A multi-scene tissue engineering composite gel with high tissue fusion and in-situ soft tissue regeneration, and a preparation method and application thereof

Abstract

The application discloses a multi-scene tissue engineering composite gel for high-tissue fusion and in-situ soft tissue regeneration as well as a preparation method and application thereof. The composite gel comprises a high-tissue fusion base gel and a functional matrix component. The functional matrix component is selected from one or more of the following: microspheres, viscoelastic sodium hyaluronate gel, collagen gel and temperature-sensitive gel. The high-tissue fusion base gel and the composite gel both satisfy the following physical property parameters: a water contact angle of 20-70 degrees; a hydrophilic group molar content of greater than or equal to 3%, wherein the hydrophilic group comprises one or more of a hydroxyl group, a carboxyl group and an amino group; a gel internal or gel interface pore size of greater than or equal to 40 microns; and an elastic modulus of 1-1000 Pa.

Description

Technical Field

[0001] This invention relates to the field of medical gel technology, and in particular to a multi-scenario tissue engineering composite gel with high tissue fusion and in-situ soft tissue regeneration, as well as its preparation method and application. Background Technology

[0002] Currently, various medical gels used in tissue engineering (including urethral filling gels, cosmetic filling gels, ophthalmic viscoelastic gels, and repair-related functional gels) generally possess excellent physical filling effects and long-lasting degradation characteristics, maintaining tissue filling morphology for a long time and reducing the frequency of repeated interventions, thus meeting the basic clinical needs for long-term material retention and morphological support. However, existing commercial gel products suffer from a common core deficiency in the industry: a complete lack of tissue integration and non-inflammatory stimulation regeneration capabilities.

[0003] Most existing conventional gel materials are bio-inert filling systems. After implantation, they can only achieve filling and support through physical stacking, and cannot form an interface bond, cell infiltration, or tissue integration with the host's own tissue. A dense fibrous barrier interface forms between the material and the host tissue. In dynamically moving soft tissue sites (urethra, subcutaneous cosmetic soft tissue, periocular soft tissue, articular cartilage interface, etc.), this interface defect can directly lead to a series of clinical problems such as material displacement, local collapse, shaping failure, encapsulation, and inflammatory stimulation. It cannot achieve true tissue regeneration and repair, and can only achieve temporary physical filling, which greatly limits the clinical application value and long-term safety of tissue engineering materials.

[0004] Currently, conventional modification solutions in the industry mostly focus on optimizing gel viscoelasticity, adjusting degradation rate, and improving mechanical support properties. These solutions can only improve the physical properties of materials and cannot overcome the technical bottleneck of "filling without integration and retention without regeneration." Among existing technologies, mainstream tissue engineering matrix materials such as microspheres (hydroxyapatite, polycaprolactone, polylactic acid, etc.), conventional sodium hyaluronate gel, collagen gel, and thermosensitive gel all have inherent problems such as insufficient tissue affinity, lack of interfacial integration ability, and inflammatory stimulation. Single use or conventional compounding cannot achieve in-situ soft tissue regeneration and cannot fundamentally solve the pain points of displacement, collapse, and repair failure.

[0005] In summary, the field of tissue engineering has long lacked a universal composite gel system that combines long-lasting filling, controllable degradation, excellent mechanical compatibility, high tissue integration, and in-situ non-inflammatory regeneration capabilities. This system cannot meet the high-end clinical needs of regenerative medicine in various scenarios and is a key technical challenge that urgently needs to be overcome in this field. Summary of the Invention

[0006] The purpose of this invention is to provide a multi-scenario tissue engineering composite gel with high tissue integration and in-situ soft tissue regeneration, as well as its preparation method and application.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A multi-scenario tissue engineering composite gel with high tissue fusion and in-situ soft tissue regeneration includes a high tissue fusion base gel and a functional matrix component, wherein the functional matrix component is selected from one or more of microspheres, viscoelastic sodium hyaluronate gel, collagen gel, and thermosensitive gel. Both the high tissue fusion substrate gel and the composite gel meet the following physical properties: water contact angle of 20~70°; molar percentage of hydrophilic groups ≥3%, wherein the hydrophilic groups include one or more of hydroxyl, carboxyl, and amino groups; pore size inside the gel or at the gel interface ≥40μm; and elastic modulus of 1~1000Pa.

[0008] The microspheres include hydroxyapatite, polycaprolactone, polylactic acid, and agarose.

[0009] The functional matrix component is used to endow the composite gel with long-lasting filling, stable support, viscoelastic adaptation, temperature-responsive molding and basic tissue repair properties; the high tissue fusion base gel is the core of regenerative modification, used to improve the overall tissue affinity and interface integration ability of the composite gel.

