 400 μm, the particle size of the composite microspheres after curing in the receiving liquid is 200 - 1000 μm, the size of the internal core material microspheres is 30 - 100 μm, the mass fraction of the internal microspheres in the mixed gel is 10 - 30%, and the mass fraction of the hyaluronic acid shell layer in the mixed gel is 1 - 20%. The shell layer of the composite microspheres for tissue filling, regeneration and repair of the present invention is hyaluronic acid, which can achieve tissue occupation and meet immediate filling. At the same time, after injection, the constituent materials of the internal core material microspheres of the composite microspheres, such as poly-L-lactic acid, polycaprolactone or racemic polylactic acid, are gradually released, stimulating the growth of subcutaneous collagen fibers, and the filling site is gradually replaced by its own dermal fibers and collagen. The degradation period of the internal microsphere material is generally 9 - 12 months. Through the composite microspheres, immediate filling and long-term effective tissue repair effects can be achieved, and autologous collagen generation and filling are induced, with a more natural effect and no feeling of a fake face. At the same time, the sizes of the shell layer and the core layer microspheres of the composite microspheres for tissue filling, regeneration and repair of the present invention are uniform, resulting in a smaller inflammatory reaction of the product in the body, and being beneficial to the injection of the product and the accurate evaluation of the injection volume, reducing discomfort.

[0056] The second technical problem solved by the present invention is to provide a preparation method for the monodisperse composite microspheres for tissue filling, regeneration and repair. It is prepared by the microfluidic method. Specifically, after stable droplets are formed from the internal phase solution, the middle phase solution and the external phase solution, they are then received by a receiving liquid, and then the monodisperse composite microspheres for tissue filling, regeneration and repair of the present invention are obtained through steps such as solvent extraction, crosslinking, washing, and dialysis. The specific steps are as follows:

[0057] A. Weigh each raw material and auxiliary material according to the internal phase solution, the middle phase solution, and the external phase solution;

[0058] B. Prepare the internal phase solution: Use a solvent to fully dissolve any one or more of poly-L-lactic acid, polycaprolactone, and racemic polylactic acid to obtain the internal phase solution;

[0059] C. Prepare the middle phase solution: After fully dissolving hyaluronic acid (HA), polyethylene glycol (PEG), and butanediol diglycidyl ether (BDDE) in a mixed solution of physiological saline and ultrapure water, add sodium hydroxide or sodium bicarbonate, stir evenly, and then let it stand in an environment of 1 - 4 °C to obtain the middle phase solution;

[0060] D. Prepare the external phase solution: Mix heavy paraffin, polyisobutylene succinimide, and butanediol diglycidyl ether to obtain it;

[0061] E. Inject the inner phase solution, the middle phase solution, and the outer phase solution into the microfluidic device respectively. After obtaining stable droplets by controlling the flow rates of the three-phase solutions, use a receiving solution to receive the O / W / O double emulsion.

[0062] F. Under the condition of 25 - 50 °C, the hyaluronic acid in the water phase of the double emulsion undergoes crosslinking for 1 - 72 h, and at the same time, the dichloromethane solvent in the oil phase volatilizes to obtain composite microspheres.

[0063] G. Add petroleum ether to soak the composite microspheres to remove the liquid on the outer layer of the composite microspheres, and then dialyze the composite microspheres in PBS buffer solution for 24 - 72 h to obtain the finished composite microspheres.

[0064] In the technical solution of the above preparation method, the addition of sodium hydroxide / sodium bicarbonate in step C is to adjust the pH value to 10 - 13.5; the purpose is to enable the crosslinking of hyaluronic acid under alkaline conditions and increase the fluidity of the middle phase.

[0065] In the technical solution of the above preparation method, the receiving solution described in step E has the same components as the outer phase solution, including components with the following volume ratio relationship:

[0066] Heavy paraffin 60 - 100% v / v

[0067] T - 154 1 - 20% v / v

[0068] BDDE 0 - 20% v / v;

[0069] Among them, the sum of the volume ratios of heavy paraffin, T - 154, and BDDE is 100% v / v.

[0070] Further preferably, the receiving solution described in step E includes components with the following volume ratio relationship:

[0071] Heavy paraffin 80 - 100% v / v

[0072] T - 154 1 - 15% v / v

[0073] BDDE 1 - 10% v / v

[0074] Among them, the sum of the volume ratios of heavy paraffin, T - 154, and BDDE is 100% v / v.

[0075] Most preferably, the receiving solution described in step E includes components with the following volume ratio relationship:

[0076] Heavy paraffin 90 - 100% v / v

[0077] T - 154 1 - 10% v / v

[0078] BDDE 1 - 5% v / v

[0079] Among them, the sum of the volume ratios of heavy paraffin, T-154, and BDDE is 100% v / v.

[0080] In the technical solution of the above preparation method, the ratio of the flow rate of the internal phase solution to the flow rate of the middle phase solution described in step E is 1:1 - 1:50, and the ratio of the flow rate of the middle phase solution to the flow rate of the external phase solution is 1:1 - 1:100.

[0081] Among them, preferably, the microemulsion is crosslinked under the condition of 25 - 50 °C in step F;

[0082] Among them, most preferably, the microemulsion is crosslinked for 48 h under the condition of 37 °C in step F;

[0083] Among them, preferably, the crosslinked microemulsion stands still under the condition of 25 °C in step G;

[0084] Among them, preferably, the composite microspheres are dialyzed for 36 - 48 h after crosslinking in step G;

[0085] Among them, most preferably, the composite microspheres are dialyzed for 48 h after crosslinking in step G;

[0086] Among them, the purpose of adding PBS solution for dialysis is to completely remove BDDE.

