Tissue phase homogenizing agent as well as preparation method and application thereof

Abstract

The invention provides a tissue phase homogenizing agent and a preparation method and application thereof, and belongs to the technical field of biological tissue engineering, the homogenizing agent is composed of hydrophilic matrixes (glucose, polyglycerol, poloxamer F127 and polyvinyl alcohol), functional additives (mannose, N-acetylcysteine, sodium hyaluronate and polyethylene glycol) and ultrapure water, and the pH value is 7.0 + / -0.2. Hydrophilic groups and water molecules form a hydrogen bond network, a continuous hydration layer is constructed on a phase interface, phase domain fusion, refractive index homogenization and molecular ordering are achieved, the problem of biological tissue phase homogenization is solved, tissue light transmittance can be improved, the swelling ratio can be stabilized, and the hydrogel has cell proliferation promoting activity and is suitable for tissue transplantation operation and bioengineering tissue preservation.

Description

Technical Field

[0001] This invention relates to the field of bio-tissue engineering technology, specifically to a tissue homogenizer, its preparation method, and its application. Background Technology

[0002] The problem of phase homogeneity is common in biological tissues, which manifests as heterogeneous microstructure. This can lead to a series of adverse consequences: enhanced light scattering leads to decreased transparency, weakened mechanical properties, and hindered mass transfer, which seriously affects the application effect of tissue engineering materials. For example, in tissue transplantation, insufficient light transmittance or structural instability may lead to surgical failure, and in tissue preservation, tissue structure may be damaged and biological activity may be lost.

[0003] Therefore, a tissue homogenizer, its preparation method, and its application are proposed. Summary of the Invention

[0004] The present invention aims to solve the problems mentioned in the background art by providing a tissue homogenizer, its preparation method and application.

[0005] The specific technical solution is as follows:

[0006] A tissue homogenizer, comprising the following components in parts by weight:

[0007] Hydrophilic matrix: 5-60 parts glucose, 10-40 parts polyglycerol, 40 parts poloxamer F1275, 1-20 parts polyvinyl alcohol;

[0008] Functional additives: 0.1-2 parts mannose, 0.5-5 parts N-acetylcysteine, 0.01-1 parts sodium hyaluronate, 0.05-1 parts polyethylene glycol;

[0009] Solvent: Ultrapure water to bring the total to 100 parts;

[0010] The pH value of the tissue homogenizer is 7.0 ± 0.2.

[0011] The tissue homogenizer using the above-mentioned technical solution uses glucose, polyglycerol, poloxamer F127, and polyvinyl alcohol as hydrophilic matrices, combined with mannose, N-acetylcysteine, sodium hyaluronate, and polyethylene glycol as functional additives, and is prepared with ultrapure water to a pH of 7.0±0.2. The polyhydroxy components in the hydrophilic matrix form a hydrogen bond network with water molecules, constructing a continuous hydration layer at the tissue phase interface, reducing the interfacial energy between phases to promote phase domain fusion, and optimizing the refractive index matching to reduce light scattering. The functional additives synergistically enhance the stability of the hydration layer, protect the tissue microstructure and maintain the orderly arrangement of phase regions, thereby improving tissue homogeneity, increasing transparency, stabilizing the swelling state, and exhibiting good biocompatibility.

[0012] In the aforementioned tissue homogenizer, the content of glucose in the hydrophilic matrix is ​​10 to 40 parts.

[0013] Using the above technical solution, the glucose content in the hydrophilic matrix is ​​limited to 10-40 parts. Glucose within this range can provide a suitable density of hydroxyl groups, which ensures that it forms a sufficient hydrogen bond network with water molecules to effectively fill the interphase gaps in the tissue and promote mutual fusion, while avoiding overhydration due to excessive content. Thus, while improving tissue transparency, it maintains the stability of the swollen state, and the natural nutritional properties of glucose can help enhance the activity of tissue cells.

[0014] In the above-mentioned tissue homogenizer, the degree of polymerization of the polyglycerol is 5-8.

[0015] Using the above technical solution, the degree of polymerization of polyglycerol is limited to 5-8. Polyglycerol molecules with this degree of polymerization have a moderate branching structure and number of hydroxyl groups. They can form a hydrogen bond network with good permeability, penetrate into the micro gaps at the tissue phase interface to promote phase domain fusion, and enhance the interaction with tissue molecules through the branching structure, thereby improving the intermolecular forces, improving the orderly arrangement of phase regions, and enhancing the structural stability of the tissue.

[0016] In the above-mentioned tissue homogenizer, the critical micelle temperature of poloxamer F127 is 27-29°C; the molecular weight of polyvinyl alcohol is 28,000-32,000, and the degree of alcoholysis is ≤85%.

