An injectable composite for soft tissue repair and its preparation method and application

Abstract

The present application relates to a kind of injectable compound for soft tissue repair and its preparation method and application, the injectable compound includes: temperature-sensitive medium;Pre-crosslinked, water-insoluble hydrophilic polymer gel microparticles dispersed in the temperature-sensitive medium, content is 5-23% of the total mass of injectable compound;And acellular matrix microparticle dispersed in the temperature-sensitive medium, content is 6-12% of the total mass of injectable compound;Wherein, the network phase formed by the hydrophilic polymer gel microparticle gelatinization with the temperature-sensitive medium at body temperature is combined, constitutes the support framework of enhancing the structural stability of the injectable compound.The injectable compound involved in the present application is flowable viscous gel when injection, rapidly converted into non-flowing semi-solid at physiological temperature, forms tissue-conforming repair layer.Among them, gelatinous particle, temperature-sensitive medium, acellular matrix microparticle three synergistically promote tissue repair.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to an injectable complex for soft tissue repair, its preparation method, and its application. Background Technology

[0002] Soft tissues are an important component of human organs and tissue systems, broadly including skin, tendons, muscles, fat, mucous membranes, and related connective tissues. Soft tissue injuries are often caused by trauma, infection, surgical procedures, ischemia and hypoxia, radiotherapy, and chemotherapy, leading to structural damage, tissue necrosis, inflammatory reactions, or fibrosis, thereby affecting tissue function and even the stability of organ systems. With modern medicine's emphasis on functional reconstruction and tissue microenvironment repair, how to safely and effectively promote soft tissue regeneration and repair has become an important direction in clinical treatment. Especially in organ surgery, tumor resection, gynecological interventions, and pelvic and abdominal surgeries, deep soft tissue injuries are common, such as those in the intermuscular spaces, pelvic tissues, endometrium, urogenital tract, and digestive tract walls. Repairing injuries in these areas is more difficult, easily leading to local adhesions, internal cavity deformation, or functional impairment, seriously affecting patients' quality of life and prognosis.

[0003] Currently, commonly used soft tissue repair materials in clinical practice include membrane dressings, biological patches, foam dressings, and block implants. Although they are widely used in the repair of superficial wounds, they have the following limitations in the repair of soft tissues, especially deep areas: (1) They have fixed shapes and are difficult to adapt to complex, irregular or deep cavity wounds; (2) They have poor retention performance and are easily discharged under the influence of tissue fluid flushing, body cavity movement or postoperative exudate, which reduces the repair effect; (3) They often have limited biological activity and are difficult to provide cell adhesion sites, regeneration signals or reconstruct the microenvironment; (4) They are highly operation-dependent and require suture fixation, making them difficult to use conveniently in minimally invasive surgery or deep cavities.

[0004] In recent years, injectable hydrogel materials have attracted widespread attention in the fields of tissue engineering and regenerative medicine due to their fluidity, conformability, and minimally invasive injection characteristics. In particular, thermoresponsive hydrogels, such as poloxamer systems, can be in solution at room temperature and rapidly gel in situ at body temperature, adapting to complex three-dimensional spaces, sealing wounds, and forming flexible scaffolds, providing a new material basis for deep tissue repair. However, single thermoresponsive hydrogels still face important challenges: (1) limited bioactivity, making it difficult to induce cell adhesion and functional repair; (2) poor gel mechanical properties, with stability and degradation cycles that are difficult to synchronize with tissue repair; (3) insufficient retention performance, making it difficult to adhere to the target tissue surface for a long time, which limits its repair of damaged sites. Decellularized matrix materials, because they retain the tissue-specific extracellular matrix components (such as collagen, glycosaminoglycans, growth factors, etc.), have been proven to provide good cell scaffolds and tissue regeneration microenvironments, and have shown potential in various soft tissue repairs. Currently, most clinically used acellular matrix products (such as acellular dermal matrix) are derived from specific tissues and exist in pre-formed forms such as membranes, granules, or sponges. Their use relies on surgical sutures or covering fixation, making minimally invasive injection and in-situ shaping impossible, thus limiting their application in complex anatomical locations such as deep tissues and cavities. More importantly, for these materials rich in bioactive proteins, mainstream terminal sterilization methods (such as gamma rays, electron beam irradiation, ethylene oxide, etc.) can easily cause protein denaturation, cross-linking, or inactivation of active factors. Therefore, existing products often rely on complex and stringent aseptic manufacturing processes, limiting their large-scale production and cost control.

[0005] Therefore, developing a soft tissue repair material that can simultaneously achieve injectability, in-situ stability, active bioactivity, and terminal sterilization compatibility has become a comprehensive technical challenge that has long remained unresolved in this field. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide an injectable complex for soft tissue repair, its preparation method, and its application.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides an injectable complex for soft tissue repair, the injectable complex comprising:

[0009] Thermosensitive medium is an aqueous solution of a biocompatible polymer with thermo-gelling properties;

[0010] The pre-crosslinked, water-insoluble hydrophilic polymeric gel microparticles dispersed in the temperature-sensitive medium constitute 5-23% of the total mass of the injectable complex.

[0011] And decellularized matrix microparticles dispersed in the thermosensitive medium, comprising 6-12% of the total mass of the injectable complex;

[0012] The hydrophilic polymer gel microparticles combine with the network formed by the gelation of the temperature-sensitive medium at a temperature of 30-37℃ to form a supporting framework that enhances the structural stability of the injectable composite.

[0013] The hydrophilic polymeric gel microparticles are obtained by granulation of cross-linked products obtained by cross-linking hyaluronic acid or its salts.

[0014] The weight-average molecular weight of the hyaluronic acid or its salt is 1500-2000 kDa;

[0015] The injectable complex is a composition sterilized by 15-25 kGy electron beam irradiation, and the vascular endothelial growth factor (VEGF) content retention rate after irradiation is not less than 90% of that before irradiation (e.g., 90%, 92%, 94%, 96%, 98%, etc.).

[0016] In this invention, decellularized matrix microparticles possess excellent tissue regeneration-promoting activity, enabling the injectable complex to release active substances and growth factors during use, thereby promoting cell adhesion, proliferation, and migration; gel microparticles are used to enhance the retention and mechanical stability of the injectable complex in humid environments; and a temperature-sensitive medium can rapidly transform the injectable complex from a solution state to a gel state under body temperature conditions.

[0017] The injectable complex for soft tissue repair involved in this invention is a fluid, viscous gel upon injection, which rapidly transforms into a non-fluid, semi-solid state at physiological temperatures, forming a tissue-conforming repair layer. In the injectable complex of this invention, decellularized matrix microparticles, gel microparticles, and a temperature-sensitive medium interact to produce a synergistic effect.

