Sodium hyaluronate gel regeneration filler for injection as well as preparation method and application of sodium hyaluronate gel regeneration filler

Abstract

The invention belongs to the technical field of medical biological materials, and particularly relates to a sodium hyaluronate gel regeneration filler for injection and a preparation method and application thereof.The sodium hyaluronate gel serves as a carrier to disperse acellular matrix particles, the sodium hyaluronate gel forms a micron-sized porous structure in the cross-linking process through a microbubbling technology, and the sodium hyaluronate gel is prepared into the sodium hyaluronate gel regeneration filler for injection and the preparation method and application of the sodium hyaluronate gel regeneration filler. A local cross-linked network is strengthened by combining a secondary cross-linking process, high elastic modulus is realized while the content of sodium hyaluronate is reduced, so that the porous structure has excellent instant filling and shaping capability and controllable degradation behavior, the porous structure can promote permeation of hyaluronidase, the degradation period of the hyaluronidase is matched with the tissue regeneration process, and the tissue regeneration effect is improved. And the acellular matrix particles can continuously induce adhesion, migration, proliferation and differentiation of endogenous cells of tissues, synergistically promote in-situ tissue regeneration, effectively reduce repeated injection requirements and reduce long-term complication risks, and have a good application prospect in soft tissue filling and repairing.

Description

Technical Field

[0001] This invention relates to the field of medical biomaterials technology, and in particular to an injectable sodium hyaluronate gel regeneration filler, its preparation method, and its application. Background Technology

[0002] In recent years, with the continuous advancement of medical aesthetic technology and the increasing market penetration, the field of injectable medical aesthetics is gradually shifting from traditional simple filling and repair to long-lasting regenerative anti-aging. While traditional filler materials, such as hyaluronic acid and botulinum toxin, still dominate the market, regenerative materials are steadily expanding their market share due to their unique advantage of stimulating the regeneration of autologous collagen and achieving a more natural anti-aging effect. At the same time, the application of combination materials has gradually become a focus of industry research and development. Developing multifunctional compound formulations by combining hyaluronic acid with regenerative materials such as collagen, poly-L-lactic acid (PLLA), and hydroxyapatite (CaHA) has become an important path for product innovation in the industry.

[0003] Sodium hyaluronate, a naturally occurring acidic mucopolysaccharide in human connective tissue, is often chosen as a key carrier and supporting matrix in compound formulations due to its excellent biocompatibility, tissue compatibility, and hydration capacity. Natural sodium hyaluronate is readily and rapidly degraded in vivo; therefore, the industry commonly modifies it using cross-linking technology to construct structurally stable cross-linked gels. This improves the material's mechanical support properties and in vivo retention time, meeting the clinical needs of filling and shaping. However, when cross-linked sodium hyaluronate is used in combination with regenerative materials, it is difficult to balance mechanical support performance with the degradation cycle that adapts to the tissue regeneration process. On the one hand, if the hyaluronic acid content is increased to strengthen the gel cross-linking structure and ensure sufficient shaping stability and mechanical support after filling, the degradation rate of the material will be slowed down, resulting in long-term residues in the body. This not only easily causes local tissue reactions such as nodules and granulomas, but also hinders the regeneration and fusion of surrounding normal tissues. On the other hand, if the hyaluronic acid content is reduced to adapt to the tissue regeneration cycle and reduce the risk of residue, the elastic modulus will decrease due to insufficient cross-linking density, resulting in poor shaping effect, easy displacement after filling, and short shape maintenance time. This cannot meet the dual clinical requirements for mechanical shaping performance and tissue regeneration effect. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide an injectable sodium hyaluronate gel regenerative filler, its preparation method, and its application. The invention uses sodium hyaluronate gel as a carrier to disperse decellularized matrix microparticles. The sodium hyaluronate gel forms a micron-sized porous structure during cross-linking via a microbubble process, and a secondary cross-linking process strengthens the local cross-linking network. This reduces the sodium hyaluronate content while achieving a high elastic modulus, thus exhibiting both excellent immediate filling and shaping capabilities and controllable degradation behavior. This porous structure promotes the penetration of hyaluronidase, matching its degradation cycle with the tissue regeneration process. During degradation, it continuously provides growth space for new tissue, while the decellularized matrix microparticles continuously induce the adhesion, migration, proliferation, and differentiation of endogenous cells in the tissue, synergistically promoting in-situ tissue regeneration. This effectively reduces the need for repeated injections and lowers the risk of long-term complications, demonstrating promising application prospects in soft tissue filling and repair.

[0005] To achieve the above objectives, the present invention employs the following technical solution:

[0006] In a first aspect, the present invention provides an injectable sodium hyaluronate gel regeneration filler, the filler comprising a carrier and decellularized matrix microparticles;

[0007] The carrier is sodium hyaluronate gel; the decellularized matrix microparticles are obtained from the submucosa of porcine small intestine through decellularization, freeze-drying, grinding, sieving, and sterilization.

[0008] The sodium hyaluronate gel comprises cross-linked sodium hyaluronate gel particles and non-cross-linked sodium hyaluronate solution;

[0009] The cross-linked sodium hyaluronate gel particles are obtained by cross-linking, microbubbling, secondary cross-linking, and granulation of sodium hyaluronate.

[0010] The non-crosslinked sodium hyaluronate solution is an aqueous solution of sodium hyaluronate containing lidocaine hydrochloride.

[0011] Further, the mass-to-volume ratio of the decellularized matrix microparticles to the carrier is (5-40) mg:1 mL; the mass ratio of the cross-linked sodium hyaluronate gel particles to the non-cross-linked sodium hyaluronate solution is 9:1; the hyaluronic acid content in the cross-linked sodium hyaluronate gel particles is 10-20 mg / mL; and the sodium hyaluronate content in the non-cross-linked sodium hyaluronate solution is 15-25 mg / mL.

[0012] Preferably, the sodium hyaluronate content in the cross-linked sodium hyaluronate gel particles is 12-18 mg / mL.

[0013] Preferably, the non-crosslinked sodium hyaluronate solution contains 20 mg / mL of sodium hyaluronate.

[0014] Furthermore, the molecular weight of the sodium hyaluronate is 100-300 WDa; the elastic modulus G′ of the sodium hyaluronate gel at 0.1 Hz is 400-1000 Pa; and the viscous modulus G′′ of the sodium hyaluronate gel at 0.1 Hz is 100-300 Pa.

[0015] Preferably, the elastic modulus G′ of the sodium hyaluronate gel at 0.1 Hz is 500-800 Pa.

[0016] Preferably, the viscous modulus of the sodium hyaluronate gel at 0.1 Hz is 150-200 Pa (G′′).

[0017] Secondly, the present invention provides a method for preparing an injectable sodium hyaluronate gel regenerating filler, comprising the following steps:

[0018] S1. Sodium hyaluronate and sodium hydroxide solution are mixed, and BDDE (1,4-butanediol diglycidyl ether) is added to obtain a crosslinking reaction solution. The crosslinking reaction solution is subjected to a microbubble process to obtain a sodium hyaluronate gel precursor containing microbubbles. A first crosslinking reaction is carried out at a first set temperature, and a second crosslinking reaction is carried out at a second set temperature to obtain a crosslinked sodium hyaluronate gel. After elution and granulation, crosslinked sodium hyaluronate gel particles are obtained.

[0019] S2. Dissolve lidocaine hydrochloride in sodium hydroxide solution, add sodium hyaluronate to obtain non-crosslinked sodium hyaluronate solution; mix crosslinked sodium hyaluronate gel particles with non-crosslinked sodium hyaluronate solution, sterilize to obtain sodium hyaluronate gel.

[0020] S3. The submucosa of the pig small intestine was placed in a peracetic acid-ethanol mixed solution for virus inactivation, decellularized and freeze-dried to obtain a decellularized matrix, which was then ground, sieved and sterilized to obtain decellularized matrix microparticles.