[0017] Using the above technical solution, the critical micelle temperature of poloxamer F127 is limited to 27-29℃, the molecular weight of polyvinyl alcohol is 28,000-32,000 and the degree of alcoholysis is ≤85%. At this critical micelle temperature, poloxamer F127 can form stable micelles, and the surface tension of hydrophobic microregions can be adjusted through the micelle structure to promote phase domain fusion. Polyvinyl alcohol with specific molecular weight and degree of alcoholysis can form a continuous thin film hydration layer, which enhances the intermolecular forces of the tissue to increase the content of α-helical structure. The two work synergistically to optimize the uniformity of the tissue phase and improve transparency and mechanical stability.

[0018] In the above-mentioned tissue homogenizer, the content of mannose in the functional additive is 0.5-1.5 parts, and the content of N-acetylcysteine ​​is 1-3 parts; the molecular weight of sodium hyaluronate is 1×106 Da, and the polyethylene glycol is PEG-4000; the weight ratio of the hydrophilic matrix to the functional additive is (8-12):1.

[0019] Using the above technical solution, the functional additives are limited to 0.5-1.5 parts mannose, 1-3 parts N-acetylcysteine, and sodium hyaluronate with a molecular weight of 1×10⁻⁶. 6Da, the polyethylene glycol is PEG-4000, and the weight ratio of hydrophilic matrix to functional additive is (8-12):1. The functional additive with this ratio and parameters can precisely assist the hydrophilic matrix: mannose enhances the stability of the hydration layer, N-acetylcysteine ​​provides antioxidant protection to protect the tissue structure, high molecular weight sodium hyaluronate provides spatial support, and PEG-4000 regulates the strength of interfacial interactions. Combined with a specific ratio of hydrophilic matrix, it synergistically improves the efficiency of hydrogen bond network, further optimizes tissue homogeneity, and enhances the cell proliferation effect.

[0020] The aforementioned tissue homogenizer, wherein the transmittance of the biological tissue treated with the homogenizer is ≥120% in the wavelength range of 380-800nm; and the swelling ratio of the biological tissue after treatment with the tissue homogenizer is 145% to 160%.

[0021] Using the above technical solution, the transmittance of biological tissues treated with homogenizers is limited to ≥120% (380-800nm), and the swelling ratio is 145%~160%. The limitation of transmittance reflects the significant improvement of tissue phase homogeneity and a substantial reduction in light scattering. The limitation of the swelling ratio indicates that during the hydration process, the tissue can both fill the interphase gaps through the hydration layer to improve transparency and avoid structural damage caused by excessive swelling, thereby maintaining structural stability while ensuring tissue function.

[0022] This invention also provides a method for preparing a tissue homogenizer, comprising the following steps:

[0023] (1) Prepare a 1% stock solution of sodium hyaluronate;

[0024] (2) Under the stirring conditions of 20-30℃ and 250-350rpm, add other components to ultrapure water in sequence and stir until completely dissolved;

[0025] (3) Adjust the pH to 7.0±0.2 using 0.05-0.15M NaOH solution;

[0026] (4) Add ultrapure water to bring the volume to 100ml, and filter through a 0.22μm cellulose acetate membrane for sterilization;

[0027] (5) Store at 4℃ in a sealed, light-protected environment;

[0028] In step (2), the stirring time is 30 to 60 minutes.

[0029] Using the above technical solution, the stirring temperature during preparation is specified as 20-30℃, the stirring speed as 250-350rpm, and the time as 30-60min. The pH is adjusted with 0.05-0.15M NaOH, and the mixture is sterilized by passing it through a 0.22μm cellulose acetate filter membrane and then sealed and stored at 4℃ in the dark. Precise stirring parameters ensure that all components are fully dissolved and form a homogeneous system, avoiding local concentration unevenness that could affect the construction of hydrogen bond networks. Appropriate pH adjustment and sterilization steps ensure the stability and sterility of the homogenizer. Sealing and storing at 4℃ in the dark can prevent component degradation or deterioration, thereby maintaining the activity of the homogenizer and ensuring its stable and lasting effect on regulating the homogeneity of tissue phases.

[0030] The application of tissue homogenizer in corneal tissue transplantation surgery in this embodiment.

[0031] Using the above technical solution, the homogenizer is applied to corneal tissue transplantation surgery. The cornea has extremely high requirements for transparency and structural stability. The homogenizer improves the uniformity of corneal tissue to increase light transmittance, stabilizes the swelling state to maintain the shape, and its good biocompatibility can reduce rejection after transplantation, promote the fusion of corneal tissue and recipient, and improve the success rate of transplantation.

[0032] The application of the tissue homogenizer in the preservation of bioengineered skin tissue in this embodiment is described. The bioengineered skin tissue is preserved in the homogenizer at 2-8°C for 7-14 days.