[0018] (1) The pre-crosslinked, water-insoluble hydrophilic polymeric gel microparticles and decellularized matrix microparticles are co-dispersed in an aqueous solution of a thermosensitive medium. Before injection, the complex is a flowable suspension; after injection into the body, under the induction of body temperature, the thermosensitive medium gels to form a continuous network, which combines with the dispersed gel microparticles—the gel microparticles act as reinforcing nodes in the network, and together they constitute a supporting framework that enhances the structural stability of the complex. Compared with a single thermosensitive gel, this composite structure containing gel microparticles significantly enhances the overall mechanical strength and structural stability of the complex in the body fluid environment, and improves its retention performance on tissue surfaces.

[0019] (2) In the composite system, the gel particles are dispersed in an aqueous solution of a temperature-sensitive medium, and the two form a stable dispersion system through physical interactions such as hydrogen bonding. Experiments show that the composite system can withstand electron beam irradiation sterilization of 15-25 kGy, and can still maintain its thermo-gelation characteristics and structural stability after sterilization.

[0020] (3) After injection, the supporting framework formed by the gel microparticles and the thermosensitive medium gelation network can delay the rapid diffusion of active substances such as endogenous growth factors in the decellularized matrix microparticles through steric hindrance, electrostatic adsorption and hydrophilicity-hydrophobicity interactions, so that they can maintain an effective concentration at the site of injury for a longer period of time, thereby continuously promoting cell migration, proliferation and tissue regeneration.

[0021] Through the synergistic effect of the above-mentioned physical enhancement, interfacial bonding and sustained-release delivery, the composite gel of the present invention achieves a balance of injectability, in-situ stability, bioactivity and sterilization resistance.

[0022] In situ stability refers to the ability of the complex to resist erosion, maintain its three-dimensional structure, and stably adhere to the tissue surface under body temperature and fluid conditions after being injected into the target site in the body.

[0023] In this invention, the content of the hydrophilic polymeric gel microparticles is 5-23% of the total mass of the injectable composite (e.g., 5%, 7%, 9%, 11%, 13%, 15%, 17%, 19%, 21%, 23%, etc.). When the content is below 5%, a continuous and effective microskeleton cannot be formed, resulting in insufficient reinforcement. When the content is above 23%, the radiation resistance of the injectable composite deteriorates, and it interferes with the gelling properties of the temperature-sensitive medium. Preferably, it is 10-20% (e.g., 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, etc.). Within this range, the optimal balance between reinforcement and operability can be achieved.

[0024] In this invention, by controlling the mass percentage of gel microparticles within the above-mentioned range, the prepared injectable composite has higher cohesion and viscosity, exhibits a more stable state after irradiation, has good gelling properties, and the gel is not easily delaminated or dissolved.

[0025] In this invention, the content of the decellularized matrix microparticles is 6-12% (e.g., 6%, 7%, 8%, 9%, 10%, 11%, 12%, etc.) of the total mass of the injectable complex, preferably 10-12% (e.g., 10%, 10.3%, 10.6%, 10.9%, 11.2%, 11.5%, 11.8%, 12%, etc.). This content range provides bioactivity while avoiding excessive microparticles from disrupting the continuity of the gel network and ensuring uniform dispersion of the microparticles in the gel.

[0026] In this invention, the mass percentage of decellularized matrix microparticles is controlled within the above-mentioned range, which enables them to better form a stable structure with gel microparticles in a temperature-sensitive medium, thereby improving the stability of the prepared thermosensitive composite gel.

[0027] In this invention, the weight-average molecular weight of the hyaluronic acid or its salt is 1500-2000 kDa (e.g., 1500 kDa, 1600 kDa, 1700 kDa, 1800 kDa, 1900 kDa, 2000 kDa, etc.). Preferably, it is 1500-1700 kDa. Raw materials in this molecular weight range, after cross-linking, can form a network with suitable cross-linking density and swelling degree, allowing it to maintain structural integrity after irradiation and preventing excessively rapid dissolution. Furthermore, the viscosity of the dissolved hyaluronic acid salt is suitable, facilitating the cross-linking reaction.

[0028] In this invention, by controlling the weight-average molecular weight of hyaluronic acid or its salt within the above-mentioned range, the gelation stability can be further improved and the dissolution and dispersion time of the sample after irradiation can be extended.

[0029] Preferably, the decellularized matrix microparticles are prepared from decellularized matrix raw materials through virus inactivation, decellularization, washing, drying, defatting, secondary drying, and grinding.

[0030] Preferably, the decellularized matrix material includes any one or a combination of at least two of the following: placental amnion, bladder basement membrane, small intestinal mucosa, skin tissue, nerve or trachea.

[0031] Preferably, the grinding temperature is -15℃ to -5℃ (e.g., -15℃, -13℃, -11℃, -9℃, -7℃, -5℃, etc.). This temperature range can effectively prevent the denaturation and inactivation of heat-sensitive proteins (such as collagen and growth factors) in the decellularized matrix, protect their biological activity, and at the same time ensure the dryness of the powder, preventing adhesion and aggregation caused by particle agglomeration due to temperature rise.

[0032] Preferably, the particle size of the decellularized matrix microparticles is 1-1000 μm, more preferably 50-300 μm (e.g., 50 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, etc.), and more preferably 50-200 μm (e.g., 50 μm, 100 μm, 150 μm, 200 μm, etc.). This particle size range ensures the mechanical stability and rheological properties of the system, avoiding the decrease in gel strength caused by excessively small particle size and the disruption of the gel network continuity caused by excessively large particle size.

[0033] Preferably, the hydrophilic polymeric gel microparticles are cross-linked hyaluronic acid salt microparticles;

[0034] Preferably, the cross-linked hyaluronic acid microparticles are prepared by a method comprising the following steps:

[0035] Hyaluronic acid or its salt is subjected to a cross-linking reaction with a cross-linking agent under alkaline conditions, followed by purification and granulation to obtain the cross-linked hyaluronic acid salt microparticles.

[0036] More preferably, the cross-linked hyaluronic acid microparticles are prepared by a preparation method comprising the following steps:

[0037] Hyaluronic acid, a cross-linking agent, and an alkaline solution are mixed to carry out a cross-linking reaction, resulting in cross-linked hyaluronic acid salts. The cross-linked hyaluronic acid salts are then subjected to dialysis with a phosphate buffer solution and extrusion sieving to obtain cross-linked hyaluronic acid salt microparticles.

[0038] Preferably, the cross-linked hyaluronic acid microparticles are cross-linked sodium hyaluronate microparticles.

[0039] Preferably, the crosslinking reaction temperature is 35-40℃ (e.g., 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, etc.); the crosslinking reaction time is 4-6 h (e.g., 4 h, 4.5 h, 5 h, 5.5 h, 6 h), preferably 5-6 h (e.g., 5 h, 5.2 h, 5.4 h, 5.6 h, 5.8 h, 6 h). These reaction conditions aim to ensure the sufficiency of the crosslinking reaction and the uniformity of the gel network while minimizing the damage to the sodium hyaluronate molecular chains caused by high temperatures under alkaline conditions, thereby preserving its biocompatibility and ability to form a stable microskeleton. The time range ensures that the reaction reaches dynamic equilibrium, avoiding excessive crosslinking and side reactions.