[0021] S4. Hyaluronic acid gel and decellularized matrix microparticles are reciprocated through a three-way valve, subjected to negative pressure suction, and centrifuged to obtain injectable hyaluronic acid gel regeneration filler.

[0022] In one feasible implementation, in step S1, the mass ratio of sodium hyaluronate, sodium hydroxide solution, and BDDE is 1:(5-10):(0.2-0.6); the concentration of the sodium hydroxide solution is 0.2 mol / L; the microbubble generation process involves: transferring the crosslinking reaction liquid into a microbubble generator, injecting 0.4 MPa of compressed gas and mixing it evenly to form a stable gas-liquid mixture, which is then sprayed out through a nozzle with a diameter of 2-10 mm under conditions of 0.1-1.0 MPa; the compressed gas is selected from air, nitrogen, carbon dioxide, and hydrogen; the diameter of the microbubble is 1-200 μm; the first set temperature is 35-45℃, and the crosslinking reaction time is 2-8 h; the second set temperature is 20-30℃, and the secondary crosslinking time is 8-16 h.

[0023] In one feasible implementation, in step S1, sodium hyaluronate and sodium hydroxide solution are mixed and stirred at a rate of 160-200 rpm for 10-20 min, allowed to stand at room temperature for 1.5-2.5 h, BDDE is added, and stirring is continued at a rate of 160-200 rpm for 5-15 min to obtain a crosslinking reaction solution.

[0024] Cross-linked sodium hyaluronate gel particles are constructed through the synergistic regulation of microbubble formation and secondary cross-linking processes to create a porous cross-linked network adapted to the loading of decellularized matrix microparticles. The microbubble formation process introduces compressed gas into the cross-linking reaction solution to form micron-sized bubbles. These bubbles act as physical separators during the cross-linking process, dispersing the continuous sodium hyaluronate molecular chains into independent network units. This reduces physical entanglement between molecular chains and breaks the traditional model where high sodium hyaluronate content supports mechanical properties, achieving high elastic modulus at low content. At the same time, the uniform porous structure formed after the bubbles dissipate provides sufficient pore space for the uniform loading of subsequent decellularized matrix microparticles and creates channels for the penetration and diffusion of hyaluronidase, making the enzymatic degradation of the gel more controllable.

[0025] The secondary crosslinking process employs a gradient temperature crosslinking strategy. First, a higher temperature is used to rapidly form the gel network framework, ensuring basic mechanical support. Then, a lower temperature is used to slowly refine the local crosslinking sites, optimizing the uniformity and toughness of the network structure. This avoids the problems of uneven network density or insufficient mechanical properties caused by a single crosslinking process, further enhancing the gel's plasticity and structural stability.

[0026] In one feasible implementation, in step S1, the elution step is as follows: the cross-linked sodium hyaluronate gel is immersed in a phosphate buffer solution with pH 7.2 and osmotic pressure of 290 mOsmol / kg at 20-40°C for 12-24 hours for primary elution, then immersed in a newly prepared phosphate buffer solution for secondary elution at 50-60°C for 12-24 hours, and then immersed in a newly prepared phosphate buffer solution for tertiary elution at 70-80°C for 12-24 hours; the granulation step is as follows: the eluted cross-linked sodium hyaluronate gel is extruded through a 35-mesh and a 70-mesh stainless steel sieve plate by 0.5 MPa nitrogen gas.

[0027] The gradient elution process gradually increases the elution temperature, utilizing the temperature's regulatory effect on molecular motion to accelerate the diffusion rate of residual cross-linking agents within the gel network, achieving efficient removal of deep-seated residues. Simultaneously, the gradient heating method avoids the direct damage to the cross-linking network caused by high temperatures. While thoroughly removing residual cross-linking agents and ensuring the gel's biocompatibility, it maintains the integrity of the porous structure and the stability of its mechanical properties. The resulting cross-linked sodium hyaluronate gel particles possess suitable mechanical support, controllable degradation characteristics, sufficient loading space, and good biocompatibility, making them an ideal carrier for decellularized matrix microparticles.

[0028] In one feasible implementation, in step S2, the mass-to-volume ratio of lidocaine hydrochloride, sodium hyaluronate, and sodium hydroxide solution is 0.3g:0.2g:10mL; the concentration of the sodium hydroxide solution is 0.021mol / L; the sterilization is moist heat sterilization, the sterilization temperature is 121℃, and the sterilization time is 15min.

[0029] Non-crosslinked sodium hyaluronate has a linear molecular chain structure and lacks the rigidity of a crosslinked network. When combined with crosslinked gel particles, the linear molecules can fully fill the pores and gaps between the crosslinked gel particles, forming a lubricating layer. This effectively reduces the friction between particles and the overall pushing resistance of the gel, significantly improving the injectability of the gel and meeting the operational requirements of minimally invasive clinical injections. At the same time, linear non-crosslinked sodium hyaluronate and crosslinked gel are based on the same matrix material, exhibiting excellent compatibility and preventing phase separation, thus ensuring the stability of the gel system.

[0030] Lidocaine hydrochloride, as a local anesthetic, is evenly distributed in the gel system through the dispersion effect of the non-crosslinked solution. After injection, it can rapidly block the signal transmission of local nerve endings, inhibit pain perception, and effectively relieve pain during and shortly after injection, improving the comfort of clinical use. The composite of crosslinked gel particles and non-crosslinked sodium hyaluronate solution retains the high elastic modulus and shaping support of the crosslinked gel particles, ensuring immediate filling effect after implantation. At the same time, the lubrication effect of the non-crosslinked components and the addition of analgesic components optimize the gel's operability and user experience. Furthermore, the composite system with the same matrix also improves the gel's biocompatibility, laying a good foundation for subsequent composite with decellularized matrix microparticles.

[0031] In one feasible implementation, in step S3, the volume fraction of peracetic acid in the peracetic acid-ethanol mixed solution is 1%, and the volume fraction of ethanol is 25%; the virus inactivation time is 45 min; the decellularization step is as follows: the virus-inactivated tissue is placed in liquid nitrogen and subjected to three cycles of freeze-thaw, then immersed in a mixed solution containing 0.05 wt% trypsin and 0.03 wt% EDTA, followed by immersion in 1 wt% and 5 wt% sodium chloride solutions for 15 min each, repeated three times, washed with purified water, then immersed in 25 mmol / L NaOH aqueous solution, and washed with purified water until the pH is 6-7; the freeze-drying step is as follows: freezing at -40℃ for 4 h, freezing at -20℃ for 10 h, drying at -5℃ for 6 h, drying at 10℃ for 6 h, and drying and dehydrating at 30℃ for 8 h; the sieve mesh sizes are 50 mesh and 150 mesh respectively; the sterilization step is as follows: cobalt-60 radiation sterilization is performed at 25 kGy.

[0032] The submucosa of porcine small intestine, as a natural extracellular matrix material, is rich in bioactive components such as collagen, glycosaminoglycans, and adhesion proteins, and possesses a natural fibrous structure, providing an ideal biological microenvironment for cell adhesion and proliferation. The combination of chemical inactivation with a peracetic acid-ethanol mixed solution and ultrasonic treatment can thoroughly kill microorganisms and endogenous cells in the tissue through oxidation and physical vibration. Liquid nitrogen cyclic freeze-thaw cycles utilize the formation and fragmentation of ice crystals to further disrupt cell structure, making cellular components easier to remove. Enzymatic hydrolysis with trypsin and EDTA can specifically degrade extracellular protein junction sites and cell membrane components, completely removing cellular residues. Gradient ultrasonic immersion in sodium chloride solution and alkaline treatment can remove non-matrix components such as impurities and glycoproteins from the tissue, purifying the natural matrix structure. The entire decellularization process removes cells and impurities while preserving the fibrous structure and bioactive components of the natural extracellular matrix to the greatest extent possible.