[0033] Using the above technical solution, the homogenizer is used for the preservation of bioengineered skin tissue. The preservation conditions are limited to 2-8℃ and a duration of 7-14 days. Under these conditions, the homogenizer can maintain the phase homogeneity of the skin tissue through a stable hydrogen bond network, inhibit excessive swelling or dehydration deformation of the tissue, and at the same time, the antioxidant and nutritional effects of the functional additives can protect the activity of skin cells, thereby prolonging the preservation time of the skin tissue, maintaining its structural and functional integrity, and meeting the needs of transplantation applications.

[0034] The application of the tissue homogenizer in the modification of cartilage tissue engineering materials in this embodiment includes increasing the α-helical structure content of the cartilage tissue material to more than 35%.

[0035] Using the above technical solution, a homogenizer is used to modify cartilage tissue engineering materials, limiting the α-helical structure content to over 35%. The increase in α-helical structure content indicates that the intermolecular forces of cartilage tissue are enhanced and the phase regions are arranged more orderly. The homogenizer enhances intermolecular interactions through hydrogen bond networks, improves the phase homogeneity of cartilage materials, and thus improves their mechanical properties and structural stability. At the same time, the proliferation-promoting effect can assist in the repair and regeneration of cartilage tissue.

[0036] The present invention has the following beneficial effects:

[0037] 1. Improve tissue homogeneity: Through hydrogen bond network construction and phase interface reconstruction, promote phase domain fusion, optimize refractive index matching, reduce light scattering, and improve tissue transparency;

[0038] 2. Stabilizes structural properties: Enhances intermolecular forces, maintains a suitable swelling state, avoids excessive swelling or structural deformation, and improves the mechanical stability of the tissue;

[0039] 3. Enhanced bioactivity: Functional additives work synergistically with the hydrophilic matrix to promote cell proliferation, and their good biocompatibility can reduce transplant rejection.

[0040] 4. Adaptable to multiple scenarios: It is suitable for various tissue engineering scenarios such as corneal transplantation, skin preservation, and cartilage material modification. It can specifically solve the phase homogeneity problem of different tissues and extend the effective service life of tissues. Attached Figure Description

[0041] Figure 1 A flowchart illustrating the preparation method of the tissue phase homogenizer provided in this embodiment of the invention;

[0042] Figure 2 The image shows the light transmittance curve of the tissue after treatment in Example 4 (H2O - tissue immersed in water, Tps - tissue grafts immersed in a phase homogenizer);

[0043] Figure 3 To compare the morphology of dry and wet tissue grafts, Case 4 was subjected to further experiments. Pig eyeballs were used to simulate the dynamic state of the human eye. The tissue was soaked in a homogenizing agent for 30 minutes and then the changes were observed.

[0044] Figure 4 To observe the tissue transparency after the tissue graft was placed in a pig eye for 0.5 hours and the changes after adding deionized water;

[0045] Figure 5 For cell experiments, live / dead staining and CCK8 (T- – tissue grafts were immersed in a homogenizing agent) bar chart were prepared.

[0046] Figure 6 These are before-and-after images showing the post-operative effects in animal experiments. Detailed Implementation

[0047] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0048] The accompanying drawings are for illustrative purposes only and are schematic diagrams, not actual images. They should not be construed as limiting the scope of this patent. To better illustrate the embodiments of the present invention, some parts in the drawings may be omitted, enlarged, or reduced, and do not represent the actual dimensions of the product. It is understandable to those skilled in the art that some well-known structures and their descriptions may be omitted in the drawings.

[0049] In the accompanying drawings of the embodiments of the present invention, the same or similar reference numerals correspond to the same or similar components. In the description of the present invention, it should be understood that if terms such as "upper," "lower," "left," "right," "inner," and "outer" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, they are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, the terms used to describe positional relationships in the drawings are only for illustrative purposes and should not be construed as limiting the present patent. For those skilled in the art, the specific meaning of the above terms can be understood according to the specific circumstances.

[0050] In the description of this invention, unless otherwise explicitly specified and limited, the term "connection" or similar designation indicating a connection between components should be interpreted broadly. For example, it can refer to a fixed connection, a detachable connection, or an integral part; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium; it can refer to the internal communication between two components or the interaction between two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0051] See attached document Figure 1-6 This specific implementation provides the following four embodiments, as detailed below:

[0052] Example 1 (Glucose-containing tissue homogenizer)

[0053] Specific composition (parts by weight):

[0054] Hydrophilic matrix: 30 parts glucose

[0055] Functional additives: 1.2 parts mannose, 1.0 part N-acetylcysteine, sodium hyaluronate (molecular weight 1×10⁻⁶) 6 0.6 parts of Da and 0.5 parts of polyethylene glycol (PEG-4000).

[0056] Solvent: Ultrapure water to bring the total to 100 parts.

[0057] pH value: 7.0±0.1

[0058] Preparation process details:

[0059] 1. Preparation of sodium hyaluronate stock solution: Accurately weigh 0.6g of sodium hyaluronate, add 59.4mL of ultrapure water, and dissolve it for 2 hours under magnetic stirring at 25℃ and 200rpm to prepare a 1% (w / v) stock solution for later use.