[0040] Preferably, the crosslinking agent comprises any one or a combination of at least two of the following: 1,4-butanediol diglycidyl ether (BDDE), ethylene glycol diglycidyl ether, 1,6-hexanediol diglycidyl ether, propylene glycol diglycidyl ether, polytetraethylene glycol diglycidyl ether, neopentyl glycol diglycidyl ether, polyglycerol diglycidyl ether, polyglycerol diglycidyl ether, glycerol polyglycidyl ether, trimethylpropane polyglycidyl ether, pentaerythritol polyglycidyl ether, sorbitol polyglycidyl ether, or 1,2-(bis(2,3-epoxypropoxy)ethylene), preferably 1,4-butanediol diglycidyl ether (BDDE).

[0041] Preferably, the amount of the crosslinking agent is 4%-5% of the mass of hyaluronic acid or its salt (e.g., 4%, 4.2%, 4.5%, 4.8%, 5%, etc.).

[0042] Preferably, the alkaline solution comprises an aqueous solution of sodium hydroxide.

[0043] Preferably, the alkaline solution contains 1-2% alkali by mass (e.g., 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, etc.).

[0044] Preferably, the dialysis treatment temperature is 50-70℃ (e.g., 50℃, 55℃, 60℃, 65℃, 70℃, etc.) to increase the diffusion rate of small molecule impurities (such as unreacted cross-linking agents, salts), thereby completing purification within a reasonable time. The total dialysis treatment time is 24-72 h (e.g., 24 h, 36 h, 48 h, 60 h, 72 h, etc.) to ensure sufficient removal of impurities to a biosafety level.

[0045] Preferably, the sieve aperture for the extrusion sieving process is 50-70 mesh (e.g., 50 mesh, 55 mesh, 60 mesh, 65 mesh, 70 mesh, etc.). This mesh range corresponds to a particle size range of approximately 224-335 micrometers or less. This range is chosen to ensure that the microparticles provide sufficient size within the gel to form effective physical support (microskeleton). Particles that are too small may approach fluidity, weakening the reinforcing effect; particles that are too large will affect injectability and dispersion uniformity.

[0046] Preferably, the temperature-sensitive medium comprises an aqueous solution of any one or a combination of at least two of chitosan / β-glycerophosphate, poly(N-isopropylacrylamide), polyethylene glycol-polylactic acid block copolymer, polyethylene glycol-polyhydroxybutyrate copolymer, or poloxamer.

[0047] Preferably, the temperature-sensitive medium is an aqueous solution of poloxamer 407.

[0048] The gel microparticles and the temperature-sensitive medium are primarily bonded through physical mixing, relying on hydrogen bonds and physical entanglement interactions. Taking cross-linked sodium hyaluronate (cross-linked HA) microparticles and poloxamer 407 as an example, the viscoelasticity of the cross-linked HA microparticles, combined with poloxamer 407, forms an interpenetrating network structure through hydrogen bonds and physical entanglement. Compared to a single poloxamer 407 gel, this physical interaction significantly enhances the mechanical properties of the composite gel, effectively preventing rapid gel loss in humid environments, thus providing a structural basis for its application in soft tissue repair.

[0049] Preferably, the concentration of poloxamer 407 in the preparation of the temperature-sensitive medium is 150-200 mg / mL (e.g., 150 mg / mL, 160 mg / mL, 170 mg / mL, 180 mg / mL, 190 mg / mL, 200 mg / mL, etc.). This concentration range is limited to ensure that the gel strength is within a reasonable range; if the concentration is too low, the gel strength at body temperature will be insufficient; if the concentration is too high, the gel will form too quickly and be difficult to inject.

[0050] In a second aspect, the present invention provides a method for preparing the injectable complex as described in the first aspect, the method comprising the following steps:

[0051] The hydrophilic polymeric gel microparticles, decellularized matrix microparticles, and the temperature-sensitive medium are mixed at 2-10℃ (e.g., 2℃, 4℃, 6℃, 7℃, 10℃) to obtain the injectable complex.

[0052] The degassing treatment is performed at a temperature of 2-10°C. This temperature range is crucial for maintaining the temperature-sensitive medium (such as poloxamer) in a low-viscosity liquid state, where bubbles are more likely to rise and escape.

[0053] Preferably, the preparation method includes the following steps:

[0054] (1) At 2-10℃, hydrophilic polymer gel microparticles are mixed with a temperature-sensitive medium, and then a first degassing treatment is performed to obtain a first mixture;

[0055] (2) At 2-10°C, the first mixture is mixed with decellularized matrix microparticles, and then a second defoaming treatment is performed to obtain the injectable complex.

[0056] Preferably, the first degassing treatment and the second degassing treatment each have an independent duration of 0.5-5 hours (e.g., 0.5 hours, 2 hours, 3.5 hours, 5 hours, etc.). This time range is designed to ensure sufficient removal of air bubbles to avoid voids after injection that could affect the repair effect. Preferably, the process of mixing the hydrophilic polymer gel microparticles, decellularized matrix microparticles, and the temperature-sensitive medium further includes electron beam irradiation treatment.

[0057] Preferably, the irradiation dose of the electron beam irradiation treatment is 15-25 KGy (e.g., 15 KGy, 18 KGy, 20 KGy, 22 KGy, 25 KGy, etc.).

[0058] The injectable composite involved in this invention exhibits excellent biocompatibility after irradiation treatment and can rapidly solidify at body temperature (37±2℃) to form a stable repair scaffold.

[0059] Thirdly, the present invention provides the use of the injectable complex described in the first aspect in the preparation of medical devices or medical materials for soft tissue repair.

[0060] Preferably, the soft tissue includes the endometrium.

[0061] The injectable complex involved in this invention improves the overall viscosity and retention time of the thermosensitive composite gel through gel microparticles, effectively delaying the diffusion and loss of active ingredients in the decellularized matrix, enhancing its local concentration and duration of action at the site of intrauterine injury, forming a three-dimensional scaffold environment, promoting the migration and proliferation of endometrial cells, and releasing regeneration signaling molecules, thereby achieving effective repair of endometrial damage.

[0062] Compared with the prior art, the present invention has the following beneficial effects:

[0063] (1) The injectable complex for soft tissue repair provided by the present invention has the advantage of constructing a "particle-supported" thermosensitive composite gel system by introducing gel microparticles into a thermosensitive medium. Compared with traditional single thermosensitive hydrogels, this structure significantly enhances the spatial stability and anti-loss ability of the gel after gelation, effectively delays its dissolution and diffusion in the body fluid environment, and improves the local retention performance of the gel in the soft tissue injury area.

[0064] (2) The injectable complex for soft tissue repair provided by the present invention has the advantage that the gel is a fluid viscous solution at room temperature, with good injectability and tissue conformability, and can penetrate deep or irregular wounds. It rapidly completes the sol-gel phase transition at physiological temperature, forming a non-fluid three-dimensional scaffold loaded with active substances that can promote tissue regeneration in situ, which significantly improves the ability to adapt to irregular tissues and support cell migration during the repair of soft tissue defects.