[0010] The composite gel enables seamless interface fusion between the material and host tissue, inducing in-situ soft tissue regeneration and forming a soft tissue mass 3-30 days after implantation. This overcomes the shortcomings of traditional tissue engineering gels, such as easy displacement, collapse, and inability to integrate with the tissue. The tensile strength of the soft tissue can reach 0.1 kPa to 1000 kPa; at a compression rate of 80%, the compressive strength can reach 0.1 kPa to 10000 kPa.

[0011] The high tissue fusion substrate gel has a mass fraction of 5% to 100%, and the functional matrix component has a mass fraction of 0% to 95%.

[0012] The viscoelastic sodium hyaluronate gel has a molecular weight of 100~3000kDa and possesses medical-grade dynamic viscoelasticity and in vivo anti-dilution stability; the collagen gel is medical-grade purified collagen without immune rejection; and the thermosensitive gel has a phase transition temperature of 30~37℃.

[0013] The preparation method of the multi-scenario tissue engineering composite gel of the present invention includes the following steps: (1) Preparation of high tissue fusion substrate gel Sodium hyaluronate was prepared into a solution, and then arginine was added to the sodium hyaluronate and dissolved completely. After stirring evenly, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride was added, and the solution was allowed to stand and solidify for 2 days. Then, impurities were removed by dialysis with pure water. (2) The high tissue fusion base gel and the functional matrix components are thoroughly mixed to obtain a mixed gel system; the free water in the system is removed by a concentration process. The concentration method can be one or a combination of vacuum filtration, vacuum drying, and polyethylene glycol osmotic pressure difference method. The removal of free water can effectively increase the molecular contact probability in the system and enhance the hydrogen bond interaction between the base gel and the functional matrix components, and finally obtain a tissue engineering composite gel that can be applied to a variety of repair scenarios.

[0014] The multi-scenario tissue engineering composite gel of this invention is used in the preparation of urethral soft tissue regeneration filling materials, male erectile dysfunction soft tissue remodeling materials, medical aesthetic soft tissue remodeling materials, ophthalmic viscoelastic protective materials, in vitro tissue culture and transplantation materials, cell-loaded cartilage repair materials, bone and joint lubrication materials, and drug-loaded osteoarthritis treatment drugs. These include: (1) Urethral tissue engineering materials: used for in situ regeneration, repair and filling of urethral mucosal atrophy, urethral relaxation, stress urinary incontinence, urethral defects after prostate surgery, and male erectile dysfunction; (2) Medical aesthetic soft tissue engineering materials: used for subcutaneous soft tissue filling, contour reshaping, and soft tissue defect regeneration and repair; (3) In vitro engineered tissue grafts: used for three-dimensional construction of tissues in vitro and autologous re-transplantation after tissue culture; (4) Joint lubricating material: implanted to reduce the coefficient of friction and increase joint lubrication.

[0015] Compared with the prior art, the outstanding effect of the present invention is as follows: (1) It broke through the industry's technical bottlenecks and pioneered a parameterized precision regeneration fusion system. Existing tissue engineering gels only focus on filling, degradation, and viscoelastic physical properties, completely neglecting the core requirements of tissue fusion and in-situ regeneration. This invention, for the first time, systematically solves the problem of the lack of tissue fusion in traditional gels by coupling and limiting four precise physical property parameters from four dimensions: interface affinity, active sites, microporous structure, and mechanical matching. It upgrades the traditional "physical filler material" to an "in-situ soft tissue regeneration material," completely breaking through the technical dilemmas of easy displacement, easy collapse, and lack of integration of fillers.

[0016] (2) It takes into account the advantages of traditional materials and the new properties of recycled materials, and the performance is synergistic and conflict-free.

[0017] This invention, through compound modification, fully retains the high-strength support of hydroxyapatite, the excellent viscoelasticity of sodium hyaluronate, the repair properties of collagen, and the injectable in-situ molding advantages of thermosensitive gel. At the same time, it endows the system with new tissue integration, cell induction, and in-situ regeneration capabilities through a highly fusion-based base gel, achieving multiple synergistic performances of "long-lasting filling + stable support + interface fusion + soft tissue regeneration," thus solving the problem of traditional materials having single performance and being unable to balance advantages and disadvantages.

[0018] (3) Eliminate clinical complications at the source and significantly improve the stability of long-term repair.

[0019] The composite gel of this invention has a highly hydrophilic interface, a permeable macroporous structure, and a soft tissue matching modulus. It can actively adsorb body proteins and induce host cell infiltration, gradually achieving seamless integration between the material and autologous tissue. It generates new soft tissue in situ, replacing the traditional physical filling mode, completely eliminating the fiber isolation interface, and fundamentally avoiding problems such as material displacement, local collapse, capsule calcification, and inflammatory failure, thus significantly improving the long-term effectiveness and safety of repair.

[0020] (4) It integrates quickly after implantation, and the fused tissue has high compressive and tensile strength.

[0021] The composite gel of this invention can achieve tissue fusion within 3-30 days after implantation. The tensile fracture strength of the fused soft tissue can reach 0.1kPa~1000kPa, and the compressive strength can reach 0.1kPa~10000kPa when the compression rate is 80%, which is similar to the strength of the original tissue.

