A preparation method of an exosome loaded photocrosslinked collagen hydrogel with hypoxic pretreatment

Abstract

This invention provides a method for preparing a hypoxia-pretreated exosome-loaded photocrosslinked collagen hydrogel, belonging to the field of exosome loading technology. This invention obtains highly active exosomes by hypoxia-pretreated bone marrow mesenchymal stem cells, prepares a photocrosslinking precursor by chemically modifying collagen with methacrylamide, constructs a positively charged fiber scaffold as a fixation substrate using electrospinning technology, breaks up exosome aggregation using ultrasonic dispersion technology, and achieves three-dimensional uniform distribution by magnetic field-assisted mixing. Key process parameters such as ultrasonic power, magnetic field strength, and mixing time are optimized through a game theory model. Layered photocrosslinking technology combined with an optical fiber transmission system ensures the uniformity and consistency of overall crosslinking. Finally, product quality is controlled by detecting the exosome distribution deviation coefficient, thus solving the technical problem of uneven exosome distribution in collagen hydrogels.

Description

Technical Field

[0001] This invention belongs to the field of exosome loading technology, and more specifically, relates to a method for preparing exosome-loaded photocrosslinked collagen hydrogels under hypoxia pretreatment. Background Technology

[0002] In the fields of tissue engineering and regenerative medicine, exosome-loaded collagen hydrogels are widely used as bioscaffold materials in treatment scenarios such as wound repair, bone defect filling, cartilage regeneration, and vascular reconstruction. Traditional preparation methods mainly employ simple physical mixing or direct addition techniques to combine exosomes with collagen precursor solutions, followed by chemical cross-linking or physical gelation to form a three-dimensional scaffold structure. However, existing preparation processes generally suffer from technical defects during the mixing of exosomes and the collagen matrix. Because exosomes themselves have nanoscale dimensions and negatively charged surfaces, they tend to aggregate easily. At the same time, the entanglement and cross-linking reactions of collagen molecular chains hinder the free diffusion of exosomes, leading to problems such as local aggregation, uneven regional distribution, and unbalanced gradient distribution of exosomes in the hydrogel matrix. This uneven distribution seriously affects the uniformity of the biological function of the hydrogel scaffold and the predictability of the therapeutic effect. In current exosome-loaded hydrogel preparation techniques, due to the lack of effective dispersion control methods and systematic process optimization approaches, exosomes often settle and aggregate under gravity, or are mechanically encapsulated by collagen fiber networks during cross-linking, forming irregular distribution patterns. This makes it impossible to achieve uniform distribution and stable fixation in three-dimensional space. In other words, existing technologies suffer from the technical problem of uneven distribution of exosomes within collagen hydrogels. Summary of the Invention

[0003] In view of this, the present invention provides a method for preparing exosome-loaded photocrosslinked collagen hydrogels under hypoxia pretreatment, which can solve the technical problem of uneven distribution of exosomes in collagen hydrogels in the prior art.

[0004] This invention is achieved as follows: A method for preparing a hypoxia-pretreated exosome-loaded photocrosslinked collagen hydrogel is provided. Bone marrow mesenchymal stem cells are cultured to confluence and then placed in a hypoxic environment for hypoxia pretreatment. The cell culture supernatant is collected. The collected cell culture supernatant is centrifuged to remove cells and cell debris, and then ultracentrifuged to collect the precipitate to obtain hypoxia-pretreated enhanced exosomes. Type I collagen is dissolved in phosphate buffer solution to prepare a collagen solution, which is then reacted with methacrylic anhydride, dialyzed, and freeze-dried to obtain a collagen methacrylamide precursor. A positively charged collagen fiber scaffold is prepared using electrospinning pretreatment technology. The collagen methacrylamide precursor is redissolved and reacted with photocrosslinked exosomes. After uniformly mixing the exosomes to form a pregel solution, an anaerobic pretreatment was added to enhance the exosomes. Ultrasonic dispersion and magnetic field-assisted mixing techniques were used to ensure uniform dispersion of the exosomes. The pregel solution loaded with exosomes was transferred to an electrospinning scaffold and layered photocrosslinking technology combined with an optical fiber transmission system was used for layered irradiation. The degree of hydrogel crosslinking and the uniformity of exosome distribution were detected until the standard requirements were met to complete the preparation. Ultrasonic dispersion technology was used to break up the exosome aggregates to achieve a monodisperse state. Magnetic field-assisted mixing technology generated three-dimensional eddy current motion to promote full mixing of exosomes and collagen precursor solutions. The positively charged fiber scaffold prepared by electrospinning formed a stable electrostatic adsorption with the negative charge on the surface of the exosomes to prevent re-aggregation.