[0087] The third technical problem solved by the present invention is to provide a composite microsphere hydrogel, which is a derivative product of the monodisperse composite microspheres for tissue filling and regeneration of the present invention.

[0088] The composite microsphere hydrogel of the present invention is prepared according to the following ratio. In each 1 ml of hydrogel:

[0089] Composite microspheres 10 - 30 mg

[0090] Hyaluronic acid 1 - 5 mg

[0091] PBS solution is made up to 1 ml.

[0092] Preferably, the composite microsphere hydrogel of the present invention is prepared according to the following ratio. In each 1 ml of hydrogel:

[0093] Composite microspheres of the present invention 18 mg

[0094] Hyaluronic acid 2 mg

[0095] PBS solution is made up to 1 ml.

[0096] The hyaluronic acid used in the composite microspheres of the present invention is uncrosslinked hyaluronic acid.

[0097] The fourth technical problem solved by the present invention is to provide a preparation method of a composite microsphere hydrogel, which is a derivative product of the monodisperse composite microspheres for tissue filling and regeneration:

[0098] (1) Weigh the raw materials according to 10 - 30 mg of the composite microspheres of the present invention and 1 - 5 mg of hyaluronic acid per 1 ml of hydrogel.

[0099] (2) Add PBS solution to the raw materials weighed in step (1) and make up the volume to 1 ml to obtain the composite microsphere hydrogel.

[0100] Preferably, in step (1), weigh the raw materials according to 18 mg of the composite microspheres of the present invention and 2 mg of hyaluronic acid per 1 ml of hydrogel.

[0101] The present invention solves the fifth technical problem by providing the use of the monodisperse composite microspheres and composite microsphere hydrogels of the present invention for tissue filling and regeneration in the preparation of soft tissue repair products.

[0102] Generally, materials such as injectable fillers are injected into the skin layer or subcutaneous tissue to restore lost volume, smooth lines, soften wrinkles and enhance facial contours. Specifically, the lost volume is supplemented in the following two ways: physical filling and stimulating the synthesis of autologous collagen fibers. The former shows an immediate filling effect after injection, but will gradually disappear in the short term; classic physical fillers include hyaluronic acid (HA) and collagen. The latter stimulates the formation of collagen by creating space and structure for the entry of fibroblasts and vascular cells, making it more effective in the later stage of injection; typical fillers for stimulating the synthesis of new collagen are composed of polymer microspheres suspended in a solution, such as polylactic acid (PLA), polycaprolactone (PCL), calcium hydroxyapatite (CaHA) and polymethyl methacrylate (PMMA). The tissue filling and regeneration repair composite microspheres developed by the present invention use microfluidic technology to uniformly and dispersedly encapsulate two classic and different types of fillers together. After the tissue filling and regeneration repair microspheres of the present invention are injected, due to the physical properties of the outer hyaluronic acid hydrogel, an immediate filling effect will be produced at the injection site. After filling, the poly-L-lactic acid (or any one of racemic polylactic acid and polycaprolactone) in the inner core material will also start to degrade and stimulate the production of collagen. The present invention provides a brand-new technical solution for the application of facial fillers. Description of the Drawings

[0103] Figure 1 HE staining diagram of the site 3 months after product injection.

[0104] Among them: A is the control product, and B is the monodisperse composite microsphere hydrogel for tissue filling and regeneration of the present invention

[0105] Figure 2 Masson staining diagram of the site 3 months after product injection.

[0106] Among them: A is the control product, and B is the monodisperse composite microsphere hydrogel for tissue filling and regeneration of the present invention Detailed implementation manners

[0107] The following will explain and illustrate the solution of the present invention in combination with embodiments, but will not limit the present invention. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the embodiments regarding specific technologies or conditions, they shall be carried out according to the technologies or conditions described in the literature in the art or according to the product specifications. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0108] The present invention provides a tissue filling and regenerative repair composite microsphere prepared by a microfluidic method with poly-L-lactic acid, polycaprolactone, and racemic poly(lactic acid) as the inner phase and hyaluronic acid as the intermediate phase. After being injected into the filling site, due to the physical properties of the outer hyaluronic acid hydrogel, an obvious filling effect will be immediately generated at the injection site. After filling, the poly-L-lactic acid (or any one of racemic poly(lactic acid) and polycaprolactone) in the internal core material also starts to degrade, stimulating the production of collagen.

[0109] The following are the abbreviations and main noun explanations in English that appear in the present invention:

[0110] 1. Poly-L-lactic acid, also known as poly(L-lactic acid), abbreviated as PLLA in English, has good biocompatibility and is mainly used in the medical fields such as surgical sutures, dentistry, ophthalmology, pharmaceutical controlled release systems, artificial skin, artificial blood vessels, fillers for bone and soft tissue defects, and bioabsorbable scaffolds.

[0111] 2. Polycaprolactone (PCL) is a biocompatible, biodegradable, and bioabsorbable polymer, which is widely used in surgical sutures, artificial blood vessels / skin, bone and soft tissue fillers, and tissue scaffolds. Similar to PLLA, the final degradation products of PCL are CO2 and H2O, which can be completely eliminated from the body.

[0112] 3. Poly(d,l-lactic acid) (PDLLA) is also a biocompatible, biodegradable, biostimulatory, and persistent material and can be used as a new subcutaneous stimulatory filler.

[0113] 4. Hyaluronic acid (HA), also known as hyaluronic acid, can keep the skin moist, smooth, delicate, tender, and elastic, and has the functions of preventing and reducing wrinkles, anti-wrinkle, beauty care, and restoring the physiological functions of the skin.