[0022] (5) It has strong versatility and covers multiple fields of organizational engineering application scenarios.

[0023] This invention's composite gel system is highly adjustable and widely adaptable, allowing for flexible formulation of matrix components to meet the needs of different scenarios, making it suitable for high-end applications in multiple fields: 1) Urethral tissue engineering: used for urethral mucosal atrophy, urethral relaxation, stress urinary incontinence, and postoperative defect regeneration and repair; 2) Medical aesthetic soft tissue engineering: used for subcutaneous soft tissue filling, contour reshaping, and soft tissue defect regeneration; 3) Ophthalmic engineering: used as a medical viscoelastic agent, suitable for minimally invasive ocular surgery protection and interface repair; 4) In vitro regeneration culture: used for three-dimensional culture of external tissues and in vitro soft tissue reconstruction and re-transplantation; 5) Cartilage repair: can be loaded with seed cells for three-dimensional culture and defect repair of articular cartilage; 6) Bone and joint repair: can be loaded with anti-inflammatory and repair drugs for targeted sustained-release treatment of osteoarthritis, covering multiple sub-sectors of regenerative medicine.

[0024] The following description, in conjunction with the accompanying drawings and specific embodiments, further illustrates the multi-scenario tissue engineering composite gel with high tissue fusion and in-situ soft tissue regeneration, its preparation method, and its applications. Attached Figure Description

[0025] Figure 1 These are photos of different experimental groups in Example 1.

[0026] Figure 2 The results show the immunoexpression of different samples.

[0027] Figure 3 Pressure values ​​for urethral leakage points were determined for different samples.

[0028] Figure 4 Skin test experiments (7 days) were conducted on different samples. Detailed Implementation

[0029] Example 1: Fusion properties of high tissue fusion substrate gel

[0030] (1) Prepare a 3% (w / v) solution of sodium hyaluronate. Take 30 mL of the solution and add arginine to the sodium hyaluronate. Dissolve the arginine completely to make the concentration 1% (w / v). Place the solution at 25°C and stir at 50 rpm. After stirring evenly, add 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM) to make the final concentration 2%. Let it stand for 2 days to solidify. Then, dialyze with pure water to remove impurities and obtain a high tissue fusion substrate gel.

[0031] (2) Prepare a 3% (w / v) solution of sodium hyaluronate. Take 30 mL of the solution and add BDDE to the sodium hyaluronate to make the concentration 1% (w / v). Place the solution at a temperature of 4~10℃ and let it stand for 5 days to solidify. Then, dialyze with pure water to remove impurities. Then, homogenize the solution with 500 mesh silk to form a particulate gel, i.e., viscoelastic sodium hyaluronate gel.

[0032] (3) The hydroxyapatite microspheres were mixed evenly with a 3 mg / mL HA solution; (4) The highly fusion-compatible base gel prepared in step (1) was implanted into the dermis of rats and observed at 3, 7 and 30 days, i.e. Experiment 1; the sodium hyaluronate gel prepared in step (2) and the hydroxyapatite microsphere solution prepared in step (3) were implanted into the dermis of rats and samples were taken for observation at 30 days, i.e. Experiment 2 and Experiment 3.

[0033] (5) HA was added to 0.5% and 0.6% succinic anhydride respectively and reacted at low temperature for 24h to modify carboxylation and increase hydrophilicity. After dialysis and freeze drying, gels were prepared according to the method in step (1) for Experiment 4 and Experiment 5.

[0034] Add 25% tetrabutylammonium hydroxide (TBA-OH) dropwise to HA, with pH around 7, and freeze-dry to obtain HA-TBA salt soluble in DMSO. Dissolve it in anhydrous DMSO, add EDC and DMAP to activate the carboxyl group at room temperature for 2h and 2.1h respectively, then add amino-based hydrophobic substrate dissolved in DMSO dropwise, and react at 50℃ in the dark for 48h. Add cold acetone to the reaction solution to precipitate the precipitate, filter and wash with acetone, dissolve the precipitate in water, put it into a 3500 cutoff dialysis bag and dialyze with pure water for 5 days. Freeze-dry the dialysate to obtain the hydrophobically modified sodium hyaluronate product. Then prepare the gel according to the method in step (1), for experiments 6 and 7.

[0035] Experimental results: Experimental results are as follows Figure 1 As shown, a represents optical images of tissue implanted subcutaneously in Experiment 1 at 3, 7, and 30 days, while b, c, d, e, f, and g represent optical images of tissue implanted subcutaneously in Experiments 2, 3, 4, 5, 6, and 7 at 30 days, respectively.

[0036] Further analysis of the four major indicators—water contact angle, hydrophilic groups, gel porosity, and elastic modulus—and the test results of the seven gel samples in experiments 1, 2, 3, 4, 5, 6, and 7 are shown in Table 1.