[0005] Specifically, the hypoxia pretreatment step involves placing the cells in a hypoxic environment containing 2% oxygen and 5% carbon dioxide and culturing them at 37°C for 24 hours to activate the intracellular hypoxia response pathway and promote the secretion of exosomes with stronger biological activity.

[0006] The hypoxia pretreatment-enhanced exosomes refer to exosomes secreted by stem cells cultured in a hypoxic environment, which have a stronger ability to promote matrix synthesis and resist apoptosis compared to exosomes under normoxic conditions.

[0007] Specifically, the step of sequential centrifugation involves centrifuging the collected cell culture supernatant sequentially at 300g for 10 minutes, 2000g for 10 minutes, and 10000g for 30 minutes to remove cells and cell debris, and then repeating the process twice at 100000g for 70 minutes.

[0008] The collagen methacrylamide precursor refers to a polymer precursor obtained by modifying type I collagen with methacrylic anhydride, which has the characteristic of undergoing free radical polymerization under the action of a photoinitiator to form a three-dimensional network structure.

[0009] Specifically, the electrospinning pretreatment process involves using a high-voltage electrostatic field to stretch the polymer solution into nanofibers and collect them to form a fiber scaffold. By adjusting the spinning voltage to 15kV and the collection distance to 12cm, a uniform fiber network structure with a fiber diameter of 500nm to 800nm ​​is formed.

[0010] Specifically, the steps for detecting the degree of crosslinking of the hydrogel and the uniformity of exosome distribution are completed when the degree of crosslinking reaches 85% or more and the exosome distribution deviation coefficient is less than 15%; otherwise, the concentration gradient distribution of the photoinitiator is adjusted and the layered photocrosslinking steps are repeated until the standard requirements are met.

[0011] The exosome distribution deviation coefficient refers to the ratio of the standard deviation of the exosome distribution density in the hydrogel to the average distribution density, and is used to evaluate the uniformity of exosome distribution.

[0012] The step of adding hypoxia pretreatment to enhance exosomes also includes a step of optimizing the mixing process parameters using a game model. The optimal combination of ultrasonic power, magnetic field strength, and mixing time parameters is determined through a game process between the upper and lower models.

[0013] The game theory model includes an upper-level model that aims to maximize exosome dispersion efficiency and a lower-level model that aims to minimize exosome activity loss.

[0014] The constraint condition of the game theory model is ultrasonic power. ∈[50, 200]W, magnetic field strength ∈[0.1, 0.8]T, mixing time ∈[60, 300]s.

[0015] The layered photocrosslinking technology refers to a method of dividing a thick hydrogel into multiple thin layers and performing photocrosslinking sequentially, ensuring overall crosslinking uniformity by controlling the thickness of each layer and the irradiation time.

[0016] The fiber optic transmission system refers to a device that uses optical fibers to guide a light source into different depths inside the hydrogel for irradiation. It uses 405nm blue light to irradiate each layer for 30 seconds, while a rotating irradiation device is used to ensure uniform illumination in all directions.

[0017] The ultrasonic dispersion technology refers to a method that uses the cavitation effect and mechanical vibration of ultrasound to disrupt the aggregation of exosomes and promote their uniform dispersion in solution.