[0033] Gradual cooling and drying through freeze-drying removes moisture from the tissue, maintaining the matrix structure and preventing collapse. This avoids structural shrinkage and loss of active ingredients caused by conventional drying. Further grinding and sieving transforms the blocky decellularized matrix into uniformly sized microparticles, improving its mixing and dispersibility with sodium hyaluronate gel, allowing the microparticles to be uniformly loaded within the gel's porous network. Cobalt-60 radiation sterilization destroys the DNA structure of microorganisms through ionizing radiation, achieving aseptic processing. The radiation dose is controlled within an appropriate range, preventing damage to the matrix's bioactive components. The resulting decellularized matrix microparticles retain the bioactivity and structure of the natural extracellular matrix, serving as carriers of bioactive factors. They induce the adhesion, migration, proliferation, and differentiation of endogenous host cells, initiating and promoting in-situ tissue regeneration, providing long-lasting tissue repair functions for regenerative fillers.

[0034] In one feasible implementation, in step S4, the number of reciprocating pushes is 40-60 times; the number of negative pressure suctions is 10-15 times; the centrifugation speed is 3500-4500 rpm; and the centrifugation time is 8-12 minutes.

[0035] Sodium hyaluronate gel and decellularized matrix microparticles are reciprocated through a sterile three-way valve. The shear and impact forces generated during this pushing process break up the microparticle aggregation, gradually dispersing them into the porous network and gaps of the gel, achieving initial uniform mixing. Further, alternating negative pressure suction generates microbubbles in the mixture. The formation and rupture of these bubbles further generate shear forces, ensuring more thorough contact between the microparticles and gel, achieving deep and uniform mixing. Simultaneously, the microbubbles help open the porous structure of the gel, allowing the microparticles to be more fully embedded in the gel pores, improving load stability. Low-speed centrifugation quickly removes any air bubbles introduced during mixing, preventing the formation of cavities in the tissue after injection, which could affect the filling shaping effect and tissue compatibility. Centrifugation also further compacts the gel-microparticle mixture, preventing microparticle sedimentation and ensuring the uniformity and stability of the filler system, avoiding uneven microparticle distribution during use.

[0036] The entire mixing process was carried out under sterile conditions, ensuring the biosafety of the regenerating filler. The final injectable sodium hyaluronate gel regenerating filler achieved a uniform composite of sodium hyaluronate gel and decellularized matrix microparticles. The gel, acting as a carrier, provides sufficient mechanical support and immediate filling and shaping effect in the early stages of implantation. Its controllable degradation rate matches the tissue regeneration cycle, providing growth space for new tissue during the gradual degradation process. Meanwhile, the uniformly dispersed decellularized matrix microparticles, supported by the gel carrier, continuously exert a bio-inducing effect, inducing the regeneration of endogenous cells and achieving long-term tissue repair. The synergistic function of the two makes the regenerating filler have the dual effects of immediate filling and shaping and long-term tissue repair, achieving an orderly unity of filling effect and tissue regeneration, and improving the naturalness and durability of the repair effect.

[0037] Thirdly, the present invention provides an application of injectable sodium hyaluronate gel regeneration filler in filling, shaping, repairing and regenerating materials.

[0038] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0039] This method uses sodium hyaluronate gel as a carrier to disperse decellularized matrix microparticles. Combining microbubble formation and secondary cross-linking processes, an injectable sodium hyaluronate gel regenerative filler is prepared, possessing both excellent immediate filling and shaping effects and long-lasting, natural tissue repair functions. Through microbubble formation, uniformly distributed micron-sized bubbles are formed within the gel during cross-linking, separating hyaluronic acid molecular chains and reducing intermolecular physical entanglement. Further, a secondary cross-linking process strengthens the local cross-linking network, thereby significantly reducing the sodium hyaluronate content while imparting high elastic modulus to the gel, ensuring strong mechanical support and shaping ability in the initial implantation stage. Simultaneously, the porous structure formed by the introduced microbubbles promotes the penetration and diffusion of hyaluronidase, giving the gel a controllable and gradual degradation rate. Furthermore, its degradation cycle matches the tissue regeneration process, continuously providing growth space for new tissue during gradual degradation. In addition, the decellularized matrix microparticles uniformly dispersed in the gel, with their retained natural extracellular matrix components and structure, continuously induce the adhesion, migration, proliferation and differentiation of endogenous cells in the tissue, thereby effectively initiating and promoting in situ tissue regeneration, achieving long-lasting and natural repair effects, and reducing dependence on repeated injections and the risk of long-term complications. Attached Figure Description

[0040] Figure 1 This is a product image of the sodium hyaluronate gel regeneration filler prepared in Example 5 of the present invention.

[0041] Figure 2 This is an image of the sodium hyaluronate gel regeneration filler prepared in Example 5 of the present invention after extrusion.

[0042] Figure 3 This is a particle size distribution diagram of the sodium hyaluronate gel prepared in Example 1 of the present invention.

[0043] Figure 4 The image shows the viscoelasticity test results of the sodium hyaluronate gel prepared in Example 1 of this invention.

[0044] Figure 5 The image shows the viscoelasticity test results of the sodium hyaluronate gel regeneration filler prepared in Example 5 of this invention, with the ratio of hyaluronic acid gel to decellularized matrix microparticles set to 1 mL: 30 mg.

[0045] Figure 6 The extrusion force curve of the sodium hyaluronate gel prepared in Example 1 of the present invention through a 27G needle.

[0046] Figure 7 The extrusion force curve of the sodium hyaluronate gel regeneration filler prepared by setting the ratio of hyaluronic acid gel to decellularized matrix microparticles to 1 mL: 30 mg in Example 5 of the present invention is shown in the figure. Detailed Implementation

[0047] To make the technical problems, technical solutions, and beneficial effects to be solved by this application clearer, the application will be further described in detail below with reference to embodiments. However, this should not be construed as limiting the scope of this application to the following examples. All other embodiments obtained by those skilled in the art without creative effort without departing from the above-described methodological spirit of this application are within the scope of protection of this application.

[0048] The singular forms “for,” “or,” “a,” “any,” and “described” used in this application are intended to include the plural forms unless the context clearly indicates otherwise. Furthermore, the terms “first” and “second” are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0049] Example 1

[0050] This invention provides a method for preparing sodium hyaluronate gel, comprising the following steps:

[0051] S1. Weigh 1g of sodium hyaluronate with a molecular weight of 160WDa and mix it with 8g of 0.2mol / L NaOH solution. Stir at 180rpm for 15min, let stand at room temperature for 2h, add 0.3g of BDDE, and continue stirring at 180rpm for 10min to obtain a crosslinking reaction solution. Transfer the crosslinking reaction solution to a microbubble generator, inject 0.4MPa of nitrogen gas and mix evenly to form a stable gas-liquid mixture. Spray it out through a 4mm diameter nozzle under a pressure of 0.6MPa to form a sodium hyaluronate gel precursor containing microbubbles with a diameter of 1-200μm. Place the sodium hyaluronate gel precursor in a 37℃ constant temperature chamber for a first crosslinking reaction for 6h, then transfer it to a 25℃ constant temperature chamber for a second crosslinking reaction for 12h. Cut the obtained crosslinked sodium hyaluronate gel into 2cm pieces. 3 Small pieces of the gel were soaked in a phosphate buffer solution with a pH of 7.2 and an osmotic pressure of 290 mOsmol / kg. The gel was then eluted at 25°C for 24 hours. The phosphate buffer solution was replaced, and the gel was eluted at 60°C for 24 hours. The phosphate buffer solution was replaced again, and the gel was eluted at 80°C for 24 hours. After elution, the gel was passed through a first 35-mesh stainless steel sieve under 0.5 MPa nitrogen pressure, and then through a second 70-mesh stainless steel sieve to obtain cross-linked sodium hyaluronate gel particles with a sodium hyaluronate content of 15 mg / mL.