[0037] Table 1. Gel water contact angle, hydrophilic groups, gel porosity, and elastic modulus

[0038] According to the experimental results, the products from experiments 1, 5, and 6 all achieved tissue fusion within 3-30 days, combining... Figure 1 As shown in Table 1, the results indicate that a gel with tissue fusion in 3 days can be prepared when the following conditions are met: water contact angle of 20-70°; molar percentage of hydrophilic groups ≥3%; pore size inside the gel or at the gel interface ≥40μm; and elastic modulus of 1-1000Pa. However, sodium hyaluronate gel (Experiment 2) and hydroxyapatite microspheres (Experiment 3) outside these ranges did not have a fusion effect.

[0039] Example 2: High tissue strength after gel fusion with high tissue fusion substrate

[0040] Gels were prepared according to step (1) in Example 1. Then, 3 mg / mL, 10 mg / mL, and 25 mg / mL gels were implanted into the dermis of rats for 3 days, 7 days, and 30 days. The soft tissue blocks that formed tissue fusion were removed, and the capsule was removed as much as possible. The blocks were then cut into approximately cylindrical shapes with a height of 5 mm and a diameter of 10 mm. The compression speed was 2 mm / min and the compression rate was 80% to test the compression strength and calculate the compression modulus.

[0041] The soft tissue block after removing the capsule was cut into a cuboid with a length of 10 mm, a width of 2 mm, and a height of 2 mm. It was stretched at a speed of 2 mm / min until it broke to test the tensile strength and calculate the tensile modulus.

[0042] The experimental results are shown in Table 2.

[0043] Table 2. Compression modulus and tensile modulus of soft tissue

[0044] As shown in Table 2, the high tissue fusion substrate gel prepared by the present invention has high tissue strength after fusion. When the compression rate reaches 80%, the compression modulus can be 0.1kPa~10000kPa, and the tensile fracture strength can be 0.1kPa~1000kPa, which has good soft tissue strength and controllable properties.

[0045] Example 3: Multi-scenario tissue engineering composite gel

[0046] The high tissue fusion substrate gel prepared by the method in step (1) of Example 1 was mixed evenly with the functional matrix component to obtain a multi-scenario tissue engineering composite gel. The functional matrix component was selected from one or more of the following: microspheres (hydroxyapatite, polycaprolactone, polylactic acid, agarose, etc.), viscoelastic sodium hyaluronate gel, collagen gel, and thermosensitive gel. The dosage ratios mentioned in this example refer to the mass percentage of the functional matrix component.

[0047] (1) Microspheres

[0048] Hydroxyapatite microspheres: A high tissue fusion substrate gel was prepared according to the preparation method in step (1) of Example 1. Hydroxyapatite microspheres were added to the substrate gel and mixed in proportions of 10%, 95%, and 95.5%. After mixing, the mixed gel system was transferred to a sand core filtration device and filtered under a negative pressure of 0.04 MPa for 40 min to remove some of the free water in the system.

[0049] Polycaprolactone: A high tissue fusion substrate gel was prepared according to the preparation method in step (1) of Example 1. Polycaprolactone was added to the substrate gel and mixed in proportions of 10%, 95%, and 95.5%. The mixed gel was spread evenly on a culture dish with a thickness of 2 mm and placed in a vacuum drying oven at a vacuum degree of 0.03 MPa and a temperature of 25 °C for 25 min to remove some of the free water in the system. Polylactic acid: A high tissue fusion substrate gel was prepared according to the preparation method in step (1) of Example 1. Polylactic acid was added to the substrate gel and mixed in proportions of 10%, 95%, and 95.5%. The mixed gel was placed into a dialysis bag (molecular weight cutoff 1.4 kDa), and the dialysis bag was immersed in a 20 wt% polyethylene glycol (PEG20000) aqueous solution and osmotically equilibrated at 25 °C for 12 h. Free water inside the gel was removed by relying on the osmotic pressure difference to permeate outward.

[0050] Agarose: A high tissue fusion substrate gel was prepared according to the preparation method in step (1) of Example 1. Agarose was added to the substrate gel and mixed in proportions of 10%, 95%, and 95.5%. A large amount of free water was first removed by vacuum filtration at 0.04 MPa for 30 min. Then, the water content of the system was further adjusted by the PEG osmotic pressure difference method and the system was osmotically treated for 6 h.

[0051] (2) Viscoelastic sodium hyaluronate gel

[0052] HA of 100 kDa and 3000 kDa were dissolved in 1% NaOH to obtain a solution of 30 mg / mL. Then, 2% BDDE was added and the mixture was reacted for 4 days before homogenization and granulation (100 μm) to obtain HA gel.

[0053] The high tissue fusion substrate gel was prepared according to the preparation method in step (1) of Example 1. HA gel was added to the substrate gel and mixed in proportions of 10%, 95%, and 95.5%. After mixing, the mixed gel system was transferred to a sand core filtration device and filtered under a negative pressure of 0.04 MPa for 40 min to remove some of the free water in the system.