[0114] 5. 1,4-Butanediol diglycidyl ether (BDDE), with the Chinese alias of 1,4-Butanediol diglycidyl ether.

[0115] 6. Polyisobutylene succinimide (T-154), an ashless dispersant prepared from high-activity polyisobutylene (molecular weight = 1000) by a thermal addition process. It has good detergency and dispersibility and can inhibit the formation of carbon deposits and paint films on engine pistons.

[0116] 7. Polyethylene glycol (PEG), a polymer with the chemical formula HO(CH2CH2O)nH. It is non-irritating, slightly bitter in taste, has good water solubility, and good compatibility with many organic components. It has excellent lubricity, moisturizing property, dispersibility, and adhesiveness.

[0117] 8. PBS solution, also known as phosphate buffer solution, is generally prepared by choosing Na2HPO4 and KH2PO4 because the sodium salt dissolves slowly. According to solutions with different pH values, different masses of phosphates are weighed, and the pH of the solution can also be adjusted with a pH meter. PBS is generally used as a supporting electrolyte. The pH value of the PBS solution is about 7.4. Take 38.0 g of sodium dihydrogen phosphate and 5.04 g of disodium hydrogen phosphate, and add water to make 1000 ml, then it is obtained.

[0118] The following are the screening experiments on the main raw materials, dosages, and process parameters of the composite microspheres and composite microsphere hydrogels of the present invention.

[0119] Example 1:

[0120]

[0121] Steps:

[0122] A. Weigh each raw material and auxiliary material according to the inner phase solution, middle phase solution, and outer phase solution;

[0123] B. Prepare the inner phase solution: Fully dissolve poly-L-lactic acid with a solvent to obtain the inner phase solution;

[0124] C. Prepare the middle phase solution: Fully dissolve hyaluronic acid (HA), polyethylene glycol (PEG), and butanediol diglycidyl ether (BDDE) in a mixed solution of physiological saline and ultrapure water for 1 h, then add sodium hydroxide, stir evenly, and continue to stand in a 4°C environment until the bubbles completely disappear to obtain the middle phase solution;

[0125] D. Prepare the outer phase solution: Mix heavy paraffin, T-154, and butanediol diglycidyl ether to obtain it (the components of the receiving solution are the same as those of the outer phase solution);

[0126] E. Inject the internal phase solution, middle phase solution, and external phase solution into the microfluidic device respectively. After controlling the flow rates of the three-phase solutions to obtain stable droplets, use a receiving solution (with the same components as the external phase solution) to receive the droplets to obtain an O / W / O composite emulsion. The flow rate of the internal phase solution is 1 ml / h, the flow rate of the middle phase solution is 4 ml / h, and the flow rate of the external phase solution is 12 ml / h.

[0127] F. The composite microemulsion is crosslinked for 2 h at 50 °C, and at the same time, the dichloromethane solvent in the oil phase volatilizes to obtain composite microspheres.

[0128] G. Add petroleum ether to completely immerse the composite microspheres, volatilize the petroleum ether to remove the liquid on the outer layer of the composite microspheres, and then dialyze the composite microspheres in PBS buffer solution for 48 h to obtain the final composite microspheres.

[0129] H. Weigh 18 mg of composite microspheres and 2 mg of non-crosslinked HA, and make up the volume to 1 mL with buffer solution to obtain a composite microsphere hydrogel product.

[0130] In this example, 1,4-butanediol diglycidyl ether (BDDE) in the formula was screened and determined. BDDE was not added to the middle phase in Formula 1, BDDE was not added to the external phase in Formula 2, and BDDE was added to both phases in Formula 3. Composite microspheres could be formed in both Formula 1 and Formula 2. However, in animal experiments, HA degraded within 2 - 3 weeks for these two formulas, while HA in Formula 3 degraded at 3 months. Although microspheres could be formed with all three formulas, in order to achieve the most preferred purpose of the present invention, which is to obtain an extended immediate filling effect, and to safely and slowly expose the internal phase gradually to achieve the purpose of long-term filling, the best choice is Formula 3, that is, the technical solution of adding 1,4-butanediol diglycidyl ether (BDDE) to both the middle phase and the external phase simultaneously.

[0131] Example 2:

[0132]

[0133] Steps:

[0134] A. Weigh each raw material and auxiliary material according to the internal phase solution, middle phase solution, and external phase solution.

[0135] B. Prepare the internal phase solution: Use a solvent to fully dissolve poly-L-lactic acid to obtain the internal phase solution.

[0136] C. Prepare the middle phase solution: Use a mixed solution of physiological saline and ultrapure water to fully dissolve hyaluronic acid (HA), polyethylene glycol (PEG), and butanediol diglycidyl ether (BDDE) for 1 h, then add sodium hydroxide, stir evenly, and continue to stand in a 4 °C environment until the bubbles completely disappear to obtain the middle phase solution.

[0137] D. Prepare the external phase solution: Mix heavy paraffin, T-154, and butanediol diglycidyl ether to obtain it (the components of the receiving solution are the same as those of the external phase solution).

[0138] E. Inject the internal phase solution, the intermediate phase solution, and the external phase solution into the microfluidic device respectively. After obtaining stable droplets by controlling the flow rates of the three-phase solutions, use the receiving solution (with the same components as the external phase solution) to receive the droplets to obtain the microemulsion. The flow rate of the internal phase solution is 1 ml / h, the flow rate of the intermediate phase solution is 4 ml / h, and the flow rate of the external phase solution is 12 ml / h.

[0139] F. Crosslink the hyaluronic acid in the water phase of the multiple emulsion at 50 °C for 2 h, and at the same time, the dichloromethane solvent in the oil phase volatilizes to obtain the composite microspheres.