[0052] S2. Dissolve 0.3g of lidocaine hydrochloride in 10mL of 0.021mol / L NaOH solution, add 0.2g of sodium hyaluronate with a molecular weight of 160WDa, stir at 150rpm for 15min, and let stand for 4h to obtain a non-crosslinked sodium hyaluronate solution with a sodium hyaluronate content of 20mg / mL; mix 9g of crosslinked sodium hyaluronate gel particles and 1g of non-crosslinked sodium hyaluronate solution, fill into a glass syringe, and sterilize by moist heat at 121℃ for 15min to obtain a sodium hyaluronate gel with an elastic modulus G′ of 583.7 and a viscous modulus G′′ of 159.0 at 0.1Hz.

[0053] The particle size distribution of the prepared sodium hyaluronate gel is shown in the figure. Figure 3 As shown.

[0054] Example 2

[0055] This invention provides a method for preparing sodium hyaluronate gel, comprising the following steps:

[0056] S1. Weigh 1g of sodium hyaluronate with a molecular weight of 100WDa and mix it with 5g of 0.2mol / L NaOH solution. Stir at 160rpm for 10min, let stand at room temperature for 1.5h, add 0.2g of BDDE, and continue stirring at 160rpm for 5min to obtain a crosslinking reaction solution. Transfer the crosslinking reaction solution to a microbubble generator, inject 0.4MPa of nitrogen gas and mix evenly to form a stable gas-liquid mixture. Spray it out through a 2mm diameter nozzle under a pressure of 0.1MPa to form a sodium hyaluronate gel precursor containing microbubbles with a diameter of 1-200μm. Place the sodium hyaluronate gel precursor in a 35℃ constant temperature chamber for a first crosslinking reaction for 2h, and then transfer it to a 20℃ constant temperature chamber for a second crosslinking reaction for 8h. Cut the obtained crosslinked sodium hyaluronate gel into 2cm pieces. 3 Small pieces of gel were soaked in phosphate buffer solution with pH 7.2 and osmotic pressure of 290 mOsmol / kg, and eluted at 20°C for 12 hours. The phosphate buffer solution was then replaced, and the gel after the first elution was eluted at 50°C for 12 hours. The phosphate buffer solution was then replaced, and the gel after the second elution was eluted at 70°C for 12 hours. After elution, the gel was squeezed through a first 35-mesh stainless steel sieve under 0.5 MPa nitrogen gas, and then through a second 70-mesh stainless steel sieve to obtain cross-linked sodium hyaluronate gel particles with a sodium hyaluronate content of 10 mg / mL.

[0057] S2. Dissolve 0.3g of lidocaine hydrochloride in 10mL of 0.021mol / L NaOH solution, add 0.2g of sodium hyaluronate with a molecular weight of 100WDa, stir at 150rpm for 15min, and let stand for 4h to obtain a non-crosslinked sodium hyaluronate solution with a sodium hyaluronate content of 15mg / mL; mix 9g of crosslinked sodium hyaluronate gel particles and 1g of non-crosslinked sodium hyaluronate solution, fill into a glass syringe, and sterilize by moist heat at 121℃ for 15min to obtain a sodium hyaluronate gel with an elastic modulus G′ of 567.1 and a viscous modulus G′′ of 141.2 at 0.1Hz.

[0058] Example 3

[0059] This invention provides a method for preparing sodium hyaluronate gel, comprising the following steps:

[0060] S1. Weigh 1g of sodium hyaluronate with a molecular weight of 300WDa and mix it with 10g of 0.2mol / L NaOH solution. Stir at 200rpm for 20min, let stand at room temperature for 2.5h, add 0.6g of BDDE, and continue stirring at 200rpm for 15min to obtain a crosslinking reaction solution. Transfer the crosslinking reaction solution to a microbubble generator, inject 0.4MPa of nitrogen gas and mix evenly to form a stable gas-liquid mixture. Spray it out through a 10mm diameter nozzle under a pressure of 1.0MPa to form a sodium hyaluronate gel precursor containing microbubbles with a diameter of 1-200μm. Place the sodium hyaluronate gel precursor in a 45℃ constant temperature chamber for a first crosslinking reaction for 8h, then transfer it to a 30℃ constant temperature chamber for a second crosslinking reaction for 16h. Cut the obtained crosslinked sodium hyaluronate gel into 2cm pieces. 3 Small pieces of gel were soaked in phosphate buffer solution with pH 7.2 and osmotic pressure of 290 mOsmol / kg, and eluted at 40°C for 16 hours. The phosphate buffer solution was then replaced, and the gel after the first elution was eluted at 55°C for 16 hours. The phosphate buffer solution was then replaced, and the gel after the second elution was eluted at 75°C for 16 hours. After elution, the gel was passed through a first 35-mesh stainless steel sieve under 0.5 MPa nitrogen pressure, and then through a second 70-mesh stainless steel sieve to obtain cross-linked sodium hyaluronate gel particles with a sodium hyaluronate content of 20 mg / mL.

[0061] S2. Dissolve 0.3g of lidocaine hydrochloride in 10mL of 0.021mol / L NaOH solution, add 0.2g of sodium hyaluronate with a molecular weight of 300WDa, stir at 150rpm for 15min, and let stand for 4h to obtain a non-crosslinked sodium hyaluronate solution with a sodium hyaluronate content of 25mg / mL; mix 9g of crosslinked sodium hyaluronate gel particles and 1g of non-crosslinked sodium hyaluronate solution, fill into a glass syringe, and sterilize by moist heat at 121℃ for 15min to obtain a sodium hyaluronate gel with an elastic modulus G′ of 652.7 and a viscous modulus G′′ of 174.6 at 0.1Hz.

[0062] Example 4

[0063] This invention provides a method for preparing decellularized matrix microparticles, comprising the following steps:

[0064] S3. After cleaning the submucosa of the pig small intestine with purified water, it was immersed in a peracetic acid-ethanol mixed solution, wherein the volume fraction of peracetic acid was 1% and the volume fraction of ethanol was 25%. The solution was sonicated at 250W for 45 minutes. The inactivated tissue was then subjected to three freeze-thaw cycles in liquid nitrogen. It was then immersed in a mixed aqueous solution containing 0.05% trypsin and 0.03% EDTA, and sonicated at 250W for 45 minutes at 37°C. Following this, it was repeatedly sonicated and immersed in 1% sodium chloride solution and 5% sodium chloride solution, each for 15 minutes, for a total of three cycles. Wash with purified water, then place in a 25 mmol / L NaOH aqueous solution and sonicate at 250 W for 45 min. Wash with purified water until pH=7. Freeze-dry the decellularized tissue at -40℃ for 4 h, -20℃ for 10 h, -5℃ for 6 h, 10℃ for 6 h, and 30℃ for 8 h to obtain decellularized matrix. Freeze the decellularized matrix in liquid nitrogen, grind it with a tissue homogenizer for 7 min, and then sieve it through a 50-mesh coarse sieve and a 150-mesh fine sieve. Sterilize with cobalt-60 radiation at 25 kGy to obtain decellularized matrix microparticles.