[0054] (3) Collagen gel

[0055] Collagen extracted from pig skin was used. 2% collagen was added to 2% glutaraldehyde and reacted for 6 hours. Then, dialysis was performed to remove impurities. After homogenization, it was found to be 50μm gel particles. The high tissue fusion base gel was prepared according to the preparation method in step (1) of Example 1. Collagen gel particles were added to the base gel and mixed in proportions of 10%, 95%, and 95.5%. The mixed gel was spread evenly on a culture dish with a thickness of 2 mm and placed in a vacuum drying oven at a vacuum degree of 0.03 MPa and a temperature of 25 °C for 25 min to remove some of the free water in the system.

[0056] (4) Thermosensitive gel

[0057] 1g of chitosan with a degree of deacetylation of 97% was dissolved in 1% acetic acid solution to obtain a 1% chitosan solution. Next, the pH of the reaction solution was adjusted to 5.5 using 0.5M NaOH solution, and 0.58g of EDC was added. The mixture was stirred until homogeneous and reacted at room temperature for 20 hours. Then, 4% and 4.5% NaOH (final concentration) were added to dissolve and disperse the solution, resulting in a homogeneous and transparent solution. The reaction temperature was then raised to 40℃, and 50mL of 1,2-epoxybutane was added, reacting for 72 hours. The pH was then adjusted to neutral, and impurities were removed by dialysis with pure water (using a dialysis bag with a molecular weight cutoff of 14000) for 4 days. A white, sponge-like substance was obtained by freeze-drying; subsequently, it dissolved to form a 1.5% thermosensitive chitosan gel with gelation temperatures of 30℃ and 37℃, which are more closely related to human body temperature.

[0058] The high tissue fusion substrate gel was prepared according to the preparation method in step (1) of Example 1. Thermosensitive chitosan gel was added to the substrate gel and mixed at 4-8°C in proportions of 10%, 95%, and 95.5%. The mixed gel was placed into a dialysis bag (molecular weight cutoff 1.4 kDa), and the dialysis bag was immersed in a 20 wt% polyethylene glycol (PEG20000) aqueous solution and osmotically equilibrated at 4°C for 12 h. Free water inside the gel was removed by relying on the osmotic pressure difference to permeate outward.

[0059] (5) Multi-component composite gel

[0060] a. The polycaprolactone microspheres from step (1) were added to 10%, 50%, and 80% of the total mass, respectively. The collagen gel from step (3) was added to 85%, 40%, and 15.5% of the total mass, respectively. The highly fused gel was added to 5%, 10%, and 4.5% of the total mass, respectively. The mixed gel was placed into a dialysis bag (molecular weight cutoff 1.4 kDa). The dialysis bag was immersed in a 20 wt% polyethylene glycol (PEG20000) aqueous solution and osmotically equilibrated at 4°C for 12 h. The free water inside the gel was removed by relying on the osmotic pressure difference to permeate outward.

[0061] b. Add the polycaprolactone microspheres from step (1) to 10%, 50%, and 40% of the total mass, respectively; the collagen gel from step (3) to 15%, 20%, and 15.5% of the total mass, respectively; the thermosensitive gel from step (4) to 70%, 20%, and 40% of the total mass, respectively; and the highly fused gel to 5%, 10%, and 4.5% of the total mass, respectively. Pack the mixed gel into a dialysis bag (molecular weight cutoff 1.4 kDa), immerse the dialysis bag in a 20 wt% polyethylene glycol (PEG20000) aqueous solution, and allow it to osmotically equilibrate at 4°C for 12 h; rely on the osmotic pressure difference to allow free water inside the gel to permeate outwards, thereby achieving the removal of free water.

[0062] (6) Control group gel

[0063] The high tissue fusion substrate gel was prepared according to the preparation method in step (1) of Example 1. The thermosensitive chitosan gel prepared in step (4) of this example was added to the substrate gel and mixed at 4~8℃ in proportions of 10%, 95%, and 95.5%. The experimental results are shown in Table 3.

[0064] Table 3. Water contact angle, hydrophilic group molar ratio, gel porosity, and elastic modulus after mixing different samples (10%, 95%, 95.5%)

[0065] In addition to the multi-component gel, the data in each column, from left to right, are the functional matrix component additions of 10%, 95%, and 95.5%, respectively. The multi-component composite gels a and b are both 95%, 90%, and 95.5%.

[0066] All the above samples were implanted subcutaneously into rats for 3-30 days for sampling and observation. The results are shown in Table 4.

[0067] Table 4. Fusion of different sample mixtures (10%, 95%, 95.5%) implanted subcutaneously for 3–30 days.