[0065] (3) Furthermore, the injectable complex for soft tissue repair provided by the present invention has the advantage that, by adjusting the amount of raw materials added and the preparation parameters of the gel microparticles, the resulting gel can be sterilized by electron beam irradiation, and the gel still has excellent thermosensitive properties and tissue retention properties after sterilization. In addition, the content of growth factors in the gel is not significantly different from that before irradiation, and it has good radiation resistance.

[0066] (4) The injectable complex for soft tissue repair provided by the present invention has the advantage that the composite structure constructed by the three components of decellularized matrix microparticles, gel microparticles and temperature-sensitive medium solves the comprehensive problems of insufficient structural support, weak biological activity, short retention time and poor adaptability in the existing injectable gel system. It exhibits better tissue adhesion, mechanical stability and regeneration support capabilities, and is particularly suitable for the repair of complex soft tissue environments such as endometrium, abdominal tissue and mucosal layer.

[0067] (5) The injectable complex for soft tissue repair provided by the present invention has the advantage that the preparation process is simple, can be terminally sterilized, and is suitable for large-scale production. Attached Figure Description

[0068] Figure 1 This is a graph showing the dispersibility test results of the injectable complexes of Example 1 and Comparative Example 1 in solution.

[0069] Figure 2 This is a graph showing the homogeneity test results of the injectable complex in Comparative Example 10.

[0070] Figure 3 This is a graph showing the homogeneity test results of the injectable complexes of Example 1 and Comparative Example 1.

[0071] Figure 4 This is a graph showing the adhesion test results of the injectable complexes of Example 1 and Comparative Example 1 on the tissue surface.

[0072] Figure 5 This is a graph showing the retention test results of the injectable complexes of Example 1 and Comparative Example 1 in the uterus.

[0073] Figure 6 This is an image of HE-stained sections of the endometrium after treatment with the injectable complex.

[0074] Figure 7 This is a statistical result chart of endometrial thickness after treatment with injectable complex.

[0075] Figure 8 This is a statistical result chart of the number of endometrial glands after treatment with injectable complex. Detailed Implementation

[0076] To further illustrate the technical means and effects of the present invention, the following describes the technical solution of the present invention in conjunction with preferred embodiments of the present invention. However, the present invention is not limited to the scope of the embodiments.

[0077] The sodium hyaluronates of different weight-average molecular weights mentioned in the following specific embodiments are all from Bloomage Biotechnology Co., Ltd.

[0078] The poloxamer 407 used in the following specific implementation method is sourced from Shanghai Xietai Chemical Co., Ltd., a first-tier distributor of BASF in Germany.

[0079] Example 1

[0080] This embodiment provides an injectable complex prepared by the following method:

[0081] (1) Preparation of decellularized matrix microparticles: The porcine bladder was pretreated to remove excess tissue and retain the submucosal matrix layer; the virus was inactivated by constant temperature shaking with 0.15% (v / v) peracetic acid solution for 120 min, followed by decellularization with a mixed solution of trypsin (0.08%, v / v) and sodium dodecyl sulfate (0.08%, v / v); after washing and freeze-drying, the bladder was degreased by soaking in ether for 10 h, and freeze-dried to obtain a lyophilized porcine bladder matrix material with intact structure and effective removal of cell components. Subsequently, 1 g of the lyophilized porcine bladder matrix was placed in a cryo-mill and ground into powder at -15℃. After grinding, the obtained decellularized matrix powder was screened through a sieve with a pore size of 200 μm to obtain decellularized matrix microparticles of the desired particle size (hereinafter referred to as UBM microparticles).

[0082] (2) Preparation of gel microparticles (cross-linked sodium hyaluronate microparticles):

[0083] Weigh 1 g of sodium hyaluronate (weight-average molecular weight 1600 kDa) into a glass bottle. Add 5.31 mL of a 0.25 mol / L NaOH aqueous solution to the bottle and stir continuously until the sodium hyaluronate is completely dissolved, forming a homogeneous gel solution. Using the same concentration of NaOH aqueous solution (0.25 mol / L) as the solvent, prepare a 0.23 g / mL BDDE solution. Slowly add 0.2 mL of the BDDE solution dropwise to the sodium hyaluronate sol and mechanically stir for 20 min to ensure uniform mixing. Place the glass bottle containing the reaction solution in a 40℃ water bath for cross-linking reaction for 6 h. After the reaction, remove the gel sample from the bottle and cut it into 0.5-1 cm pieces. 3 The gel fragments were dialyzed using PBS solution, with the dialysate changed every 2 hours for a total dialysis time of 48 hours. After dialysis, the gel particles were sieved through a 60-mesh sieve to obtain cross-linked sodium hyaluronate microparticles of the desired particle size.

[0084] (3) Preparation of thermosensitive material (poloxamer solution):

[0085] A poloxamer 407 (P407) solution with a concentration of 180 mg / mL was prepared using ultrapure water.

[0086] (4) Preparation of injectable complex:

[0087] The poloxamer solution obtained in step (3) was mixed with the cross-linked sodium hyaluronate microparticles obtained in step (2). The mixture was then pushed back and forth with a syringe to ensure that the two were fully mixed. The mixture was then left to stand in a refrigerator at 4°C for 2 hours to remove bubbles, thus obtaining a sodium hyaluronate-poloxamer mixed solution.

[0088] The UBM microparticles obtained in step (1) were placed in a new injection tube and mixed with a sodium hyaluronate-poloxam mixed solution. The mixture was then pushed together with a syringe to allow the UBM microparticles to fully swell and disperse evenly in the sodium hyaluronate-poloxam mixed solution. The mixture was then placed in a 4°C refrigerator for degassing for 2 hours. In the final system, the mass percentage of UBM microparticles was 10% and the mass percentage of cross-linked sodium hyaluronate microparticles was 20%.

[0089] (5) Irradiation sterilization treatment:

[0090] The gel system obtained in step (4) was subjected to electron beam irradiation treatment with an irradiation dose of 15 KGy to obtain an irradiated sterilized injectable complex.

[0091] Example 2

[0092] This embodiment provides an injectable complex prepared by the following method:

[0093] (1) Preparation of decellularized matrix microparticles:

[0094] The porcine bladder was pretreated to remove excess tissue, preserving the submucosal matrix layer. Viruses were inactivated by constant-temperature shaking with 0.15% (v / v) peracetic acid solution for 120 min. Subsequently, decellularization was performed using a mixed solution (0.08%, v / v) containing trypsin and sodium dodecyl sulfate. After washing and freeze-drying, the bladder was degreased by immersion in ether for 10 h. Freeze-drying yielded a structurally intact, cell-free, lyophilized porcine bladder matrix material. Then, 1 g of the lyophilized porcine bladder matrix was placed in a cryo-mill and ground into powder at -10°C. The resulting decellularized matrix powder was then sieved through a 400 μm sieve to obtain decellularized matrix microparticles of the desired particle size (hereinafter referred to as UBM microparticles).