[0060] 2. Mixing and dissolving: In a sterile operating table, add 30g glucose, 1.2g mannose and 1.0g N-acetylcysteine ​​to 50mL of ultrapure water in sequence, and stir magnetically at 300rpm for 15 minutes until completely dissolved; then slowly add 60mL of sodium hyaluronate stock solution (containing 0.6g sodium hyaluronate) and 0.5g polyethylene glycol, and continue stirring for 30 minutes (25℃) to form a homogeneous solution.

[0061] 3. pH adjustment: Slowly add 0.1M NaOH solution to adjust the pH, stirring for 3 minutes after each addition. Repeat the operation until the pH stabilizes at 7.0±0.1.

[0062] 4. Volume adjustment and sterilization: Add ultrapure water to 100 mL and stir for 5 minutes to mix well; sterilize by negative pressure filtration using a 0.22 μm cellulose acetate membrane (25 mm in diameter) and collect the filtrate.

[0063] 5. Storage conditions: Dispense into sterile glass reagent bottles, seal and store at 4°C protected from light. Shelf life is 6 months.

[0064] Performance characteristics: The swelling ratio of the treated tissue grafts was 168.4%, the transmittance at 550nm wavelength was 110.2%, and it had a significant proliferative effect on corneal epithelial cells (the OD value detected by CCK-8 was 18% higher than that of the blank group).

[0065] The formulation consists of glucose as the core of a hydrophilic matrix, combined with mannose, N-acetylcysteine, sodium hyaluronate and polyethylene glycol, and prepared into a homogenizer with a pH of 7.0±0.1 using ultrapure water.

[0066] Working principle: Glucose molecules contain multiple hydroxyl groups (-OH), which form a dense hydrogen bond network with water molecules. They function through a "hydrogen bond-mediated phase interface reconstruction" mechanism: on one hand, the hydroxyl groups interact with hydrophobic microdomains in the tissue, reducing interfacial energy and promoting phase domain fusion; on the other hand, glucose hydration fills the interphase gaps in the tissue, optimizing refractive index matching and reducing light scattering. Simultaneously, mannose in the functional additives helps enhance the stability of the hydration layer, N-acetylcysteine ​​protects the tissue microstructure through its antioxidant effect, and sodium hyaluronate maintains the ordered arrangement of phase regions through the spatial support of its polymer chains.

[0067] Technical effects: Through the strong hydrophilicity of glucose and the synergistic effect of functional additives, the uniformity of tissue microstructure can be improved and the transparency of tissue can be enhanced. At the same time, as a natural sugar, glucose can provide nutritional support for tissue cells, and with the protective effect of N-acetylcysteine, it can promote cell proliferation. In addition, the stable structure of the hydrogen bond network can inhibit excessive swelling of tissue and maintain an appropriate swelling state.

[0068] Experimental data: The swelling ratio of the treated tissue grafts was 168.4%, the transparency was 110.2%, and cell viability testing showed that it had a proliferative effect.

[0069] Example 2 (Texture homogenizer containing polyvinyl alcohol)

[0070] Specific composition (parts by weight):

[0071] Hydrophilic matrix: 5 parts polyvinyl alcohol (Mw 30,000, degree of alcoholysis 88%)

[0072] Functional additives: 1.2 parts mannose, 1.0 part N-acetylcysteine, sodium hyaluronate (molecular weight 1×10⁻⁶) 6 0.6 parts of Da and 0.5 parts of polyethylene glycol (PEG-4000).

[0073] Solvent: Ultrapure water to bring the total to 100 parts.

[0074] pH value: 7.0±0.1

[0075] Preparation process details:

[0076] 1. Preparation of sodium hyaluronate stock solution: Same as in Example 1.

[0077] 2. Polyvinyl alcohol pretreatment: Weigh 5g of polyvinyl alcohol, add 30mL of ultrapure water, heat in a 60℃ water bath and stir (200rpm) until completely dissolved, then cool to 25℃ for later use.

[0078] 3. Mixing and dissolving: Under sterile conditions, the above polyvinyl alcohol solution, 1.2 g mannose, 1.0 g N-acetylcysteine, 60 mL sodium hyaluronate stock solution, and 0.5 g polyethylene glycol were added sequentially to the remaining ultrapure water and stirred at 300 rpm for 45 minutes until a transparent solution was formed.

[0079] 4. pH adjustment and subsequent treatment: Same as in Example 1, with the final pH controlled at 7.0±0.1.

[0080] Performance characteristics: The swelling ratio of the treated tissue grafts is 154.3%, the transmittance at 550nm wavelength is 114.5%, and the content of α-helical structure is increased by 22% compared with the untreated group. It is suitable for tissue engineering materials that require enhanced mechanical stability.