[0068] The data in Tables 3 and 4 show that when the base gel is mixed with other gels, the mixture fails to fuse when the content of the other gels exceeds 95%. This may be because the base gel loses its fusion effect. Furthermore, without a concentration process, even if the mixed gel meets the following conditions: water contact angle of 20–70°; hydrophilic group molar percentage ≥3%; internal or interfacial pore size ≥40 μm; and elastic modulus of 1–1000 Pa, the fusion will not occur. However, when the functional matrix component accounts for 0–95% of the mixed gel, regardless of whether it is a composite of multiple gels, as long as the above four physicochemical properties are met, a concentration process can be used to remove some free water from the system. Concentration methods can include filtration, vacuum drying, and polyethylene glycol osmotic pressure difference, or a combination thereof. Removal of free water effectively increases the probability of molecular contact within the system, enhances the hydrogen bonding interaction between the base gel and the functional matrix component, and ultimately yields a tissue-engineered composite gel applicable to various repair scenarios. All gels can fuse within 3–30 days.

[0069] Example 4: Non-inflammatory tissue fusion and regeneration

[0070] (1) Regulation of gel immunomodulatory activity: Gels and hydroxyapatite prepared by step (1) of Example 1 and Example 3 (10% hydroxyapatite addition) were implanted subcutaneously in rats for 30 days. The tissues were homogenized at 10,000 rpm for 10 minutes and centrifuged at 10,000 rpm for 10 minutes. The supernatant was collected, and the fluorescence results in the supernatant were detected using TNF-α and IL-10 kits. Blank tissues were used as the control group. The experimental results are as follows: Figure 2 As shown. From Figure 2 It can be found that the samples of the present invention exhibit non-inflammatory tissue fusion after implantation and have macrophage M2 type induction effect. The composite gel also increases macrophage M2 type expression compared with hydroxyapatite microspheres (step (3) of Example 1).

[0071] (2) Promoting the production of type I and type III collagen: Twenty Wistar rats were randomly divided into experimental groups, namely, a sham control group, experimental group 1 (gel prepared in step (1) of Example 1), experimental group 2 (10% hydroxyapatite composite gel prepared by the method of Example 3), and an aging control group of 5 rats each; except for the sham control group (no treatment group), Wistar rats in each experimental group were subcutaneously injected with D-galactose (D-gal) at a dose of 125 mg / kg·d for 6 consecutive weeks to induce a rat skin aging model. On the 18th day after the injection of D-galactose (D-gal), each rat was anesthetized with chloral hydrate (0.25 ml per 100 g body weight) and its buttock area was disinfected. A 3cm circular tattoo area was pre-made on both sides of the rat's buttocks. Then, under general anesthesia, the rats were treated with microspheres (0.2ml each) once a week for 4 weeks. The experimental groups 1-4 were treated with microspheres (0.2ml each). The aging control group and the sham control group underwent the same surgical procedure but did not receive any gel injection treatment. Forty-two days after treatment, all animals were euthanized. The expression levels of skin wounds and type I collagen, type III collagen and MMP-1 were compared by histological and quantitative real-time PCR tests, as shown in Table 5 below.

[0072] Table 5 Regeneration effect of different samples

[0073] The results showed that, compared with the sham control group, aging skin exhibited reduced thickness in both the epidermis and dermis. The number of cell layers in the aging epidermis decreased, and dermal collagen fibers became sparse, elongated, or broken. However, compared with the aging control group, these histological changes were significantly improved after injection of microspheres 1–2 in the experimental group, with significantly higher epidermal and dermal thickness and collagen fiber density.

[0074] The above demonstrates that the samples of the present invention exhibit non-inflammatory tissue fusion and regeneration effects.

[0075] Example 5: Urethral Filler

[0076] Twenty-four female SD rats were randomly divided into three groups of eight each. The normal control group underwent sham surgery (normal group); the other two groups underwent vaginal balloon dilation and bilateral oophorectomy to establish a stress urinary incontinence model (successful modeling was confirmed 4 weeks post-surgery). In the two modeling groups, one group was injected with the gel prepared in step (1) of Example 1, and the other group was injected with physiological saline (physiological saline group). Leakage pressure was measured at week 1 and week 4 post-injection. The detection procedure was briefly described as follows: A 90-gauge polyethylene catheter was inserted through the top of the bladder, and sterile physiological saline containing methylene blue was continuously instilled into the bladder. Simultaneously, a pressure transducer (Ed Instruments, Castle Hill, New South Wales, Australia) was used to record the increase in bladder pressure in real time until urine leakage occurred; the bladder pressure at the instant of urine leakage was the leakage pressure.

[0077] The results are as follows Figure 3 As shown, the sample using the present invention has a good urethral pressure enhancement effect and can reach the leakage pressure point consistent with that of normal rats.