[0095] (2) Preparation of gel microparticles (cross-linked sodium hyaluronate microparticles):

[0096] Weigh 1 g of sodium hyaluronate (weight-average molecular weight 1500 kDa) into a glass bottle. Add 5.31 mL of a 0.25 mol / L NaOH aqueous solution to the bottle and stir continuously until the sodium hyaluronate is completely dissolved, forming a homogeneous gel solution. Using the same concentration of NaOH aqueous solution (0.25 mol / L) as a solvent, prepare a 0.23 g / mL BDDE solution. Slowly add 0.2 mL of the BDDE solution dropwise to the sodium hyaluronate sol and mechanically stir for 30 min to ensure uniform mixing. Place the glass bottle containing the reaction solution in a 35℃ water bath for cross-linking reaction for 5 h. After the reaction, remove the gel sample from the bottle and cut it into 0.5-1 cm pieces. 3 The gel fragments were dialyzed using PBS solution, with the dialysate changed every 2 hours for a total dialysis time of 72 hours. After dialysis, the gel particles were sieved through a 40-mesh sieve to obtain cross-linked sodium hyaluronate microparticles of the desired particle size.

[0097] (3) Preparation of thermosensitive material (poloxamer solution):

[0098] A 150 mg / mL solution of poloxamer 407 (P407) was prepared using ultrapure water.

[0099] (4) Preparation of injectable complex:

[0100] The poloxamer solution obtained in step (3) was mixed with the cross-linked sodium hyaluronate microparticles obtained in step (2). The mixture was then pushed back and forth with a syringe to ensure that the two were fully mixed. The mixture was then left to stand in a refrigerator at 4°C for 0.5 h to remove bubbles, thus obtaining a sodium hyaluronate-poloxamer mixed solution.

[0101] The UBM microparticles obtained in step (1) were placed in a new injection tube and mixed with a sodium hyaluronate-poloxam mixed solution. The mixture was then injected with a syringe to allow the UBM microparticles to fully swell and disperse evenly in the sodium hyaluronate-poloxam mixed solution. The mixture was then placed in a 4°C refrigerator for degassing for 0.5 h. In the final system, the mass percentage of UBM microparticles was 11% and the mass percentage of cross-linked sodium hyaluronate microparticles was 18%.

[0102] (5) Irradiation sterilization treatment:

[0103] The gel system obtained in step (4) was subjected to electron beam irradiation treatment with an irradiation dose of 15 KGy to obtain an irradiated sterilized injectable complex.

[0104] Example 3

[0105] This embodiment provides an injectable complex prepared by the following method:

[0106] (1) Preparation of decellularized matrix microparticles:

[0107] The porcine bladder was pretreated to remove excess tissue, preserving the submucosal matrix layer. Viruses were inactivated by constant-temperature shaking with 0.15% (v / v) peracetic acid solution for 120 min. Subsequently, decellularization was performed using a mixed solution (0.08%, v / v) containing trypsin and sodium dodecyl sulfate. After washing and freeze-drying, the bladder was degreased by immersion in ether for 10 h. Freeze-drying yielded a lyophilized porcine bladder matrix material with intact structure and effective removal of cellular components. Then, 1 g of the lyophilized porcine bladder matrix was placed in a cryo-mill and ground into powder at -5°C. The resulting decellularized matrix powder was then sieved through a 100 μm sieve to obtain decellularized matrix microparticles of the desired particle size (hereinafter referred to as UBM microparticles).

[0108] (2) Preparation of gel microparticles (cross-linked sodium hyaluronate microparticles):

[0109] Weigh 1 g of sodium hyaluronate (weight-average molecular weight 1700 kDa) into a glass bottle. Add 5.31 mL of a 0.25 mol / L NaOH aqueous solution to the bottle and stir continuously until the sodium hyaluronate is completely dissolved, forming a homogeneous gel solution. Using the same concentration of NaOH aqueous solution (0.25 mol / L) as a solvent, prepare a 0.23 g / mL BDDE solution. Slowly add 0.2 mL of the BDDE solution dropwise to the sodium hyaluronate sol and mechanically stir for 15 min to ensure uniform mixing. Place the glass bottle containing the reaction solution in a 38℃ water bath for cross-linking reaction for 4 h. After the reaction, remove the gel sample from the bottle and cut it into 0.5-1 cm pieces. 3 The gel fragments were dialyzed using PBS solution, with the dialysate changed every 2 hours for a total dialysis time of 24 hours. After dialysis, the gel particles were sieved through an 80-mesh sieve to obtain cross-linked sodium hyaluronate microparticles of the desired particle size.

[0110] (3) Preparation of thermosensitive material (poloxamer solution):

[0111] A 200 mg / mL solution of poloxamer 407 (P407) was prepared using ultrapure water.

[0112] (4) Preparation of injectable complex:

[0113] The poloxamer solution obtained in step (3) was mixed with the cross-linked sodium hyaluronate microparticles obtained in step (2). The mixture was then pushed back and forth with a syringe to ensure that the two were fully mixed. The mixture was then left to stand in a refrigerator at 4°C for 5 hours to remove bubbles, thus obtaining a sodium hyaluronate-poloxamer mixed solution.

[0114] The UBM microparticles obtained in step (1) were placed in a new injection tube and mixed with a sodium hyaluronate-poloxam mixed solution. The mixture was then pushed together with a syringe to allow the UBM microparticles to fully swell and disperse evenly in the sodium hyaluronate-poloxam mixed solution. The mixture was then placed in a 4°C refrigerator for degassing for 5 h. In the final system, the mass percentage of UBM microparticles was 12% and the mass percentage of cross-linked sodium hyaluronate microparticles was 22%.

[0115] (5) Irradiation sterilization treatment:

[0116] The gel system obtained in step (4) was subjected to electron beam irradiation treatment with an irradiation dose of 15 KGy to obtain an irradiated sterilized injectable complex.

[0117] Example 4

[0118] This embodiment provides an injectable complex, which differs from Example 1 only in that the mass percentage of cross-linked sodium hyaluronate particles in the final system of step (4) is adjusted to 5%, while the remaining steps are consistent with Example 1.

[0119] Example 5

[0120] This embodiment provides an injectable complex, which differs from Example 1 only in that the mass percentage of cross-linked sodium hyaluronate microparticles in the final system of step (4) is adjusted to 10%, while the remaining steps are consistent with Example 1.

[0121] Example 6

[0122] This embodiment provides an injectable complex, which differs from Example 1 only in that the mass percentage of cross-linked sodium hyaluronate microparticles in the final system of step (4) is adjusted to 15%, while the remaining steps are consistent with Example 1.

[0123] Example 7

[0124] This embodiment provides an injectable complex, which differs from Example 1 only in that the mass percentage of UBM microparticles in the final system of step (4) is adjusted to 6%, while the remaining steps are consistent with Example 1.

[0125] Example 8

[0126] This embodiment provides an injectable complex, which differs from Example 1 only in that the mass percentage of UBM microparticles in the final system of step (4) is adjusted to 12%, while the remaining steps are consistent with Example 1.