[0140] G. Add petroleum ether to completely immerse the composite microspheres, volatilize the petroleum ether to remove the liquid on the outer layer of the composite microspheres, and then dialyze the composite microspheres in PBS buffer solution for 48 h to obtain the final composite microspheres.

[0141] H. Weigh 18 mg of the composite microspheres and 2 mg of non-crosslinked HA, and make up to 1 mL with buffer solution to obtain the composite microsphere hydrogel product.

[0142] In this example, the molecular weight of hyaluronic acid (HA) in the formula was screened and determined. Formula 1 was HA with a molecular weight of 1.2 million, Formula 2 was HA with a molecular weight of 1.3 million, and Formula 3 was HA with a molecular weight of 1.5 million. Microspheres could be formed with all three formulas. However, in animal experiments, the degradation times of Formula 1 and Formula 2 were shorter than that of Formula 3. Since this patent aims to obtain a longer immediate filling effect and safely and slowly expose the internal phase, the optimal choice is Formula 3, using hyaluronic acid with a larger molecular weight.

[0143] Example 3

[0144]

[0145] Steps:

[0146] A. Weigh each raw material and auxiliary material according to the internal phase solution, the intermediate phase solution, and the external phase solution.

[0147] B. Prepare the internal phase solution: Dissolve poly-L-lactic acid with a solvent to obtain the internal phase solution.

[0148] C. Prepare the intermediate phase solution: Dissolve hyaluronic acid (HA), polyethylene glycol (PEG), and butanediol diglycidyl ether (BDDE) in a mixed solution of physiological saline and ultrapure water for 1 h, then add sodium bicarbonate, stir evenly, and continue to stand in a 4 °C environment until the bubbles completely disappear to obtain the intermediate phase solution.

[0149] D. Prepare the external phase solution: Mix heavy paraffin, T-154, and butanediol diglycidyl ether to obtain it (the components of the receiving solution are the same as those of the external phase solution).

[0150] E. Inject the internal phase solution, intermediate phase solution, and external phase solution into the microfluidic device respectively. After obtaining stable droplets by controlling the flow rates of the three-phase solutions, use the receiving solution (with the same components as the external phase solution) to receive the droplets to obtain the microemulsion. The flow rate of the internal phase solution is 1 ml / h, the flow rate of the intermediate phase solution is 4 ml / h, and the flow rate of the external phase solution is 12 ml / h.

[0151] F. Crosslink the hyaluronic acid in the water phase of the multiple emulsion for 2 h at 50 °C, and at the same time, volatilize the dichloromethane solvent in the oil phase to obtain the composite microspheres, thus obtaining the composite microspheres.

[0152] G. Add petroleum ether to completely immerse the composite microspheres, volatilize the petroleum ether to remove the liquid on the outer layer of the composite microspheres, and then dialyze the composite microspheres in PBS buffer solution for 48 h to obtain the final composite microspheres.

[0153] H. Weigh 18 mg of the composite microspheres and 2 mg of non-crosslinked HA, and make up the volume to 1 mL with a buffer solution to obtain the composite microsphere hydrogel product.

[0154] In this example, the molecular weight of poly-L-lactic acid (PLLA) in the formula was screened and determined. Formula 1 was PLLA with a molecular weight of 180,000, formula 2 was PLLA with a molecular weight of 280,000, and formula 3 was PLLA with a molecular weight of 400,000. Microspheres could be formed with all three formulas. However, in animal experiments, the collagen-stimulating ability of formula 1 was not strong and the degradation time was fast, and the degradation time of formula 3 was 9 months with the possibility of causing fibrous nodules. Therefore, formula 2 was the optimal solution.

[0155] According to the above screening experiments, the monodisperse composite microspheres of the present invention for tissue filling and regeneration are prepared by a microfluidic method from an internal phase solution, an intermediate phase solution, and an external phase solution:

[0156] Based on 100% w / v of the total amount of the internal phase solution, it includes components with the following weight ratio relationship:

[0157] Any one or more of poly-L-lactic acid, polycaprolactone, and racemic poly-lactic acid 1-20% w / v

[0158] The solvent is the balance as the solvent;

[0159] Further preferably, based on 100% w / v of the total amount of the internal phase solution, it includes components with the following weight ratio relationship:

[0160] Any one or more of poly-L-lactic acid, polycaprolactone, and racemic poly-lactic acid 1-10% w / v

[0161] The balance of the solvent is the solvent.

[0162] Most preferably, based on the total amount of the internal phase solution being 100% w / v, it includes components with the following weight ratio relationships:

[0163] Any one or more of poly-L-lactic acid, polycaprolactone, and racemic poly-lactic acid 4-8% w / v

[0164] The balance of the solvent is the solvent.

[0165] When poly-L-lactic acid, polycaprolactone, and racemic poly-lactic acid are used as raw materials, the molecular weight is 40,000 - 400,000; preferably the molecular weight is 70,000 - 140,000

[0166] The solvent is any one of dichloromethane and chloroform.

[0167] Moreover, if the mass-volume ratio of any one or more of poly-L-lactic acid, polycaprolactone, and racemic poly-lactic acid is less than 2% w / v, the yield of the composite microspheres will be too low, and if it is higher than 8% w / v, the difficulty of microfluidic preparation of the composite microspheres will increase greatly.

[0168] Based on the total amount of the middle phase solution being 100% w / v, it includes components with the following weight ratio relationships:

[0169]

[0170]

[0171] The balance of the mixed solution of normal saline and ultrapure water is the mixed solution of normal saline and ultrapure water.