[0078] Example 6: Cosmetic Fillers

[0079] The gel prepared using step (1) of Example 1 and the gel prepared using the method of Example 3 (a composite gel containing 10% thermosensitive chitosan gel) was used as the test product. It was filled into 1.0 ml BD tubes and sterilized by moist heat. Four rabbits (weighing 4-6 kg, half male and half female) were prepared for skin testing. The rabbits' fur was removed, and the epidermis was wiped with 75% alcohol. Microspheres of each batch were injected (0.2 ml / point, n=3) into the superficial dermis of the rabbits, creating a hemispherical protrusion approximately 0.3-0.7 mm high and 0.5-1.5 mm in diameter. The rabbits were then returned to their cages and allowed to move freely. Seven days after the injection, the experimental rabbits were quickly euthanized by intravenous air injection. The epidermis of the injection area was then cut open with a scalpel to examine the degradation and suppuration of the injected area. The results were as follows: Figure 4 As shown.

[0080] from Figure 4It can be seen that the gels of Examples 1 and 3 (containing 10% thermosensitive chitosan gel) showed no obvious inflammatory reaction after being injected into rabbits for 7 days. The inflammatory reaction was initially severe from day 1 to 3, and basically ended from day 3 to 7. As shown in the control group, if there was inflammation, the tissue on day 7 would show severe redness, swelling, yellowing, and purulent discharge. However, the gels prepared in Examples 1 and 3 (containing 10% thermosensitive chitosan gel) of this invention showed no obvious redness, swelling, or lumps on the skin surface after injection, and the gel itself did not show yellowing or purulent discharge, indicating that it has excellent biocompatibility. It also showed obvious swelling and certain support. The good biocompatibility and slow degradation rate make this gel system a potential tissue filler material. At the same time, it has complete integration after 7 days and can be used for facial filling, which can be applied in the medical aesthetics field.

[0081] Example 7: In Vitro Engineered Tissue Grafts

[0082] Rabbit articular chondrocytes or bone marrow mesenchymal stem cells (BMSCs) were added to the high tissue fusion substrate gel prepared in step (1) of Example 1 before crosslinking. Then, DMTMM was added to carry out the crosslinking reaction. Cartilage induction medium was added, and after static culture for 3 days, the cells were transferred to a bioreactor for dynamic culture for 2 to 4 weeks. The cells proliferated in large quantities and secreted glycosaminoglycans and type II collagen, forming a dense white engineered cartilage tissue block in the scaffold.

[0083] Rabbits were anesthetized and their skin prepared. The knee joint cavity was opened, and a standard full-thickness cartilage defect was created using a chisel on the femoral trochlea. A pre-prepared mature cartilage tissue block was removed, trimmed to perfectly match the defect cavity, and embedded into the defect. The joint capsule and skin were then sutured layer by layer. Animals were euthanized in batches at 4, 8, and 12 weeks of routine feeding, and knee joint specimens were harvested, fixed, and decalcified. Gross appearance was then assessed. The assessment criteria are as follows: 1. Degree of filling defects (0–2 points) 2 points: The defect is completely filled and level with the surrounding normal cartilage; 1 point: The defect is partially filled and the depression is obvious; 0 points: There is almost no new tissue and the defect is severely depressed.

[0084] 2. Repairing tissue edge integration (0–2 points)

[0085] 2 points: The newly formed tissue is seamlessly integrated with the surrounding natural cartilage, and the boundary is blurred; 1 point: The boundary is clear, with slight cracks; 0 points: There is obvious separation and lifting, and the boundary cracks are large.

[0086] 3. Improve tissue surface smoothness (0–2 points)

[0087] 2 points: Smooth and flat surface, no cracks or fiber protrusions; 1 point: Rough surface, a few small cracks; 0 points: Numerous cracks, fibrosis, unevenness.

[0088] 4. Repairing tissue color (0–2 points)

[0089] 2 points: Milky white and translucent, consistent with the color of normal cartilage; 1 point: White / slightly yellow, with decreased transparency; 0 points: Cloudy white, obvious fibrosis, dark red granulation color.

[0090] 5. Improve tissue firmness (tactile sensation upon pressing, 0–2 points)

[0091] 2 points: Elasticity and hardness are close to normal cartilage; 1 point: Too soft or too hard, insufficient elasticity; 0 points: Soft and easily broken / Hard, fibrotic and inelastic.

[0092] The results showed that the samples of the present invention scored 2, 2, 1, 2, and 1 in five categories at 12 weeks, for a total of 8 points, indicating a good repair effect.

[0093] Example 8: Joint Lubricant

[0094] Application of bone and joint lubricant in the field of orthopedics: The high tissue fusion base gel and composite gel products prepared by the above-mentioned Example 1 step (1) and Example 3 (mixed with 10% hydroxyapatite) were used as test samples. The friction coefficient of the samples was detected by atomic force microscopy (AFM). The gels of Example 1 and Example 3 were dissolved in PBS to prepare a lubricating solution of 10.0 mg / ml. Sodium hyaluronate solution of the same concentration and hydroxyapatite microsphere lubricating solution of the same particle size and concentration (without free water on the surface) were used as control group 1. In AFM operation, a rectangular cantilever beam probe with 10.0 μm polystyrene microspheres is used. A polished silicon wafer fixed on a glass slide is fixed on an operating table equipped with a liquid tank. The contact mode is selected to test the friction force. After calibrating the probe using the thermal calibration method in atmospheric mode, the solution to be measured is injected into the liquid tank. The probe under the thermally calibrated liquid phase is used. The transverse force mode is used to apply a set load to the probe and perform friction test. The friction coefficient is calculated by the ratio of transverse force to load. The results are shown in Table 6 below.