[0081] The formulation consists of polyvinyl alcohol (Mw30,000, degree of hydrolysis 88%) as the hydrophilic matrix, combined with functional additives of the same type and proportion as in Example 1, and ultrapure water as the solvent, with a pH of 7.0 ± 0.1.

[0082] Working principle: The hydroxyl groups on the polyvinyl alcohol (PVA) molecular chain can form stable hydrogen bonds, and its linear polymer structure can form a continuous thin-film hydration layer at the tissue phase interface, exerting its effect through three-fold regulation: First, the entanglement of the polymer chains enhances the intermolecular forces in the tissue, increases the content of α-helical structures, and improves the orderly arrangement of phase regions; second, the hydration layer fills the interphase gaps, reduces refractive index differences, and reduces light scattering; third, the film-forming properties of PVA coat the tissue surface, inhibiting excessive swelling. In the functional additives, sodium hyaluronate forms a synergistic network with PVA, further stabilizing the phase interface structure, while N-acetylcysteine ​​reduces the damage of free radicals to the tissue structure.

[0083] Technical effects: The high molecular weight properties of polyvinyl alcohol provide stronger mechanical support to tissues. Combined with the synergistic effect of functional additives, it can improve the mechanical stability of tissues. At the same time, the optimization of the hydration layer improves tissue transparency. Furthermore, the biocompatibility of polyvinyl alcohol combined with the nutritional support of functional additives ensures the bioactivity of tissues, making it suitable for tissue engineering scenarios with high requirements for mechanical performance.

[0084] Experimental data: The swelling ratio of the treated tissue grafts was 154.3%, the transparency was 114.5%, the content of α-helical structures was increased compared with the untreated group, and cell activity detection showed that it had a proliferative effect.

[0085] Example 3 (Tissue homogenizer containing poloxamer F127)

[0086] Specific composition (parts by weight):

[0087] Hydrophilic matrix: 20 parts of poloxamer F127 (critical micelle temperature 28℃)

[0088] Functional additives: 0.6 parts mannose, 0.5 parts N-acetylcysteine, sodium hyaluronate (molecular weight 1×10⁻⁶) 6 0.3 parts of Da and 0.25 parts of polyethylene glycol (PEG-4000).

[0089] Solvent: Ultrapure water to bring the total to 100 parts.

[0090] pH value: 7.0±0.2

[0091] Preparation process details:

[0092] 1. Preparation of sodium hyaluronate stock solution: Weigh 0.3g of sodium hyaluronate, add 29.7mL of ultrapure water, and stir at 25℃ for 1.5 hours to prepare a 1% stock solution.

[0093] 2. Mixing and dissolving: In a 28°C constant temperature water bath, add 20g of poloxamer F127 to 60mL of ultrapure water and stir at 300rpm for 30 minutes until dissolved; then add 0.6g of mannose, 0.5g of N-acetylcysteine, 30mL of sodium hyaluronate stock solution, and 0.25g of polyethylene glycol in sequence, and continue stirring for 20 minutes until homogeneous.

[0094] 3. pH adjustment: Adjust the pH to 7.0±0.2 with 0.1M NaOH. After each adjustment, let it stand for 2 minutes and then retest.

[0095] 4. Volume adjustment and sterilization: Add ultrapure water to 100 mL, filter through a 0.22 μm filter membrane, and store in a sealed container at 4 °C.

[0096] Performance characteristics: The swelling ratio of the treated tissue graft is 148.1%, the average transmittance in the wavelength range of 380-800nm ​​is 118.7%, and in the rabbit corneal transplantation model, the transmittance retention rate is over 90% 14 days after surgery.

[0097] The formulation consists of a homogenizer with pH 7.0±0.2, prepared by using poloxamer F127 (critical micelle temperature 28℃) as the hydrophilic matrix, combined with low proportions of mannose, N-acetylcysteine, sodium hyaluronate and polyethylene glycol, and ultrapure water.

[0098] Working Principle: Poloxamer F127 is a nonionic block copolymer that forms a micelle structure near its critical micelle temperature. Its hydrophilic segments form hydrogen bonds with water molecules, while its hydrophobic segments interact with hydrophobic regions in the tissue, functioning through a "micelle-mediated phase fusion" mechanism: the micelle structure penetrates to the tissue phase interface, reducing the surface tension of hydrophobic microdomains and promoting a reduction in phase domain size. Simultaneously, the hydrated layer of the micelles is uniformly distributed in the interphase spaces, optimizing refractive index uniformity and reducing light scattering. Among the functional additives, polyethylene glycol helps regulate micelle size, and sodium hyaluronate enhances the adhesion of the hydrated layer, making the phase interface reconstruction effect more durable.

[0099] Technical effects: The thermosensitive micelle structure of poloxamer F127 can adaptively adjust the intensity of action according to the ambient temperature. It can stably exert a phase homogenization effect at physiological temperature, which significantly improves tissue transparency. At the same time, the encapsulation effect of micelles inhibits excessive tissue swelling. With the synergistic effect of functional additives, it can maintain the structural stability of tissues for a long time, making it suitable for tissue scenarios that require long-term preservation or transplantation.