[0172] Further preferably, based on the total amount of the middle phase solution being 100% w / v, it includes components with the following weight ratio relationships:

[0173]

[0174] Most preferably, based on the total amount of the middle phase solution being 100% w / v, it includes components with the following weight ratio relationships:

[0175]

[0176] The middle phase solution in the above technical solution:

[0177] The molecular weight of the HA is 400,000 - 3,000,000, preferably the molecular weight is 1,000,000 - 2,000,000, and it is used as the shell layer of the composite microspheres for occupancy filling during injection in the present invention.

[0178] The volume ratio of the mixed solution of physiological saline and ultrapure water is 1:1 - 3.

[0179] Preferably, the volume ratio of the mixed solution of physiological saline and ultrapure water is 1:1.5

[0180] The outer phase solution comprises components with the following volume ratio relationship:

[0181] Heavy paraffin wax 60 - 100% v / v

[0182] T - 154 1 - 20% v / v

[0183] Butanediol diglycidyl ether (BDDE) 0 - 20% v / v;

[0184] Among them, the sum of the volume ratios of heavy paraffin wax, T - 154, and BDDE is 100% v / v.

[0185] More preferably, the outer phase solution described in step D comprises components with the following volume ratio relationship:

[0186] Heavy paraffin wax 80 - 100% v / v

[0187] T - 154 1 - 15% v / v

[0188] Butanediol diglycidyl ether (BDDE) 1 - 10% v / v

[0189] Among them, the sum of the volume ratios of heavy paraffin wax, T - 154, and BDDE is 100% v / v.

[0190] Most preferably, the outer phase solution described in step D comprises components with the following volume ratio relationship:

[0191] Heavy paraffin wax 90 - 100% v / v

[0192] T - 154 1 - 10% v / v

[0193] Butanediol diglycidyl ether (BDDE) 1 - 5% v / v

[0194] Among them, the sum of the volume ratios of heavy paraffin wax, T - 154, and BDDE is 100% v / v.

[0195] The preparation method of the monodisperse composite microspheres for tissue filling and regeneration of the present invention adopts the microfluidic method. Specifically, after stable droplets are formed using an inner phase solution, an intermediate phase solution, and an outer phase solution, they are then received by a receiving solution, and then obtained through steps such as drying, washing, dialysis, and adjusting the osmotic pressure to obtain the monodisperse composite microspheres for tissue filling and regeneration of the present invention.

[0196] The specific steps are as follows:

[0197] A. Weigh each raw material and auxiliary material according to the internal phase solution, intermediate phase solution, and external phase solution;

[0198] B. Prepare the internal phase solution: Use a solvent to fully dissolve any one or more of poly-L-lactic acid, polycaprolactone, and racemic poly-lactic acid to obtain the internal phase solution;

[0199] C. Prepare the intermediate phase solution: Use a mixed solution of physiological saline and ultrapure water to fully dissolve hyaluronic acid (HA), polyethylene glycol (PEG), and butanediol diglycidyl ether (BDDE), then add sodium hydroxide or sodium bicarbonate, stir evenly, and leave it to stand in an environment of 1 - 4 °C to obtain the intermediate phase solution;

[0200] D. Prepare the external phase solution: Mix heavy paraffin, T - 154, and butanediol diglycidyl ether to obtain it;

[0201] E. Inject the internal phase solution, intermediate phase solution, and external phase solution into a microfluidic device respectively. By controlling the flow rates of the three-phase solutions, after obtaining stable droplets, use a receiving liquid to receive and obtain an O / W / O double emulsion;

[0202] F. Under the condition of 25 - 50 °C, the hyaluronic acid in the water phase of the double emulsion undergoes cross-linking (the cross-linking time is 1 - 72 h), and at the same time, the dichloromethane solvent in the oil phase volatilizes to obtain composite microspheres;

[0203] G. Add petroleum ether to completely immerse the composite microspheres to remove the liquid on the outer layer of the composite microspheres, and then dialyze the composite microspheres in PBS buffer solution for 24 - 72 h to obtain the final composite microspheres.

[0204] In the technical solution of the above preparation method, the addition of sodium hydroxide / sodium bicarbonate in step C is to adjust the pH value to 10 - 13.5; the purpose is to make hyaluronic acid cross-link under alkaline conditions and increase the fluidity of the intermediate phase.

[0205] In the technical solution of the above preparation method, the receiving liquid described in step E has the same components as the external phase solution, including components with the following volume ratio relationship:

[0206] Heavy paraffin 60 - 100% v / v

[0207] T - 154 1 - 20% v / v

[0208] BDDE 0 - 20% v / v;

[0209] Among them, the sum of the volume ratios of heavy paraffin, T - 154, and BDDE is 100% v / v.

[0210] Further preferably, the receiving in step E includes components with the following volume ratio relationship:

[0211] Heavy paraffin 80 - 100% v / v

[0212] T - 154 1 - 15% v / v

[0213] BDDE 1 - 10% v / v

[0214] Among them, the sum of the volume ratios of heavy paraffin, T - 154, and BDDE is 100% v / v.

[0215] Most preferably, the receiving solution described in step E comprises components with the following volume ratio relationship:

[0216] Heavy paraffin 90 - 100% v / v

[0217] T - 154 1 - 10% v / v

[0218] BDDE 1 - 5% v / v

[0219] Among them, the sum of the volume ratios of heavy paraffin, T - 154, and BDDE is 100% v / v.

[0220] In the technical solution of the above preparation method, the flow rate ratio of the inner phase solution to the middle phase solution described in step E is 1:1 - 1:50, and the flow rate ratio of the middle phase solution to the outer phase solution is 1:1 - 1:100.