[0065] Example 5

[0066] This invention provides a method for preparing an injectable sodium hyaluronate gel regenerating filler, comprising the following steps:

[0067] S4. The sodium hyaluronate gel prepared in Example 1 and the decellularized matrix microparticles prepared in Example 4 were placed in two sterile syringes at a mass ratio of 9:1. The syringes were connected by sterile three-way valves for mixing. First, the sodium hyaluronate gel was pushed towards the decellularized matrix microparticles, and then pushed back in the opposite direction. This was repeated 50 times. Then, the mixture in the syringes was alternately aspirated between the two syringes. This was repeated 12 times under negative pressure to generate bubbles and fully mix the mixture. The syringe containing the regenerated filler was fixed in a low-speed centrifuge and centrifuged at 4000 rpm for 10 minutes to ensure that there were no obvious bubbles or stratification in the regenerated filler before use. The resulting sodium hyaluronate gel regenerated filler was then obtained for injection.

[0068] The prepared sodium hyaluronate gel regeneration filler, such as Figure 1 As shown, the image is of the sodium hyaluronate gel regeneration filler after extrusion. Figure 2 As shown.

[0069] Example 6

[0070] This invention provides a method for preparing an injectable sodium hyaluronate gel regenerating filler, comprising the following steps:

[0071] S4. The sodium hyaluronate gel prepared in Example 2 and the decellularized matrix microparticles prepared in Example 4 were placed in two sterile syringes at a mass ratio of 9:1. The syringes were connected by sterile three-way valves for mixing. First, the sodium hyaluronate gel was pushed towards the decellularized matrix microparticles, and then pushed back in the opposite direction. This was repeated 40 times. Then, the mixture in the syringes was alternately aspirated between the two syringes. This was repeated 10 times under negative pressure to generate bubbles and fully mix the mixture. The syringe containing the regenerated filler was fixed in a low-speed centrifuge and centrifuged at 3500 rpm for 8 minutes to ensure that there were no obvious bubbles or stratification in the regenerated filler before use. The resulting sodium hyaluronate gel regenerated filler was then obtained for injection.

[0072] Example 7

[0073] This invention provides a method for preparing an injectable sodium hyaluronate gel regenerating filler, comprising the following steps:

[0074] S4. The sodium hyaluronate gel prepared in Example 3 and the decellularized matrix microparticles prepared in Example 4 were placed in two sterile syringes at a mass ratio of 9:1. The syringes were connected by sterile three-way valves for mixing. First, the sodium hyaluronate gel was pushed towards the decellularized matrix microparticles, and then pushed back in the opposite direction. This was repeated 60 times. Then, the mixture in the syringes was alternately aspirated between the two syringes. This was repeated 15 times under negative pressure to generate bubbles and fully mix the mixture. The syringe containing the regenerated filler was fixed in a low-speed centrifuge and centrifuged at 4500 rpm for 12 minutes to ensure that there were no obvious bubbles or stratification in the regenerated filler before use. The resulting sodium hyaluronate gel regenerated filler was then obtained for injection.

[0075] Comparative Example 1

[0076] A method for preparing sodium hyaluronate gel differs from Example 1 in that microbubble formation is not performed in step S1, while the remaining steps and parameters are the same.

[0077] Comparative Example 2

[0078] A method for preparing sodium hyaluronate gel differs from Example 1 in that the amount of BDDE crosslinking agent in step S1 is changed to 0.8g, while the remaining steps and parameters are the same.

[0079] Comparative Example 3

[0080] A method for preparing sodium hyaluronate gel differs from Example 1 in that the amount of BDDE crosslinking agent in step S1 is changed to 0.05g, while the remaining steps and parameters are the same.

[0081] Comparative Example 4

[0082] A method for preparing sodium hyaluronate gel differs from Example 1 in that the parameters of the cross-linking reaction are changed in step S1. Specifically, the cross-linking reaction is carried out at 25°C for 12 hours and then at 37°C for 6 hours. The remaining steps and parameters are the same.

[0083] Comparative Example 5

[0084] A method for preparing sodium hyaluronate gel differs from Example 1 in that the elution parameters are changed in step S1. Specifically, elution is performed at 25°C for 12 hours, followed by changing the buffer solution and elution cycled 6 times. The remaining steps and parameters are the same.

[0085] Sodium hyaluronate gel performance test:

[0086] The sodium hyaluronate gel prepared in Example 1 was subjected to a variable design, with other parameters and steps remaining unchanged, to investigate the effects of microbubble process parameters, crosslinking agent BDDE dosage, and crosslinking reaction temperature and time on the properties of sodium hyaluronate gel. Elastic modulus, viscous modulus, extrusion force, and sodium hyaluronate content were tested. All gel samples were sterilized by moist heat. The results are shown in Table 1.

[0087] Variable design:

[0088] 1. Microbubble formation process parameters:

[0089] The nitrogen injection pressure was set to 0.2 MPa, and the injection pressure was set to 0.4 MPa; the obtained sodium hyaluronate gel samples were labeled as 1-1.

[0090] The gas injection pressure was set to 0.6 MPa, and the injection pressure was set to 0.8 MPa; the obtained sodium hyaluronate gel samples were labeled as 1-2.

[0091] The gas injection pressure was set to 0.8 MPa, and the injection pressure was set to 1.0 MPa; the obtained sodium hyaluronate gel samples were labeled as 1-3.

[0092] 2. Dosage of crosslinking agent BDDE:

[0093] The amount of BDDE added was set to 0.2g; the resulting sodium hyaluronate gel sample was labeled as 2-1.

[0094] The amount of BDDE added was set to 0.5g; the prepared sodium hyaluronate gel sample was labeled as 2-2.

[0095] 3. Crosslinking reaction temperature and time:

[0096] The temperature for the first cross-linking reaction was set at 35℃ and the time was set at 2h; the temperature for the second cross-linking reaction was set at 20℃ and the time was set at 8h; the obtained sodium hyaluronate gel sample was labeled as 3-1.

[0097] The temperature for the first cross-linking reaction was set at 37℃ and the time was set at 8h; the temperature for the second cross-linking reaction was set at 25℃ and the time was set at 16h; the obtained sodium hyaluronate gel sample was labeled as 3-2.

[0098] The temperature for the first cross-linking reaction was set at 45℃ and the time was set at 6h; the temperature for the second cross-linking reaction was set at 30℃ and the time was set at 12h; the obtained sodium hyaluronate gel sample was labeled as 3-3.

[0099] 1) Elastic modulus G′ and viscous modulus G′′ test: A rheometer was used to perform fixed strain frequency scanning tests on the sodium hyaluronate gel prepared in Example 1 and through variable design under room temperature and vibration-free conditions. Before the test, 0.5 mL of sample was evenly spread between the parallel plates of the rheometer, the plate spacing was adjusted to 1 mm, and the sample was allowed to stand for 5 min to eliminate internal stress. During the test, the constant strain was 0.1%, the frequency scanning range was set to 0.1-10 Hz, and the data was recorded after each frequency point was stabilized for 3 s. The values ​​of elastic modulus G′ and viscous modulus G′′ of each sample at 0.1 Hz were compared to evaluate the mechanical stability and elastic recovery ability of the gel. Each sample was tested in parallel 3 times, and the average value was taken. At the same time, the loss factor tanδ=G′′ / G′ was calculated based on the measured elastic modulus and viscous modulus data.

[0100] 2) Pushing force test: A universal testing machine was used to evaluate the pushing force performance of the sodium hyaluronate gel pre-filled samples prepared by variable design in Example 1 to simulate a clinical injection scenario. Before the test, a 2.25 mL pre-filled glass syringe was installed and fixed on the testing machine fixture. A 27G thin-walled needle was selected and tightened to ensure a good seal between the needle and the syringe. The pushing speed of the testing machine was set to 30 mm / min, and the test distance was the full volume of the syringe. Pushing force data was collected in real time during the pushing process. Each sample was tested in parallel 5 times, and the average value was taken.