[0095] Table 6

[0096] As can be seen from Table 6, both the base gel and the composite gel prepared by this invention have the effect of reducing friction, with a friction coefficient of less than 0.02, and have a good lubrication effect. After intra-articular injection, they can reduce the friction between damaged tissues and have good application in the field of orthopedics. In contrast, the friction coefficient of the control group is larger, which may be related to the integrability of this invention.

[0097] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A multi-scenario tissue engineering composite gel with high tissue integration and in-situ soft tissue regeneration, characterized in that: It includes a high tissue fusion substrate gel and a functional matrix component, wherein the functional matrix component is selected from one or more of microspheres, viscoelastic sodium hyaluronate gel, collagen gel, and thermosensitive gel; Both the high tissue fusion substrate gel and the composite gel meet the following physical properties: water contact angle of 20~70°; molar percentage of hydrophilic groups ≥3%, wherein the hydrophilic groups include one or more of hydroxyl, carboxyl, and amino groups; pore size inside the gel or at the gel interface ≥40μm; and elastic modulus of 1~1000Pa.

2. The multi-scenario tissue engineering composite gel with high tissue fusion and in-situ soft tissue regeneration according to claim 1, characterized in that: The microspheres include hydroxyapatite, polycaprolactone, polylactic acid, and agarose.

3. The multi-scenario tissue engineering composite gel with high tissue fusion and in-situ soft tissue regeneration according to claim 1, characterized in that: The functional matrix components are used to endow the composite gel with long-lasting filling, stable support, viscoelastic adaptation, temperature-responsive molding and basic tissue repair properties; the high tissue fusion base gel is used to improve the overall tissue affinity and interface integration ability of the composite gel.

4. The multi-scenario tissue engineering composite gel with high tissue fusion and in-situ soft tissue regeneration according to claim 1, characterized in that: The composite gel enables seamless interface fusion between the material and the host tissue, inducing in-situ soft tissue regeneration and forming a soft tissue mass 3-30 days after implantation.

5. The multi-scenario tissue engineering composite gel with high tissue fusion and in-situ soft tissue regeneration according to claim 4, characterized in that: The tensile fracture strength of the soft tissue can reach 0.1 kPa to 1000 kPa; when the compression ratio is 80%, the compressive strength can reach 0.1 kPa to 10000 kPa.

6. The multi-scenario tissue engineering composite gel with high tissue fusion and in-situ soft tissue regeneration according to claim 1, characterized in that: The mass fraction of the high tissue fusion substrate gel is 5% to 100%, and the mass fraction of the functional matrix component is 0% to 95%.

7. The multi-scenario tissue engineering composite gel with high tissue fusion and in-situ soft tissue regeneration according to claim 1, characterized in that: The viscoelastic sodium hyaluronate gel has a molecular weight of 100~3000kDa; the collagen gel is medical-grade purified collagen without immune rejection; and the phase transition temperature of the thermosensitive gel is 30~37℃.

8. The method for preparing the multi-scenario tissue engineering composite gel according to any one of claims 1-7, characterized in that, Includes the following steps: (1) Preparation of high tissue fusion substrate gel Sodium hyaluronate was prepared into a solution, and then arginine was added to the sodium hyaluronate and dissolved completely. After stirring evenly, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride was added, and the solution was allowed to stand and solidify for 2 days. Then, impurities were removed by dialysis with pure water. (2) The high tissue fusion substrate gel and the functional matrix components are thoroughly mixed to obtain a mixed gel system; the free water in the system is removed by a concentration process. The concentration method is one or a combination of vacuum filtration, vacuum drying, and polyethylene glycol osmotic pressure difference method to finally obtain a multi-scenario tissue engineering composite gel.

9. The method for preparing the multi-scenario tissue engineering composite gel according to claim 8, characterized in that: The functional matrix component is selected from one or more of microspheres, viscoelastic sodium hyaluronate gel, collagen gel, and thermosensitive gel.

10. The application of the multi-scenario tissue engineering composite gel according to any one of claims 1-7 in the preparation of urethral soft tissue regeneration filling materials, male erectile dysfunction soft tissue remodeling materials, medical aesthetic soft tissue remodeling materials, ophthalmic viscoelastic protective materials, in vitro tissue culture transplantation materials, cell-loaded cartilage repair materials, bone and joint lubrication materials, and drug-loaded osteoarthritis treatment drugs.

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