[0127] Example 9

[0128] This embodiment provides an injectable complex, which differs from Example 1 only in that the weight-average molecular weight of sodium hyaluronate is adjusted to 2000 kDa in step (1), while the remaining steps are consistent with Example 1.

[0129] Example 10

[0130] This embodiment provides an injectable complex, which differs from Example 1 only in that the weight-average molecular weight of sodium hyaluronate is adjusted to 1200 kDa in step (1), while the remaining steps are consistent with Example 1.

[0131] Example 11

[0132] This embodiment provides an injectable complex, which differs from Example 1 only in that the crosslinking reaction time in step (2) is adjusted to 4 h, while the other steps are consistent with Example 1.

[0133] Example 12

[0134] This embodiment provides an injectable complex, which differs from Example 1 only in that the sodium hyaluronate raw material used in the crosslinking reaction in step (2) is replaced with an equal amount of carboxymethyl chitosan, while the remaining steps are consistent with Example 1.

[0135] Example 13

[0136] This embodiment provides an injectable complex, which differs from Example 1 only in that it does not undergo the irradiation sterilization treatment in step (5).

[0137] Example 14

[0138] This embodiment provides an injectable complex, which differs from Example 1 only in that the irradiation dose is adjusted to 25 KGy in the irradiation sterilization process in step (5).

[0139] Example 15

[0140] This embodiment provides an injectable complex, which differs from Example 2 only in that the irradiation dose is adjusted to 25 KGy in the irradiation sterilization process in step (5).

[0141] Example 16

[0142] This embodiment provides an injectable complex, which differs from Embodiment 3 only in that the irradiation dose is adjusted to 25 KGy in the irradiation sterilization process in step (5).

[0143] Comparative Example 1

[0144] This comparative example provides an injectable complex that differs from Example 1 only in that cross-linked sodium hyaluronate microparticles are not added in step (4), while the remaining steps are consistent with Example 1.

[0145] Comparative Example 2

[0146] This comparative example provides an injectable complex that differs from Example 1 only in that UBM microparticles are not added in step (4), while the remaining steps are consistent with Example 1.

[0147] Comparative Example 3

[0148] This comparative example provides an injectable complex prepared by the following method:

[0149] A poloxamer 407 (P407) solution with a concentration of 180 mg / mL was prepared using ultrapure water and subjected to electron beam irradiation at a dose of 15 KGy to obtain an irradiated sterilized injectable complex.

[0150] Comparative Example 4

[0151] This comparative example provides an injectable complex that differs from Example 1 only in that the mass percentage of UBM microparticles in the final system of step (4) is adjusted to 3%, while the remaining steps are consistent with Example 1.

[0152] Comparative Example 5

[0153] This comparative example provides an injectable complex that differs from Example 1 only in that the mass percentage of UBM microparticles in the final system of step (4) is adjusted to 5%, while the remaining steps are consistent with Example 1.

[0154] Comparative Example 6

[0155] This comparative example provides an injectable complex that differs from Example 1 only in that the mass percentage of UBM microparticles in the final system of step (4) is adjusted to 13%, while the remaining steps are consistent with Example 1.

[0156] Comparative Example 7

[0157] This comparative example provides an injectable complex that differs from Example 1 only in that the mass percentage of cross-linked sodium hyaluronate particles in the final system of step (4) is adjusted to 2.5%, while the remaining steps are consistent with Example 1.

[0158] Comparative Example 8

[0159] This comparative example provides an injectable complex that differs from Example 1 only in that the mass percentage of cross-linked sodium hyaluronate microparticles in the final system of step (4) is adjusted to 4%, while the remaining steps are consistent with Example 1.

[0160] Comparative Example 9

[0161] This comparative example provides an injectable complex that differs from Example 1 only in that the mass percentage of cross-linked sodium hyaluronate particles in the final system of step (4) is adjusted to 24%, while the remaining steps are consistent with Example 1.

[0162] Comparative Example 10

[0163] This comparative example provides an injectable complex that differs from Example 1 only in that the mass percentage of cross-linked sodium hyaluronate particles in the final system of step (4) is adjusted to 30%, while the remaining steps are consistent with Example 1.

[0164] Comparative Example 11

[0165] This comparative example provides an injectable complex that differs from Example 1 only in that the preparation of cross-linked sodium hyaluronate microparticles in step (2) is not performed, and the cross-linked sodium hyaluronate microparticles in step (4) are replaced with a 1 mg / mL sodium hyaluronate solution.

[0166] Test Example 1

[0167] Post-irradiation stability test:

[0168] (1) Samples to be tested: injectable complexes obtained from each example and comparative example.

[0169] (2) Test method:

[0170] (2.1) Sample gelation performance test:

[0171] Inject 0.5 mL of each sample into a 6-well plate, incubate the plate at 37°C for 5 min, then hold the plate vertically and observe whether the sample slides off. If the sample flows down in a liquid state due to gravity, it is considered non-gelling; if the sample adheres stably to the plate or falls off in a block, it is considered gelling.

[0172] (2.2) Sample dissolution and dispersion time test:

[0173] Inject 0.5 mL of each sample into a 6-well plate, incubate the plate at 37°C for 5 min, add 7 mL of preheated physiological saline solution per well to immerse the gel sample, then return the plate to 37°C for incubation, shake the plate, and determine the sample dispersion when the sample is completely dispersed into particles, take a picture and record the sample dispersion time.

[0174] (2.3) Dynamic viscosity test:

[0175] The dynamic viscosity of the gel sample at room temperature was tested using a rotational viscometer. 3 mL of sample was injected into the outer cylinder of the coaxial cylindrical rotational viscometer. The rotor was then immersed in the sample, and the rotational speed was set to 1 r / min for 5 min, with a shear rate of 0.4 / s. The sample was rotated at a constant speed, and the dynamic viscosity value was recorded after the test.

[0176] (3) Test results:

[0177] Table 1 shows the gelling properties, dissolution and dispersion time, and dynamic viscosity test results of each sample after irradiation. A comparison diagram of the dispersibility of the samples from Example 1 and Comparative Example 1 in solution is shown below. Figure 1 As shown.

[0178] Table 1

[0179]

[0180] Comparison of data from Examples 1 and 4 and Comparative Examples 1, 3, and 7-8 shows that controlling the mass percentage of cross-linked sodium hyaluronate microparticles to above 5% can promote the uniformity of the sample. Through the three-dimensional structure and viscous properties of cross-linked sodium hyaluronate, the cohesion and viscosity of the composite gel are increased, which slows down the dissolution rate of the individual poloxamer gel in the solution, thereby prolonging the dissolution and dispersion time of the sample.

[0181] A comparison of the data from Examples 1, 4-6, and Comparative Examples 9-10 shows that as the mass percentage of cross-linked sodium hyaluronate microparticles increases, the gel dynamic viscosity increases, further promoting the cohesion of the sample. When the mass percentage of cross-linked sodium hyaluronate microparticles increases to over 23%, the excessive cross-linked sodium hyaluronate microparticles lead to increased degradation after irradiation, resulting in poor sample uniformity and delamination at the top. Figure 2 As shown.