[0100] Experimental data: The swelling ratio of the treated tissue grafts was 148.1%, and the transparency in the 380-800nm ​​wavelength range was better than that of the control group. The transparency remained good 14 days after the operation in the rabbit corneal transplantation model.

[0101] Example 4 (A tissue homogenizer containing polyglycerol)

[0102] Specific composition (parts by weight):

[0103] Hydrophilic matrix: 15 parts polyglycerol (degree of polymerization 3)

[0104] Functional additives: 0.6 parts mannose, 0.5 parts N-acetylcysteine, sodium hyaluronate (molecular weight 1×10⁻⁶) 6 0.3 parts of Da and 0.25 parts of polyethylene glycol (PEG-4000).

[0105] Solvent: Ultrapure water to bring the total to 100 parts.

[0106] pH value: 7.0±0.1

[0107] Preparation process details:

[0108] 1. Preparation of sodium hyaluronate stock solution: Same as in Example 3.

[0109] 2. Mixing and dissolving: At 25°C, add 15g of polyglycerol (degree of polymerization 3) to 50mL of ultrapure water and stir at 250rpm for 10 minutes; then add 0.6g of mannose, 0.5g of N-acetylcysteine, 30mL of sodium hyaluronate stock solution, and 0.25g of polyethylene glycol in sequence, and continue stirring for 30 minutes until completely dissolved (the solution is clear and free of particles).

[0110] 3. pH adjustment: Finely adjust the pH to 7.0±0.1 with 0.1M NaOH, with each addition not exceeding 0.1mL to avoid localized over-alkalinity.

[0111] 4. Volume adjustment and storage: After adjusting the volume to 100mL, filter to sterilize and store at 4℃ away from light. Before use, allow to return to room temperature (25℃) and shake gently.

[0112] Performance characteristics: The swelling ratio of the treated tissue grafts was 144.7%, and the transmittance at 800 nm wavelength reached 126.3% (significantly higher than the control group, p<0.01). The pig eye simulation experiment showed that the transparency of the tissue grafts did not decrease significantly within 30 minutes in a moist environment in the eye, and the proliferation rate of fibroblasts reached 23% (detected by CCK-8 method).

[0113] The formulation consists of a homogenizer with pH 7.0 ± 0.1 prepared by using polyglycerol (degree of polymerization 3) as the hydrophilic matrix, combined with functional additives of the same type and proportion as in Example 3, and ultrapure water.

[0114] Working principle: Polyglycerol (degree of polymerization 3) molecules contain multiple hydroxyl groups and exhibit a branched structure. The hydrogen bond network formed with water molecules has higher flexibility and permeability, allowing it to penetrate deep into the tiny gaps at the tissue phase interface. It functions through a "branched hydrogen bond network reconstruction" mechanism: the hydroxyl groups in the branched structure can simultaneously bind with multiple water molecules to form a three-dimensional hydration network, efficiently filling phase gaps and promoting phase domain fusion. Simultaneously, the stronger interaction between the branched structure and tissue molecules enhances intermolecular forces, increases the content of α-helical structures, and improves the ordered arrangement of phase regions. Among the functional additives, mannose helps enhance the stability of the hydration network, N-acetylcysteine ​​reduces oxidative damage, and sodium hyaluronate further optimizes the phase structure through the spatial support of the polymer chain.

[0115] Technical effects: The branched structure of polyglycerol makes its hydration efficiency higher, which can more thoroughly improve tissue homogeneity and thus significantly improve tissue transparency; the stability of the branched hydrogen bond network can effectively control tissue swelling and maintain a suitable swelling state; at the same time, the combination of the biocompatibility of polyglycerol and the proliferation-promoting effect of functional additives can enhance the cell activity of tissues, making it suitable for tissue transplantation scenarios with high requirements for transparency and bioactivity.

[0116] Experimental data: The swelling ratio of the treated tissue grafts was 144.7%, and the transparency was significantly better than that of the control group (p<0.01). In the pig eye simulation experiment, the tissue transparency remained stable in a humid environment, and cell experiments showed that it had a proliferative effect.

[0117] Among them, the phase homogenization regulating factor Φ in the tissue phase homogenizer satisfies:

[0118]

[0119] in:

[0120] k: Tissue type constant (1.0 for corneal tissue, 0.8 for skin tissue, and 1.2 for cartilage tissue);

[0121] α: Hydrophilic matrix hydrogen bond donor density coefficient (glucose: 0.35, polyglycerol: 0.42, poloxamer F127: 0.28, polyvinyl alcohol: 0.31);

[0122] [HM]: Total concentration of hydrophilic matrix (g / L);

[0123] β: Synergistic coefficient of functional additives (mannose: 0.15, N-acetylcysteine: 0.22, sodium hyaluronate: 0.18, polyethylene glycol: 0.12);

[0124] [FA]: Total concentration of functional additives (g / L);

[0125] c0: Reference concentration (10 g / L);

[0126] E a Activation energy of hydrogen bond network (4.2 kJ / mol);

[0127] R: gas constant (8.314 J / mol·K);

[0128] T: Processing temperature (K);

[0129] When Φ≥0.75, the tissue transmittance increases by ≥20%, and the α-helical structure content is ≥35%.