[0221] Among them, preferably, the microemulsion in step F is crosslinked under the condition of 25 - 50 °C;

[0222] Among them, most preferably, the microemulsion in step F is crosslinked at 37 °C for 48 h;

[0223] Among them, preferably, the crosslinked microemulsion in step G is allowed to stand at 25 °C;

[0224] Among them, preferably, the composite microspheres after crosslinking in step G are dialyzed for 36 - 48 h;

[0225] Among them, most preferably, the crosslinked composite microspheres in step G are dialyzed for 48 h;

[0226] Among them, the purpose of adding PBS solution for dialysis is to completely remove BDDE.

[0227] The derivative product composite microsphere hydrogel of the present invention for tissue filling and regenerating monodisperse composite microspheres is prepared according to the following ratio. In each 1 ml of hydrogel:

[0228] Composite microspheres 10 - 30 mg

[0229] Hyaluronic acid 1 - 5 mg

[0230] PBS solution Make up to 1 ml.

[0231] Preferably, the composite microsphere hydrogel of the present invention is prepared according to the following ratio. In each 1 ml of hydrogel:

[0232] Composite microspheres 18 mg

[0233] Hyaluronic acid 2 mg

[0234] PBS solution is made up to 1 ml.

[0235] The hyaluronic acid used in the composite microspheres of the present invention is uncrosslinked hyaluronic acid.

[0236] Preparation method of the composite microsphere hydrogel of the present invention:

[0237] (1) Weigh the raw materials according to 10 - 30 mg of the composite microspheres of the present invention and 1 - 5 mg of hyaluronic acid in each 1 ml of hydrogel;

[0238] (2) Add PBS solution to the raw materials weighed in step (1) and make up to 1 ml to obtain the composite microsphere hydrogel.

[0239] Preferably, in step (1), the raw materials are weighed according to 18 mg of the composite microspheres of the present invention and 2 mg of hyaluronic acid in each 1 ml of hydrogel.

[0240] In this patent, SD rats were used as the animal model. Combining with the clinical usage method, the in - vivo degradation situation, the degree of inflammatory reaction (safety), and the situation of stimulating collagen regeneration of the composite microsphere hydrogel of the present invention and the commercially available approved products were evaluated by subcutaneous injection of the test articles.

[0241] Experimental samples:

[0242] Product: In this experiment, the composite microsphere hydrogel of the present invention was prepared according to the method of formulation two in Example three.

[0243] Control: The commercially available sculptra product from GALDRMA, batch number 2J1813.

[0244] The experimental animals were 20 female SD rats, 7 - 8 weeks old, with a body weight of 180 - 220 g.

[0245] Before injection, inject 5 ml of normal saline into each vial, shake for 5 min and then let stand for 30 min. Then gently shake to disperse the product evenly, ensuring no layering. Quickly draw an appropriate amount of suspension into a 1-ml sterile syringe with the needle of a 5-ml syringe, and then change to a 26G needle for injection into the subcutaneous tissue of the rats. After the animals have adapted for 3 - 5 days, randomly select 16 for the experiment. One day before injection, use a razor to remove the hair at the injection site. With the rat's spine as the center, mark 2 injection sites on each side, with a center distance of 2 cm between each point on the same side. Inject 0.2 ml at each point, with 2 animals at each time point, for a total of 6 time points, namely 1 week, 2 weeks, 3 weeks, 1 month, 2 months, and 3 months after injection.

[0246] At 1 week, 2 weeks, 3 weeks, 1 month, 2 months, and 3 months after injection, dissect to observe the soft tissue distribution at the filler site and take pictures for record (Dissection operation procedure: Cut the filling site and its surrounding tissues in areas as shown in the injection diagram and fix them on a specimen measurement plate. Take pictures with a single-lens reflex camera, which should be fixed on a top-down desktop bracket and keep the shooting position, angle, and lighting consistent each time. Relevant information should be marked on the measurement plate during photography), and finally observe under a microscope after sectioning.

[0247] Perform HE staining on the tissues at the filling site, and statistically analyze the scoring data of the inflammatory response, fibrosis, and total tissue response to evaluate the irritation grade of the test substance to the skin tissue; perform MASSON staining / Sirius red staining on the tissues at the filling site. Masson staining: For each section, first observe the entire tissue under low magnification, and then select 2 regions at 200 times magnification according to the tissue size and expression for image acquisition. Use the Image-Pro Plus 6.0 image analysis system to measure the fibrous tissue area (Area) in the acquired pictures, and calculate the percentage of fibrous tissue expression area = fibrous tissue area / field area (pixel area). Sirius red staining: Use the Panthera digital trinocular camera microscopic imaging plus filter system produced by Macauley Industrial Group Co., Ltd. to perform image acquisition on the sections. For each section, first observe the entire tissue under low magnification, and then select 2 representative regions at 200 times magnification according to the tissue size and expression for image acquisition. Use the data image analysis system (India labs U.S.A) to measure the expression percentages of type I collagen and type III collagen in the acquired pictures.

[0248] According to the results of the animal experiment:

[0249] Three months after injection, erythema and scratching were observed on the backs of SD rats in the control group (competitive product), while there were not many abnormal phenomena in the composite microsphere hydrogel of the present invention. This may be because there are differences in the structure of the composite microspheres of the present invention and the control group. The heterogeneous PLLA particles (with a particle size of 20 - 100 μm) of the control product can cause an increase in the degree of inflammation and the incidence of granuloma. The particle size of the internal microspheres of the composite microspheres of the present invention is 40 - 63 μm, which greatly controls the predictability of treatment. In Table 1, the particle size of the control product is 19 - 75 μm, and the particle size of the internal microspheres of the composite microspheres in the composite microsphere hydrogel of the present invention is 50 - 55 μm. In terms of the uniformity and range of the particle size of the product, the safety of the monodisperse composite microspheres and composite microsphere hydrogel products for tissue filling and regeneration of the present invention is higher, and the occurrence of inflammation is significantly reduced, which proves the safety and reliability of the monodisperse composite microspheres and composite microsphere hydrogel for tissue filling and regeneration of the present invention.