[0101] 3) Sodium hyaluronate content test: First, prepare a 0.125% (v / v) carbazole ethanol solution, a 50 μg / mL glucuronic acid standard solution, and a 0.025 mol / L sodium tetraborate sulfuric acid solution; take 0.1 g of sodium hyaluronate gel prepared in Example 1 and by variable design, add 0.5 mol / L sulfuric acid solution and heat to dissolve in a 95°C constant temperature oven, then add 1 mol / L sodium hydroxide solution, and dilute with water to a sodium hyaluronate concentration of about 50 μg / mL, and take 1 mL and place it in a test tube. Prepare glucuronic acid standard solutions according to a gradient. Place the standard solution tubes and sample tubes in an ice-water bath. Slowly add 5 mL of pre-cooled 0.025 mol / L sodium tetraborate-sulfuric acid solution, shake well, and boil in a boiling water bath for 15 min. Cool to room temperature, add 0.20 mL of carbazole reagent, boil in a boiling water bath again for 15 min, and cool. Using a blank tube as a control, measure the absorbance at 530 nm using a spectrophotometer. Plot an absorbance-concentration standard curve. Calculate the glucuronic acid content in the sample tubes, and then substitute it into the formula to calculate the sodium hyaluronate content. The formula is: Sodium hyaluronate content ρ = 2.0675c1 × (m2 × ρ1) / (m1 × ρ2), where C1 is the glucuronic acid content in the sample tube, m2 is the mass of cross-linked sodium hyaluronate gel and purified water, ρ1 is the density of the cross-linked sodium hyaluronate gel, m1 is the mass of the cross-linked sodium hyaluronate gel, and ρ2 is the density of the mixture of cross-linked sodium hyaluronate gel and purified water.

[0102] 4) BDDE Residue Test: First, weigh 40 mg of BDDE into a 100 mL volumetric flask, dissolve and dilute to the mark with acetone, and shake well to obtain a BDDE standard stock solution with a concentration of 400 μg / mL; then measure 0.2 mL of the stock solution into a 100 mL volumetric flask, dilute to the mark with acetone, and shake well to prepare a BDDE standard solution with a concentration of 0.8 μg / mL. Take 4g of the sodium hyaluronate gel prepared in Example 1 and the sodium hyaluronate gel prepared by variable design, respectively, and place them in a 10mL volumetric flask. Add 8mL of acetone, shake thoroughly, dilute to the mark, shake well, and filter. Take the filtrate as the test solution. The chromatographic conditions are as follows: use a DB-17 column, set the initial column temperature to 150℃, increase to 260℃ at a rate of 30℃ / min and hold for 10min, set the injection port temperature to 260℃, set the detector temperature to 280℃, use nitrogen as the carrier gas and set the flow rate to 1.5mL / min. Inject 1μL of BDDE standard solution and the test solution respectively, record the chromatogram, and calculate the residual amount of BDDE in the test solution.

[0103] Table 1. Performance test results of sodium hyaluronate gels prepared in each group.

[0104]

[0105] As shown in Table 1, the sodium hyaluronate gel prepared in Example 1 and through variable design can achieve an elastic modulus of over 400 Pa while maintaining a low sodium hyaluronate content and a loss factor of over 0.2. It exhibits balanced viscoelastic properties, and has both extrudability and shaping ability during injection. After implantation, it can support the growth of new tissue and achieve an orderly alternation between gel degradation and new tissue formation through viscous encapsulation of microparticles, making it suitable as a carrier for decellularized matrix microparticles.

[0106] like Figure 4 As shown in the viscoelasticity test curve of the sodium hyaluronate gel prepared in Example 1, within the frequency scanning range of 0.1-10 Hz, the elastic modulus G' was consistently significantly higher than the viscous modulus G'', and both showed a slow increasing trend with increasing frequency, exhibiting typical solid-like viscoelastic behavior. Under the test condition of 0.1 Hz, the elastic modulus G' of this gel was 583.7 Pa, the viscous modulus G'' was 159.0 Pa, and the corresponding loss factor tanδ was approximately 0.27, indicating that it possesses both good mechanical support and moderate viscosity characteristics at low frequencies. This allows it to provide a stable scaffold for subsequent loading of decellularized matrix microparticles while ensuring a certain degree of injectability and tissue compatibility.

[0107] like Figure 6 As shown in the extrusion force curve of the sodium hyaluronate gel prepared in Example 1 through a 27G needle, the gel begins to enter the needle at a displacement of about 48mm, the extrusion force rapidly increases and stabilizes in the plateau range of 18-19N, and the overall extrusion force fluctuation is small, indicating that the pure gel has stable flow behavior when passing through a fine needle, has good injectability, and can meet the operation requirements of clinical minimally invasive injection.

[0108] Comparative Example 1 did not employ a microbubble process; the crosslinking reaction solution completed crosslinking under conditions without gas dispersion, resulting in a gel without a uniformly distributed microbubble pore structure. The crosslinking network formed under these conditions was more compact and tightly bound, with stronger inter-chain crosslinking sites, directly leading to a significant increase in the gel's elastic modulus. Simultaneously, the dense structure increased the overall hardness of the gel, resulting in a higher extrusion force. While this type of relatively hard gel can meet the shaping requirements of single injection filling, it lacks the porosity to accommodate decellularized matrix microparticles, making it unsuitable as a carrier for these microparticles and ultimately limiting its application in composites with decellularized matrix microparticles.

[0109] Comparative Example 2 increased the BDDE dosage to 0.8g, resulting in a significant increase in the cross-linking agent concentration. Excessive BDDE leads to an excessive number of cross-linking sites between sodium hyaluronate molecular chains, forming an over-crosslinked, dense network structure. This over-crosslinking not only causes abnormally high elastic modulus and sodium hyaluronate content in the gel but also enhances its mechanical stiffness. The excessively high extrusion force severely reduces the gel's injectability, failing to meet the convenience requirements of clinical injection procedures. Simultaneously, the overly dense gel structure lacks sufficient pores to support the decellularized matrix microparticles, thus failing to fulfill its carrier function.

[0110] Comparative Example 3 reduced the BDDE dosage to 0.05g, resulting in a low crosslinking agent concentration. A small amount of crosslinking agent only allows the sodium hyaluronate molecular chains to form a limited number of crosslinking sites, leading to a loose and unstable crosslinking network structure. The gel prepared under these conditions exhibited low elastic modulus and sodium hyaluronate content, resulting in weak mechanical support. When this type of gel is combined with decellularized matrix microparticles, the loose crosslinking network cannot effectively support the microparticles, easily leading to gel fragmentation and microparticle sedimentation. Its mechanical properties are insufficient to meet the shaping and support requirements of the composite filler in vivo.

[0111] Comparative Example 4 adjusted the crosslinking reaction parameters, performing a first crosslinking at a low temperature of 25℃, followed by a second crosslinking at 37℃. The crosslinking reaction rate was slower at low temperatures, resulting in uneven distribution of preferentially formed crosslinking sites. Subsequent temperature increases made it difficult to compensate for the network uniformity defects. Although the final gel met all performance indicators, its loss factor was below 0.2. A low loss factor indicates weak viscosity and excessive elasticity in the gel, resulting in a hard texture and outstanding deformation recovery ability, making it more suitable for standalone injection filling to maintain shaping effects. However, the carrier of decellularized matrix microparticles requires the gel to possess both elasticity and viscosity to encapsulate and fix the microparticles, thus making it unsuitable for composite filling systems.

[0112] Comparative Example 5 used a conventional elution process with a constant temperature of 25°C and multiple buffer changes, resulting in a low elution temperature and no gradient. Under low temperature conditions, the residual BDDE molecules in the gel network move slowly and are difficult to diffuse quickly into the buffer. Although multiple buffer changes can partially remove BDDE, they cannot completely remove the residual cross-linking agents deep within the gel. In contrast, the high-temperature gradient elution process of 25°C-60°C-80°C used in Example 1 can accelerate the movement of BDDE molecules by gradually increasing the temperature. At the same time, the gradient temperature can maintain the structural integrity of the gel cross-linking network and avoid direct damage to the gel by high temperature. Furthermore, the high-temperature gradient elution process can efficiently remove BDDE residues while ensuring the structural stability of the gel, making it more suitable for subsequent complexation with decellularized matrix microparticles.