[0130] Example:

[0131] Calculation of homogenizer for corneal transplantation (Formulation of Example 1):

[0132] Total concentration of hydrophilic matrix [HM] = 30 g / L (glucose)

[0133] Total concentration of functional additives [FA] = 3.3 g / L

[0134] With α (glucose) = 0.35, β (mean) = 0.17, and T = 298K,

[0135] Substitute into the equation:

[0136]

[0137] Results verification: The measured transmittance was 110.2% (an increase of 22.2%), which is consistent with the prediction of Φ≥0.75.

[0138] Technical effect

[0139] 1. Precise regulation:

[0140] The Φ value quantifies the efficiency of the hydrogen bond network; when Φ ≥ 0.75, light scattering decreases by >40%.

[0141] Guiding formulation optimization: For example, increasing the polyglycerol concentration can increase α and Φ value growth rate by 30% (vs. glucose).

[0142] 2. Temperature adaptability:

[0143] The exponential term ensures that the Φ value fluctuates by <5% when stored at 2-8℃ (T=275-281K), maintaining the stability of the swelling ratio.

[0144] 3. Component synergy:

[0145] The logarithmic term ln(1+[HM] / c0) inhibits excessive hydration at high concentrations, preventing a swelling ratio >160%.

[0146] Working principle and process

[0147] Step 1: Hydrogen bond network generation

[0148] The hydrophilic matrix ([HM]) forms a three-dimensional network with water molecules through hydroxyl groups, and the network density is controlled by α·ln(1+[HM] / c0) → filling the interphase gaps in the tissue.

[0149] Step 2: Synergistic effect of functional additives

[0150] Mannose / sodium hyaluronate (β term) enhances network stability;

[0151] N-acetylcysteine ​​decreases E a This makes hydrogen bonds easier to form (experimentally measured E). a (Reduced by 15%)

[0152] Step 3: Interface Reconstruction

[0153] When T≈28℃ (poloxamer critical temperature): micelle permeability increases, and hydrophobic micro-region fusion efficiency increases;

[0154] When T = 4℃ (storage temperature): network rigidity increases, suppressing structural deformation.

[0155] Swelling performance test: Tissue grafts were immersed in the solutions of each example for 30 min, and the swelling ratio was measured. The results are shown in Table 1:

[0156] Element Example 1 Example 2 Example 3 Example 4 Comparative Example 1 glucose 30 0 0 0 0 Polyvinyl alcohol 0 5 0 0 0 Polyether F127 0 0 20 0 0 Polyglycerol 0 0 0 15 0 Mannose 1.2 1.2 0.6 0.6 0 N-acetylcysteine 1 1 0.5 0.5 0 Sodium hyaluronate 0.6 0.6 0.3 0.3 0 polyethylene glycol 0.5 0.5 0.25 0.25 0 Ultrapure water margin margin margin margin 100 swelling ratio 168.4％ 154.3％ 148.1％ 144.7％ 231.3％ Cell activity Promote proliferation Promote proliferation Non-toxic Non-toxic Non-toxic transparency 110.2％ 114.5％ 118.7％ 126.3％ 100％

[0157] Note: All experimental data are the mean ± standard deviation of three independent experiments. Statistical analysis was performed using the t-test, and p < 0.05 was considered statistically significant.

[0158] In summary, the tissue homogenizer, its preparation method, and its application provided in this embodiment have the following advantages:

[0159] 1. Improve tissue homogeneity: Through hydrogen bond network construction and phase interface reconstruction, promote phase domain fusion, optimize refractive index matching, reduce light scattering, and improve tissue transparency;

[0160] 2. Stabilizes structural properties: Enhances intermolecular forces, maintains a suitable swelling state, avoids excessive swelling or structural deformation, and improves the mechanical stability of the tissue;

[0161] 3. Enhanced bioactivity: Functional additives work synergistically with the hydrophilic matrix to promote cell proliferation, and their good biocompatibility can reduce transplant rejection.

[0162] 4. Adaptable to multiple scenarios: It is suitable for various tissue engineering scenarios such as corneal transplantation, skin preservation, and cartilage material modification. It can specifically solve the phase homogeneity problem of different tissues and extend the effective service life of tissues.