[0250] Table 1. Particle size of internal microparticles of the product

[0251]

[0252] The HE staining sections of the injection site are as Figure 1 shown. The tissue paraffin sections of the injection site three months after injection were stained with hematoxylin - eosin staining method to observe the inflammatory reaction at the injection site. The degree of inflammation can be judged from the blue - purple degree of the injection site. The darker the purple, the more basophilic granulocytes, which can be understood as the more severe the inflammation. Figure 1 It is significantly indicated that the degree of inflammation of the control product is still much higher than that of the composite microsphere hydrogel of the present invention three months after injection. After statistical analysis by professional software, the stimulation of the control product three months after injection is severe stimulation, and the stimulation of the composite microsphere hydrogel of the present invention is moderate stimulation, with a 46.7% reduction in degree, and the difference is extremely significant (P < 0.01).

[0253] Figure 2 The Masson staining three months after injection is used to evaluate the collagen regeneration of the composite microsphere hydrogel of the present invention and the control product after injection. After Masson section staining, muscle fibers are red, and collagen fibers are green or blue, mainly used to distinguish collagen fibers and muscle fibers. As Figure 2 shown, the composite microsphere hydrogel of the present invention is significantly superior to the control product in terms of the amount of collagen generated. The collagen fibers represented by blue are abundantly expressed within the visual field, proving that the filling effect of the composite microsphere hydrogel of the present invention is more superior. After statistical analysis by professional software, the expression amount of newly generated collagen of the competitive product three months after injection accounts for 0.38% of the total collagen, and the expression amount of newly generated collagen of our product accounts for 2.70% of the total collagen, with a highly significant difference (P < 0.01).

[0254] In summary, the inventors observed the time point when hyaluronic acid was basically degraded 3 months after injection. The PLLA in the core of the composite microspheres of the present invention was initially presented, starting to stimulate and cause subclinical inflammatory reactions and initiate the activity of stimulating collagen regeneration. Judging from the experimental results, compared with the existing commercially available products, the monodisperse composite microspheres and their derivative product composite microsphere hydrogels of the present invention for tissue filling and regeneration have smaller wound surfaces, more stable uniformity, more accurate injection doses, stronger safety, and higher collagen expression levels in the third month (the early stage of the regeneration phase). The expression of both type I / III collagen was extremely significantly higher (P < 0.01) than that of competing products, achieving the goal of faster onset and better short-term effects.

Claims

1. A monodisperse composite microsphere for tissue filling, regeneration and repair, characterized in that: Prepared by the microfluidic method, prepared by shearing the inner phase solution, the middle phase solution, and the outer phase solution into an emulsion and then curing. The composite microsphere core material is any one or more of poly(L-lactic acid), polycaprolactone, and racemic poly(lactic acid), and the shell layer is hyaluronic acid: The total amount of the inner phase solution is calculated as 100% w / v and includes components with the following weight ratio relationships: Any one of poly(L-lactic acid), polycaprolactone, and racemic poly(lactic acid) 1-20% w / v The balance of the solvent is the solvent; The total amount of the middle phase solution is calculated as 100% w / v and includes components with the following weight ratio relationships:

2. The composite microspheres according to claim 1, characterized in that: At least meet any one of the following: In the inner phase solution, the molecular weights of poly(L-lactic acid), polycaprolactone, and racemic poly(lactic acid) are 40,000-400,000; Preferably, in the inner phase solution, the molecular weights of poly(L-lactic acid), polycaprolactone, and racemic poly(lactic acid) are 70,000-140,000; In the inner phase solution, the solvent is at least one of dichloromethane and chloroform; The total amount of the inner phase solution is calculated as 100% w / v and includes components with the following weight ratio relationships: Any one of poly(L-lactic acid), polycaprolactone, and racemic poly(lactic acid) 1-10% w / v The balance of the solvent is the solvent; Most preferably, the total amount of the inner phase solution is calculated as 100% w / v and includes components with the following weight ratio relationships: Any one of poly(L-lactic acid), polycaprolactone, and racemic poly(lactic acid) 2-8% w / v The balance of the solvent is the solvent; In the middle phase solution, the molecular weight of hyaluronic acid is 400,000-3,000,000; In the middle phase solution, the molecular weight of hyaluronic acid is 1,000,000-2,000,000; In the middle phase solution, the volume ratio of the mixed solution of physiological saline and ultrapure water is 1:1-3; Preferably, in the middle phase solution, the volume ratio of the mixed solution of physiological saline and ultrapure water is 1:1.5; The total amount of the middle phase solution is calculated as 100% w / v and includes components with the following weight ratio relationships: Most preferably, the total amount of the middle phase solution is calculated as 100% w / v and includes components with the following weight ratio relationships: The outer phase solution includes components with the following volume ratio relationships: Heavy paraffin 60-100% v / v Polyisobutylene succinimide 1-20% v / v Butanediol diglycidyl ether 0-20% v / v; The sum of the volume ratios of heavy paraffin, polyisobutylene succinimide, and butanediol diglycidyl ether is 100% v / v; Further preferably, the outer phase solution includes components with the following volume ratio relationships: Heavy paraffin 80-100% v / v Polyisobutylene succinimide 1-15% v / v Butanediol diglycidyl ether 1-10% v / v The sum of the volume ratios of heavy paraffin, polyisobutylene succinimide, and butanediol diglycidyl ether is 100% v / v; Most preferably, the outer phase solution includes components with the following volume ratio relationships: Heavy paraffin 90-100% v / v Polyisobutylene succinimide 1-10% v / v Butanediol diglycidyl ether 1-5% v / v The sum of the volume ratios of heavy paraffin, polyisobutylene succinimide, and butanediol diglycidyl ether is 100% v / v.