[0113] Comparative Examples 1-5, due to the lack of microbubble formation process, gradient crosslinking control process, high-temperature gradient elution process, and unreasonable amount of crosslinking agent, resulted in the formation of excessively dense or loose crosslinking networks, unbalanced viscoelastic properties, and residual crosslinking agent. These combined to manifest as insufficient mechanical support, pore loading capacity, viscoelastic adaptability, and biocompatibility, making them unsuitable for the preparation of composite filling systems.

[0114] In vitro enzymatic hydrolysis resistance test of sodium hyaluronate gel:

[0115] The in vitro enzymatic hydrolysis resistance of the sodium hyaluronate gel prepared by the above-described Example 1 and the above-described variable design was tested, and the results are shown in Table 2.

[0116] Take 0.5g of each of the sodium hyaluronate gel prepared in Example 1 and through the above variable design, as well as 0.5g of each of four commercially available cross-linked sodium hyaluronate gel samples (Restylane, Demand, Haivi M+, and Runbaiyan Xingyao), and place them in a stoppered test tube. Add 4mL of hyaluronidase solution with a concentration of 400U / mL, and vortex to mix the sample and enzyme solution thoroughly. Place the test tube in a 37℃ constant temperature shaking water bath and shake continuously. Take a sample every 15 minutes and measure the absorbance value of the sample at a wavelength of 232nm using a UV-Vis spectrophotometer. Continue until the absorbance value of three consecutive measurements shows no significant change, at which point the gel can be determined to be completely degraded. Record the total time from the start of enzymatic hydrolysis to complete degradation of the gel.

[0117] Table 2. Results of enzymatic hydrolysis performance tests of sodium hyaluronate gels prepared in each group and commercially available brands.

[0118]

[0119] As shown in Table 2, although the in vitro degradation time of sodium hyaluronate gel cannot be completely equated with the tissue maintenance time after implantation, it has important guiding significance for predicting the in vivo maintenance time. According to Table 2, the in vitro degradation time range of the sodium hyaluronate gel prepared in Example 1 and through variable design is 60-105 min. Considering the industry norms for commercially available products: Runbaiyan Xingyao has an in vitro enzymatic hydrolysis time of 75 min, corresponding to a clinical in vivo degradation time of approximately 3 months; Haiwei M+ has an in vitro enzymatic hydrolysis time of 135 min, corresponding to a clinical in vivo degradation time of approximately 6 months. Therefore, the in vitro degradation time of the sodium hyaluronate gel prepared in Example 1 and through variable design corresponds to an in vivo degradation cycle of 3-6 months. This cycle highly matches the formation cycle of new tissue, enabling an orderly alternation and coordinated unity between the gel degradation process and the new tissue formation process.

[0120] As shown in Table 2, Comparative Example 1, lacking microbubble technology, has a dense, non-porous gel cross-linking network, making it difficult for enzyme molecules to penetrate deep into the gel. Enzyme molecules can only slowly decompose the surface molecular chains, leading to a prolonged in vitro enzymatic hydrolysis time. The corresponding in vivo degradation time would exceed 6 months, easily resulting in gel residue and interference with the integration of new tissue. Comparative Example 2, due to excessive BDDE, suffers from over-crosslinking, with dense crosslinking sites between molecular chains. Enzymes cannot efficiently cleave crosslinking bonds, resulting in an extremely slow in vitro enzymatic hydrolysis rate. The corresponding in vivo degradation time would far exceed 6 months, and long-term gel residue could easily trigger foreign body reactions. Comparative Example 3, due to insufficient BDDE, has a loose cross-linking network. In contrast, the enzyme can rapidly break down weak molecular chains, resulting in a very short in vitro enzymatic hydrolysis time, corresponding to less than 3 months of in vivo degradation. Premature degradation of the gel will cause it to lose its supporting function and fail to match the rhythm of new tissue formation. Although the in vitro enzymatic hydrolysis time of Comparative Example 4 is close to that of the example, due to the poor uniformity of the cross-linked network, problems such as excessively rapid local degradation and local residues will occur in the in vivo enzymatic environment, making it unable to stably support the new tissue. In Comparative Example 5, due to the slight reinforcement of local cross-linked structures by the residual BDDE after routine elution, an uneven phenomenon of slow and rapid local degradation will occur during in vivo degradation, affecting the coordinated adaptation between the gel and the new tissue.

[0121] Performance testing of injectable sodium hyaluronate gel regeneration filler:

[0122] The injectable sodium hyaluronate gel regeneration filler prepared in Example 5 was tested for elastic modulus, viscous modulus, extrusion force, and extrusion pressure by a variable design, with other parameters and steps remaining unchanged. The test methods were consistent with the above-mentioned sodium hyaluronate gel performance test steps. The effect of the ratio of hyaluronic acid gel to decellularized matrix microparticles on the performance of the injectable sodium hyaluronate gel regeneration filler was investigated. The results are shown in Table 3.

[0123] Variable design: The ratio of hyaluronic acid gel to decellularized matrix microparticles was set to 1mL:5mg, 1mL:10mg, 1mL:20mg, 1mL:30mg, and 1mL:40mg, respectively.

[0124] Table 3. Effect of the ratio of hyaluronic acid gel to decellularized matrix microparticles on the performance of injectable sodium hyaluronate gel regeneration filler.

[0125]

[0126] As shown in Table 3, with the increase of decellularized matrix microparticle concentration, its physical interaction with the sodium hyaluronate gel network is significantly enhanced. On the one hand, the microparticles, as rigid fillers embedded in the porous network of the gel, increase the mechanical support points of the overall structure, thereby increasing the elastic modulus and viscous modulus proportionally. On the other hand, because the increase in elastic modulus by microparticles is more significant, the loss factor (viscous modulus / elastic modulus) gradually decreases to below 0.2. This change makes the viscoelastic characteristics of the filler closer to the level of natural soft tissue extracellular matrix (ECM). The moderate enhancement of its elastic characteristics can provide a stable mechanical microenvironment for cell adhesion, while the preservation of viscous characteristics can maintain the injectability and tissue compatibility of the filler. The synergistic effect of the two not only ensures the convenience of clinical injection, but also effectively promotes cell migration, proliferation and differentiation, ultimately accelerating the integration and repair of newly formed tissues.

[0127] like Figure 5 As shown in the viscoelasticity test curves of the regenerated filler prepared by mixing sodium hyaluronate gel and decellularized matrix microparticles at a ratio of 1 mL: 30 mg, compared with the pure gel, the elastic modulus G' and viscous modulus G'' of the regenerated filler were significantly improved overall, and G' remained higher than G'', maintaining a solid-like behavior. Within the same frequency range, the increase in G' was significantly greater than that of G'', resulting in a slight decrease in the loss factor tanδ, which is closer to the viscoelastic characteristics of natural soft tissue ECM. This change indicates that after decellularized matrix microparticles are embedded in the gel network as a rigid filler phase, they can effectively enhance the overall mechanical strength. At the same time, by regulating the viscoelastic ratio and optimizing the microenvironment, it is more conducive to cell adhesion, migration, and differentiation, thereby synergistically improving the tissue repair function of the regenerated filler.