[0163] Working principle

[0164] The phase homogenizer functions based on a "hydrogen bond-mediated phase interface reconstruction" mechanism:

[0165] The hydrophilic matrix (glucose, polyglycerol, poloxamer F127, polyvinyl alcohol) contains a large number of hydrophilic groups (-OH), which form a stable hydrogen bond network with water molecules and construct a continuous hydration layer at the tissue interface.

[0166] Functional additives (mannose, N-acetylcysteine, sodium hyaluronate, etc.) work synergistically. Mannose enhances the stability of the hydration layer, N-acetylcysteine ​​protects the tissue structure through antioxidant effects, and sodium hyaluronate supports the phase region arrangement with its polymer chains, together achieving triple regulation.

[0167] 1. Reduce interfacial energy between phases, promote the fusion of hydrophobic / hydrophilic microdomains, and reduce the size of phase domains;

[0168] 2. Fill the phase gaps to optimize refractive index matching and reduce light scattering;

[0169] 3. Enhances intermolecular forces, increases the content of α-helical structures, and improves the orderly arrangement of phase regions.

[0170] How to use

[0171] 1. Preparation:

[0172] Prepare a 1% stock solution of sodium hyaluronate;

[0173] Under stirring conditions of 20-30℃ and 250-350rpm, other components are added sequentially to ultrapure water and stirred for 30-60 minutes until completely dissolved;

[0174] Adjust the pH to 7.0±0.2 with 0.05-0.15M NaOH, and add ultrapure water to bring the volume to 100ml;

[0175] Sterilize by filtration through a 0.22μm cellulose acetate membrane and store in a sealed container at 4℃ away from light.

[0176] 2. Application Scenarios:

[0177] In corneal tissue transplantation surgery, it is used to process the transplanted corneal tissue;

[0178] When preserving bioengineered skin tissue, the tissue is placed in a homogenizer and stored at 2-8℃ for 7-14 days;

[0179] When modifying cartilage tissue engineering materials, immersion treatment is used to improve material properties.

[0180] The above are merely preferred embodiments of the present invention and are not intended to limit the implementation methods and protection scope of the present invention. Those skilled in the art should recognize that any equivalent substitutions and obvious changes made based on the description and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. A tissue homogenizer, characterized in that, Composed of the following components in parts by weight: Hydrophilic matrix: 5-60 parts glucose, 10-40 parts polyglycerol, 40 parts poloxamer F1275, 1-20 parts polyvinyl alcohol; Functional additives: 0.1-2 parts mannose, 0.5-5 parts N-acetylcysteine, 0.01-1 parts sodium hyaluronate, 0.05-1 parts polyethylene glycol; Solvent: Ultrapure water to bring the total to 100 parts; The pH value of the tissue homogenizer is 7.0 ± 0.

2.

2. The tissue homogenizer according to claim 1, characterized in that, The hydrophilic matrix contains 10 to 40 parts of glucose.

3. The tissue homogenizer according to claim 1, characterized in that, The degree of polymerization of the polyglycerol is 5-8.

4. The tissue homogenizer according to claim 1, characterized in that, The critical micelle temperature of the poloxamer F127 is 27-29°C; the molecular weight of the polyvinyl alcohol is 28,000-32,000, and the degree of alcoholysis is ≤85%.

5. The tissue homogenizer according to claim 1, characterized in that, The functional additive contains 0.5 to 1.5 parts of mannose and 1 to 3 parts of N-acetylcysteine; the sodium hyaluronate has a molecular weight of 1×106 Da and the polyethylene glycol is PEG-4000; the weight ratio of the hydrophilic matrix to the functional additive is (8-12):

1.

6. The tissue homogenizer according to claim 1, characterized in that, The transmittance of biological tissue treated with the homogenizing agent is ≥120% in the wavelength range of 380-800nm; the swelling ratio of biological tissue treated with the tissue homogenizing agent is 145%~160%.

7. The method for preparing the tissue homogenizer according to any one of claims 1-6, characterized in that, Includes the following steps: (1) Prepare a 1% stock solution of sodium hyaluronate; (2) Under the stirring conditions of 20-30℃ and 250-350rpm, add other components to ultrapure water in sequence and stir until completely dissolved; (3) Adjust the pH to 7.0±0.2 using 0.05-0.15M NaOH solution; (4) Add ultrapure water to bring the volume to 100ml, and filter through a 0.22μm cellulose acetate membrane for sterilization; (5) Store at 4℃ in a sealed, light-protected environment; In step (2), the stirring time is 30 to 60 minutes.

8. The use of the tissue homogenizer according to any one of claims 1-6 in corneal tissue transplantation surgery.

9. The application of the tissue homogenizer according to any one of claims 1-6 in the preservation of bioengineered skin tissue, characterized in that, The bioengineered skin tissue was stored in the homogenizer at 2-8°C for 7-14 days.

10. The application of the tissue homogenizer according to any one of claims 1-6 in the modification of cartilage tissue engineering materials, characterized in that, The modification includes increasing the α-helical structure content of cartilage tissue material to over 35%.

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