[0182] A comparison of the data from Examples 1, 7-8, and 4-6 shows that reducing the mass percentage of decellularized matrix microparticles decreases sample viscosity and shortens dissolution and dispersion time; reducing the mass percentage of decellularized matrix microparticles to below 6% worsens sample homogeneity; further controlling the mass percentage of decellularized matrix microparticles at 10-12% increases sample viscosity and prolongs dissolution and dispersion time, indicating that decellularized matrix microparticles can increase the cohesion of different molecules in the gel. When the mass percentage of decellularized matrix microparticles exceeds 12%, the internal continuity structure of the sample is disrupted, and the dissolution and dispersion time is shortened.

[0183] A comparison of the data from Examples 1 and 9-10 shows that controlling the molecular weight of hyaluronic acid to 1200-2000 kDa during the preparation of sodium hyaluronate microparticles can further improve the viscosity of the sample.

[0184] A comparison of the data from Example 1 and Example 11 shows that controlling the crosslinking time to 5-6 h during the preparation of crosslinked sodium hyaluronate microparticles can further improve the dynamic viscosity of the sample, prolong the dissolution and dispersion time, and improve the overall structure of the sample.

[0185] A comparison of the data from Example 1 and Example 12 shows that replacing the cross-linked sodium hyaluronate microparticles with cross-linked carboxymethyl chitosan microparticles resulted in a decrease in both the sample dissolution and dispersion time and viscosity, but it still improved the sample dissolution and dispersion time compared to Comparative Example 1.

[0186] A comparison of the data from Example 1 and Example 13 shows that after irradiation, the dynamic viscosity of the sample decreases due to the degradation of cross-linked sodium hyaluronate gel and decellularized matrix microparticles, but the sample dissolution and dispersion time still meets the requirements for use.

[0187] A comparison of the data from Examples 1-3 and Examples 14-16 shows that increasing the irradiation dose decreases the dynamic viscosity of the sample, but the sample dissolution and dispersion time still meets the requirements for use.

[0188] A comparison of the data from Example 1 and Comparative Example 11 shows that when the cross-linked sodium hyaluronate microparticles are replaced with non-cross-linked sodium hyaluronate solution, the radiation resistance of the composite gel deteriorates, resulting in a significant decrease in sample viscosity that cannot be measured. The decellularized matrix microparticles lack support and settle.

[0189] Test Example 2

[0190] Uniformity test:

[0191] (1) Sample to be tested: the injectable complex obtained in Example 1 and Comparative Example 1.

[0192] (2) Test method:

[0193] Each sample was placed in a 4°C refrigerator for 72 hours, and the separation of the samples was observed.

[0194] (3) Test results:

[0195] Uniformity test results as follows Figure 3 As shown, the sample in Comparative Example 1 exhibited obvious stratification, while the sample in Example 1 showed good homogeneity with no obvious stratification. This result indicates that adding sodium hyaluronate gel microparticles can significantly improve the homogeneity of the sample.

[0196] Test Example 3

[0197] Osmotic pressure test:

[0198] (1) Samples to be tested: injectable complexes obtained in Examples 1 and 9 and Comparative Examples 2 and 3.

[0199] (2) Test method:

[0200] The osmotic pressure of each sample was tested using a Fiske Model 210 microsample osmometer manufactured by Advanced Systems, Inc. Each sample was thoroughly shaken and mixed before being aspirated, and then 20 μL of the sample was placed into a dedicated osmotic tube for testing.

[0201] (3) Test results:

[0202] The osmotic pressure test results are shown in Table 2. All samples tested met the osmotic pressure molar concentration range of 200 mOsmol / kg-400 mOsmol / kg defined in YY / T 0962-2021 standard for cross-linked sodium hyaluronate gel for orthopedic use.

[0203] Table 2

[0204]

[0205] As can be seen from the data in Table 2, the injectable complex composite osmotic pressure molar concentration range standard of the present invention.

[0206] Test Example 4

[0207] Adhesion test:

[0208] (1) Sample to be tested: the injectable complex obtained in Example 1 and Comparative Example 1.

[0209] (2) Test method:

[0210] Fresh pig bladders were cut and flattened, with the endometrium facing upwards, and placed on a tray to simulate the inner surface tissue of the uterus. The endometrial surface was washed with physiological saline. 1 mL of each sample was squeezed onto the surface of the pig bladder endometrium. The pig bladder tissue was then folded to wrap around the sample to simulate the narrow environment inside the uterus, and incubated in a 37°C water bath for 30 min. After 30 min, the pig bladders were removed. The bladders were unfolded from their folded state back to their flat state, and the tray was tilted at 45° to facilitate liquid flow. Physiological saline preheated to 35±2°C was used to rinse the gel sample laterally back and forth about 1 cm above it at a rinsing speed of about 0.5 mL / s, allowing the liquid to flow down by gravity and fully wet the gel surface. The adhesion and retention of the sample on the tissue surface were observed.

[0211] (3) Test results:

[0212] Adhesion test results as follows Figure 4 As shown, the sample of Example 1 exhibits significantly improved adhesion of the gel to the tissue surface due to the addition of sodium hyaluronate gel microparticles. Simultaneously, the introduction of cross-linked sodium hyaluronate microparticles enhances the cohesiveness of the sample, preventing the gel from spreading rapidly upon injection into the tissue site. Instead, it forms a uniform gel-like mass with a three-dimensional structure according to the injection trajectory.

[0213] Test Example 5

[0214] Active factor test:

[0215] (1) Samples to be tested: Samples of the injectable composites obtained in Examples 1-3 and Comparative Example 1 before irradiation, after 15 KGy irradiation, and after 25 KGy irradiation.

[0216] (2) Test method: Prepare 2 M urea solution, take 50 mL and add 125 mg of heparin sodium, stir evenly to obtain heparin sodium-urea solution; weigh 0.1 g of the corresponding gel into centrifuge tubes, add 1 mL of heparin sodium-urea solution, homogenize at high speed and low temperature for 5 min; centrifuge the homogenized sample at 9000 rpm for 5 min, extract the supernatant, and then perform the detection operation according to the instructions of the ELISA kit, and read the OD value at 450 nm.

[0217] (3) Test results:

[0218] The results of the growth factor content test are shown in Table 3. It can be concluded that the growth factor retention rates of the injectable complexes in Examples 1-3 after 15 KGy irradiation were 96.9%, 93.1%, and 97.5%, respectively, and after 25 KGy irradiation, the growth factor retention rates were 93.5%, 90.1%, and 94.1%, respectively, indicating that the retention rate of active factors is relatively unaffected by the irradiation dose. In contrast, in Comparative Example 1 without cross-linked sodium hyaluronate microparticles, the growth factor retention rate decreased to 61.1% after 15 KGy irradiation and to 52.4% after 25 KGy irradiation, indicating that the biological activity of the sample decreased after irradiation without the support of the cross-linked HA microscaffold.

[0219] Table 3

[0220]

[0221] Test Example 6

[0222] Intrauterine sample retention test:

[0223] (1) Sample to be tested: the injectable complex obtained in Example 1 and Comparative Example 1.