3. The preparation method of the monodisperse composite microspheres for tissue filling, regeneration and repair according to claim 1 or 2, characterized in that: Including the following steps: A. Weigh each raw material and auxiliary material according to the internal phase solution, intermediate phase solution, and external phase solution; B. Prepare the internal phase solution: Use a solvent to fully dissolve any one or more of poly(L-lactic acid), polycaprolactone, and racemic poly(lactic acid) to obtain the internal phase solution; C. Prepare the intermediate phase solution: Use a mixed solution of physiological saline and ultrapure water to fully dissolve hyaluronic acid, polyethylene glycol, and butanediol diglycidyl ether, then add sodium hydroxide or sodium bicarbonate, stir evenly, and let it stand in an environment of 1 - 4 °C to obtain the intermediate phase solution; D. Prepare the external phase solution: Mix heavy paraffin, polyisobutylene succinimide, and butanediol diglycidyl ether to obtain it; E. Inject the internal phase solution, intermediate phase solution, and external phase solution into the microfluidic device respectively. By controlling the flow rates of the three-phase solutions, after obtaining stable droplets, use a receiving liquid to receive to obtain the O / W / O double emulsion; F. Under the condition of 25 - 50 °C, the hyaluronic acid in the aqueous phase of the double emulsion undergoes crosslinking for 1 - 72 h, and at the same time, the dichloromethane solvent in the oil phase volatilizes to obtain the composite microspheres; G. Add petroleum ether to soak the composite microspheres to remove the liquid on the outer layer of the composite microspheres, and then dialyze the composite microspheres in PBS buffer solution for 24 - 72 h to obtain the finished composite microspheres.

4. The preparation method of the composite microspheres according to claim 3, characterized in that: At least meet any one of the following: In step C, the addition of sodium hydroxide / sodium bicarbonate is to adjust the pH value to 10 - 13.5; The receiving liquid described in step E includes components with the following volume ratio relationship: Heavy paraffin 60 - 100% v / v Polyisobutylene succinimide 1 - 20% v / v Butanediol diglycidyl ether 0 - 20% v / v; The sum of the volume ratios of heavy paraffin, polyisobutylene succinimide, and butanediol diglycidyl ether is 100% v / v; Further preferably, the receiving described in step E includes components with the following volume ratio relationship: Heavy paraffin 80 - 100% v / v Polyisobutylene succinimide 1 - 15% v / v Butanediol diglycidyl ether 1 - 10% v / v The sum of the volume ratios of heavy paraffin, polyisobutylene succinimide, and butanediol diglycidyl ether is 100% v / v; Most preferably, the receiving liquid described in step E includes components with the following volume ratio relationship: Heavy paraffin 90 - 100% v / v Polyisobutylene succinimide 1 - 10% v / v Butanediol diglycidyl ether 1 - 5% v / v The sum of the volume ratios of heavy paraffin, polyisobutylene succinimide, and butanediol diglycidyl ether is 100% v / v; The ratio of the flow rate of the internal phase solution to the flow rate of the intermediate phase solution described in step E is 1:1 - 1:50, and the ratio of the flow rate of the intermediate phase solution to the flow rate of the external phase solution is 1:1 - 1:100; The double emulsion in step F undergoes crosslinking under the condition of 25 - 50 °C; Most preferably, the double emulsion in step F undergoes crosslinking at 37 °C for 48 h; The crosslinked double emulsion in step G stands still at 25 °C; Preferably, the crosslinked composite microspheres in step G are dialyzed for 36 - 48 h; Most preferably, the crosslinked composite microspheres in step G are dialyzed for 48 h.

5. The derivative product composite microsphere hydrogel for the preparation of tissue filling and regeneration of monodisperse composite microspheres according to claim 1 or 2, characterized in that: The composite microsphere hydrogel is prepared according to the following ratio. In every 1 ml of hydrogel: Composite microspheres 10 - 30 mg Hyaluronic acid 1 - 5 mg The PBS solution was made up to 1 ml.

6. The derivative product composite microsphere hydrogel for the preparation of monodisperse composite microspheres for tissue filling and regeneration according to claim 5, characterized in that: Prepare according to the following ratio. In every 1 ml of hydrogel: Composite microspheres 18 mg Hyaluronic acid 2 mg The PBS solution was made up to 1 ml.

7. The preparation method of the composite microsphere hydrogel according to claim 5 or 6, characterized in that: It includes the following steps: (1) Weigh the raw materials according to 10 - 30 mg of the composite microspheres of the present invention and 1 - 5 mg of hyaluronic acid in every 1 ml of hydrogel; (2) Add the PBS solution to the raw materials weighed in step (1) and make up to 1 ml to obtain the composite microsphere hydrogel.

8. The preparation method of the composite microsphere hydrogel according to claim 7, wherein: In step (1), weigh the raw materials according to 18 mg of the composite microspheres of the present invention and 2 mg of hyaluronic acid in every 1 ml of hydrogel.

9. Use of the monodisperse composite microspheres for tissue filling and regeneration as claimed in claim 1 or 2 in the preparation of soft tissue repair products.

10. Use of the composite microsphere hydrogel as claimed in claim 5 or 6 in the preparation of soft tissue repair products.

Images