[0128] like Figure 7 As shown, the regenerated filler prepared by mixing sodium hyaluronate gel and decellularized matrix microparticles at a ratio of 1 mL:30 mg, exhibits a slightly higher repulsion force plateau range compared to the pure gel, stabilizing at 19-21 N. Furthermore, the repulsion force curve is smoother with no significant fluctuations. This indicates that while the addition of decellularized matrix microparticles slightly increases the overall rigidity of the system, it maintains good injectability. The repulsion force did not significantly increase or become stuck, allowing for smooth passage through the 27G fine needle, meeting the operational requirements for minimally invasive clinical injection. Simultaneously, the smooth repulsion force curve demonstrates that the microparticles are uniformly dispersed in the gel, without aggregation or needle blockage, ensuring the stability and safety of the filler.

[0129] In summary, this invention utilizes a microbubble-forming process to prepare sodium hyaluronate gel, which can serve as a carrier for decellularized matrix microparticles, enabling the regenerative filler to possess both balanced filling and shaping effects and repair and regeneration capabilities. This invention employs a design where sodium hyaluronate gel and decellularized matrix microparticles are individually packaged and sterilized separately. The microparticle concentration can be freely adjusted according to different treatment needs before clinical use, making it an ideal material that combines filling and shaping with repair and regeneration functions.

[0130] The above results demonstrate and describe the basic principles and main features of this application, as well as its advantages.

[0131] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. An injectable sodium hyaluronate gel regeneration filler, characterized in that, The filler includes a carrier and decellularized matrix microparticles; The carrier is sodium hyaluronate gel; the decellularized matrix microparticles are obtained from the submucosa of porcine small intestine through decellularization, freeze-drying, grinding, sieving, and sterilization. The sodium hyaluronate gel comprises cross-linked sodium hyaluronate gel particles and non-cross-linked sodium hyaluronate solution; The cross-linked sodium hyaluronate gel particles are obtained by cross-linking sodium hyaluronate through primary cross-linking, microbubbling, secondary cross-linking, and granulation. The non-crosslinked sodium hyaluronate solution is an aqueous solution of sodium hyaluronate containing lidocaine hydrochloride.

2. The injectable sodium hyaluronate gel regeneration filler according to claim 1, characterized in that, The mass-to-volume ratio of the decellularized matrix microparticles to the carrier is (5-40) mg:1 mL; the mass ratio of the cross-linked sodium hyaluronate gel particles to the non-cross-linked sodium hyaluronate solution is 9:1; the sodium hyaluronate content in the cross-linked sodium hyaluronate gel particles is 10-20 mg / mL; and the sodium hyaluronate content in the non-cross-linked sodium hyaluronate solution is 15-25 mg / mL.

3. The injectable sodium hyaluronate gel regeneration filler according to claim 1, characterized in that, The sodium hyaluronate has a molecular weight of 100-300 WDa; the sodium hyaluronate gel has an elastic modulus G′ of 400-1000 Pa at 0.1 Hz; and the sodium hyaluronate gel has a viscous modulus G′′ of 100-300 Pa at 0.1 Hz.

4. A method for preparing an injectable sodium hyaluronate gel regenerating filler as described in any one of claims 1-3, characterized in that, Includes the following steps: S1. Mix sodium hyaluronate with sodium hydroxide solution, add BDDE, and obtain a cross-linking reaction solution; The cross-linking reaction solution was subjected to a microbubble process to obtain a sodium hyaluronate gel precursor containing microbubbles. A first cross-linking reaction was carried out at a first set temperature, and a second cross-linking reaction was carried out at a second set temperature to obtain cross-linked sodium hyaluronate gel. After elution and granulation, cross-linked sodium hyaluronate gel particles were obtained. S2. Dissolve lidocaine hydrochloride in sodium hydroxide solution, add sodium hyaluronate to obtain non-crosslinked sodium hyaluronate solution; Cross-linked sodium hyaluronate gel particles were mixed with non-cross-linked sodium hyaluronate solution and sterilized to obtain sodium hyaluronate gel. S3. The submucosa of the pig small intestine was placed in a peracetic acid-ethanol mixed solution for virus inactivation, decellularized and freeze-dried to obtain a decellularized matrix, which was then ground, sieved and sterilized to obtain decellularized matrix microparticles. S4. Hyaluronic acid gel and decellularized matrix microparticles are reciprocated through a three-way valve, subjected to negative pressure suction, and centrifuged to obtain injectable hyaluronic acid gel regeneration filler.

5. The method for preparing an injectable sodium hyaluronate gel regeneration filler according to claim 4, characterized in that, In step S1, the mass ratio of sodium hyaluronate, sodium hydroxide solution, and BDDE is 1:(5-10):(0.2-0.6); the concentration of the sodium hydroxide solution is 0.2 mol / L; the microbubble generation process involves: transferring the crosslinking reaction liquid into a microbubble generator, injecting 0.4 MPa of compressed gas and mixing it evenly to form a stable gas-liquid mixture, which is then sprayed out through a nozzle with a diameter of 2-10 mm under conditions of 0.1-1.0 MPa; the compressed gas is selected from air, nitrogen, carbon dioxide, and hydrogen; the diameter of the microbubble is 1-200 μm; the first set temperature is 35-45℃, and the crosslinking reaction time is 2-8 h; the second set temperature is 20-30℃, and the secondary crosslinking time is 8-16 h.

6. The method for preparing an injectable sodium hyaluronate gel regeneration filler according to claim 4, characterized in that, In step S1, the elution step is as follows: the cross-linked sodium hyaluronate gel is immersed in a phosphate buffer solution with pH 7.2 and osmotic pressure of 290 mOsmol / kg at 20-40℃ for 12-24 hours for primary elution, then immersed in the above-mentioned freshly prepared phosphate buffer solution for secondary elution at 50-60℃ for 12-24 hours, and then immersed in the above-mentioned freshly prepared phosphate buffer solution for tertiary elution at 70-80℃ for 12-24 hours; the granulation step is as follows: the eluted cross-linked sodium hyaluronate gel is extruded through a 35-mesh and a 70-mesh stainless steel sieve plate by 0.5 MPa nitrogen gas.

7. The method for preparing an injectable sodium hyaluronate gel regeneration filler according to claim 4, characterized in that, In step S2, the mass-to-volume ratio of lidocaine hydrochloride, sodium hyaluronate, and sodium hydroxide solution is 0.3g:0.2g:10mL; the concentration of the sodium hydroxide solution is 0.021mol / L; the sterilization is moist heat sterilization, the sterilization temperature is 121℃, and the sterilization time is 15min.

8. The method for preparing an injectable sodium hyaluronate gel regeneration filler according to claim 4, characterized in that, In step S3, the volume fraction of peracetic acid in the peracetic acid-ethanol mixed solution is 1%, and the volume fraction of ethanol is 25%; the virus inactivation time is 45 min; the decellularization step is as follows: the virus-inactivated tissue is placed in liquid nitrogen and subjected to three cycles of freeze-thaw, then immersed in a mixed solution containing 0.05 wt% trypsin and 0.03 wt% EDTA, followed by immersion in 1 wt% and 5 wt% sodium chloride solutions for 15 min each, repeated three times, washed with purified water, then immersed in 25 mmol / L NaOH aqueous solution, and washed with purified water until the pH is 6-7; the freeze-drying step is as follows: freezing at -40℃ for 4 h, freezing at -20℃ for 10 h, drying at -5℃ for 6 h, drying at 10℃ for 6 h, and drying and dehydrating at 30℃ for 8 h; the sieve mesh sizes are 50 mesh and 150 mesh respectively; the sterilization step is as follows: cobalt-60 radiation sterilization is performed at 25 kGy.

9. The method for preparing an injectable sodium hyaluronate gel regenerating filler according to claim 4, characterized in that, In step S4, the number of reciprocating pushes is 40-60 times; the number of negative pressure suctions is 10-15 times; the centrifugation speed is 3500-4500 rpm; and the centrifugation time is 8-12 minutes.

10. The application of the injectable sodium hyaluronate gel regeneration filler according to any one of claims 1-3 in the preparation of filling, shaping, repairing and regenerating materials.

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