[0224] (2) Test method:

[0225] Six female New Zealand rabbits weighing 2.0-2.5 kg (purchased from Hangzhou Yuhang Kelian Rabbit Industry Professional Cooperative) were anesthetized. The abdomen was opened to expose both uteruses. A small incision was made approximately 2 mm above the cervix. Using a curette, the endometrial tissue was repeatedly scraped 2-3 cm into the incision until the uterine wall thinned and turned red. Next, a syringe catheter was inserted through the incision, and the sample to be tested was injected into the uterus until the cavity was full (approximately 0.5 mL). The catheter was then carefully withdrawn, and the uterine and abdominal incisions were sutured with non-absorbable sutures. Four hours post-surgery, an autopsy was performed, and both uteruses were removed and longitudinally cut with surgical scissors to observe the retention of the sample within the uterus.

[0226] (3) Test results:

[0227] Results of intrauterine sample retention test as follows Figure 5 As shown in the figure. Experimental results indicate that after the uterus was cut open, the sample of Comparative Example 1 showed virtually no visible sample residue, indicating that the sample failed to remain in the injection site; while the sample of Example 1 showed clear sample retention upon uterine incision. It is speculated that the gel sample of Comparative Example 1 had poor adhesion and was prone to flow, thus flowing along the uterine wall to other areas. These results demonstrate that adding cross-linked sodium hyaluronate microparticles can significantly improve the retention of the gel inside the uterus.

[0228] Test Example 7

[0229] Endometrial repair effect test:

[0230] (1) Sample to be tested: the injectable complex obtained in Example 1.

[0231] (2) Test method:

[0232] New Zealand rabbits aged 5-6 months were subcutaneously injected with 50 units of pregnant mare serum gonadotropin (PMSG). Changes in vulvar condition were observed for 3 consecutive days. After confirming estrus, a model of intrauterine adhesions was constructed.

[0233] Animals were fasted for 12 hours and deprived of water for 6 hours prior to surgery. After anesthesia, the abdomen was opened to expose both uteruses. An incision was made in the mid-segment of each uterus, and a hemostatic clip was applied 3 cm from the incision. Using a curette, the endometrial tissue was circularly scraped away from the incision, completely destroying the endometrium from the incision to the hemostatic clip segment and damaging the myometrium up to a length of 3 cm. A lipopolysaccharide solution of 1 mg / kg (2 mg / mL) was injected into the damaged segment, and the incision was sealed with a hemostatic clip, leaving it in place at the damaged site for 10 minutes.

[0234] The hemostatic clip was then opened, and one side of the incision was sutured as the model group; 0.5 mL of the injectable complex obtained in Example 1 was injected into the other side of the incision, and the incision was sutured as the treatment group.

[0235] Fourteen days after injection, the animals were dissected, and the bilateral damaged uterine segments were removed and fixed. Samples were subjected to hematologic staining (HE) to observe the endometrial repair and intrauterine adhesions.

[0236] (3) Test results:

[0237] HE section staining results are as follows: Figure 6 As shown in the figure, the degree of uterine adhesions was significantly reduced in the treatment group compared to the model group. Histopathological data were analyzed, and all data are expressed as mean ± standard deviation. The statistical results of endometrial thickness are shown below. Figure 7 As shown, the average endometrial thickness in the treatment group was 1699.35 ± 44.59 μm, significantly higher than that in the model group (657.51 ± 93.44 μm) (p < 0.01). The statistical results of the number of endometrial glands are as follows... Figure 8As shown, the mean number of glands in the treatment group was 101 ± 4.36, significantly higher than the 47 ± 4.3 in the model group (p < 0.01). One-way ANOVA and Tukey HSD post-hoc tests were performed on more than two variables. *p < 0.05 was considered statistically significant, **p < 0.01, ***p < 0.001, and ns indicated no statistically significant difference. All these results indicate that the treatment group can increase endometrial thickness and gland number, promoting endometrial repair.

[0238] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0239] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0240] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. An injectable complex for soft tissue repair, characterized in that, The injectable complex comprises: Thermosensitive medium is an aqueous solution of a biocompatible polymer with thermo-gelling properties; The pre-crosslinked, water-insoluble hydrophilic polymeric gel microparticles dispersed in the temperature-sensitive medium constitute 17-23% of the total mass of the injectable complex. And decellularized matrix microparticles dispersed in the temperature-sensitive medium, comprising 10-12% of the total mass of the injectable complex; The hydrophilic polymer gel microparticles combine with the network formed by the gelation of the temperature-sensitive medium at a temperature of 30-37℃ to form a supporting framework that enhances the structural stability of the injectable composite. The hydrophilic polymeric gel microparticles are obtained by granulation of cross-linked products obtained by cross-linking hyaluronic acid or its salts. The weight-average molecular weight of the hyaluronic acid or its salt is 1500-2000 kDa; The injectable complex is a composition sterilized by 15-25 kGy electron beam irradiation, and the vascular endothelial growth factor content retention rate after irradiation is not less than 90% of that before irradiation.

2. The injectable complex according to claim 1, characterized in that, The decellularized matrix microparticles are prepared from decellularized matrix raw materials through virus inactivation, decellularization, washing, drying, defatting, secondary drying, and grinding. The particle size of the decellularized matrix microparticles is 1-1000 μm.

3. The injectable complex according to claim 1, characterized in that, The hydrophilic polymeric gel microparticles are cross-linked hyaluronic acid salt microparticles; The cross-linked hyaluronic acid microparticles are prepared by a method comprising the following steps: Hyaluronic acid or its salt is subjected to a cross-linking reaction with a cross-linking agent under alkaline conditions, followed by purification and granulation to obtain the cross-linked hyaluronic acid salt microparticles.

4. The injectable complex according to claim 3, characterized in that, The cross-linking reaction is carried out at a temperature of 35-40°C for 4-6 hours, and the amount of cross-linking agent used is 4%-5% of the mass of hyaluronic acid or its salt.

5. The injectable complex according to claim 1, characterized in that, The temperature-sensitive medium includes an aqueous solution of any one or a combination of at least two of the following: chitosan / sodium β-glycerophosphate, poly(N-isopropylacrylamide), polyethylene glycol-polylactic acid block copolymer, polyethylene glycol-polyhydroxybutyrate copolymer, or poloxamer.

6. The injectable complex according to claim 5, characterized in that, The temperature-sensitive medium is an aqueous solution of poloxamer 407, and the concentration of poloxamer 407 in the preparation of the temperature-sensitive medium is 150-200 mg / mL.

7. A method for preparing an injectable complex as described in any one of claims 1-6, characterized in that, The preparation method includes the following steps: The hydrophilic polymeric gel microparticles, decellularized matrix microparticles, and the temperature-sensitive medium are mixed at 2-10°C to obtain the injectable complex.

8. Use of the injectable complex according to any one of claims 1-6 in the preparation of medical devices or medical materials for soft tissue repair.

9. The application according to claim 8, characterized in that, The soft tissue includes the endometrium.

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