[0018] The degree of crosslinking refers to the percentage of monomers that have undergone crosslinking reactions in the hydrogel, and is used to characterize the completeness of crosslinking in the hydrogel.

[0019] The magnetic field-assisted mixing technology refers to the technique of using a magnetic stirrer to generate eddy currents under the action of a magnetic field to promote thorough mixing of exosomes and pregelation solutions.

[0020] The photoinitiator concentration gradient distribution refers to a method of setting different concentrations of photoinitiator at different depths of the hydrogel to compensate for light intensity attenuation and ensure uniform crosslinking.

[0021] This invention effectively solves the key technical challenge of uneven exosome distribution in collagen hydrogels by employing a comprehensive technical solution: ultrasonic dispersion to disrupt exosome aggregates, magnetic field-assisted mixing to promote three-dimensional uniform distribution, and electrospinning to prepare positively charged fiber scaffolds for stable fixation. The invention innovatively introduces a game theory model to optimize mixing process parameters. By establishing a mathematical relationship between maximizing exosome dispersion efficiency and minimizing activity loss, it achieves precise control of key parameters such as ultrasonic power, magnetic field strength, and mixing time, ensuring that exosomes achieve an ideal uniform distribution throughout the gel system. Simultaneously, layered photocrosslinking technology combined with an optical fiber transmission system ensures consistent crosslinking at different depths, further stabilizing the spatial distribution pattern of exosomes. In summary, this invention solves the technical problem of uneven exosome distribution in collagen hydrogels mentioned in the background art. Attached Figure Description

[0022] Figure 1 This is a flowchart of the method of the present invention.

[0023] Figure 2 The graph shows the relationship between exosome dispersion efficiency and process parameters in the example.

[0024] Figure 3 The graph shows the change in the degree of crosslinking of the hydrogel in the examples. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0026] like Figure 1 The diagram shows a flowchart of a method for preparing a hypoxia-pretreated exosome-loaded photocrosslinked collagen hydrogel according to the present invention. This method includes the following steps:

[0027] S01. After culturing bone marrow mesenchymal stem cells to 70%–80% confluence, they were placed in a hypoxic environment containing 2% oxygen and 5% carbon dioxide at 37°C for 24 hours for hypoxic pretreatment, and the cell culture supernatant was collected.

[0028] S02. The collected cell culture supernatant was centrifuged sequentially at 300g for 10 minutes, 2000g for 10 minutes, and 10000g for 30 minutes to remove cells and cell debris. Then, it was ultracentrifuged at 100000g for 70 minutes twice. The precipitate was collected to obtain hypoxia pretreatment enhanced exosomes.

[0029] S03. Type I collagen was dissolved in phosphate buffer solution to prepare a 10% (w / w) collagen solution. Methacrylic anhydride was slowly added at 70°C to react with the amino and hydroxyl groups of collagen. After the reaction was complete, the solution was dialyzed and freeze-dried to obtain collagen methacrylamide precursor.

[0030] S04. A positively charged collagen fiber scaffold is prepared by electrospinning pretreatment technology. By adjusting the spinning voltage to 15kV and the collection distance to 12cm, a uniform fiber network structure with a fiber diameter of 500nm to 800nm ​​is formed.

[0031] S05. The collagen methacrylamide precursor prepared in step S03 is redissolved and uniformly mixed with the photoinitiator to form a pregel solution. 200 μg of the hypoxia-pretreated enhanced exosomes prepared in step S02 is added and ultrasonic dispersion and magnetic field-assisted mixing technology are used to ensure uniform dispersion of the exosomes. The step also includes a step of optimizing the mixing process parameters using a game model.

[0032] S06. Transfer the pregel solution loaded with exosomes to the electrospinning scaffold prepared in step S04. Use layered photocrosslinking technology combined with an optical fiber transmission system to irradiate each layer with 405nm blue light for 30 seconds, while using a rotating irradiation device to ensure uniform illumination in all directions.

[0033] S07. Detect the degree of crosslinking of the hydrogel and the uniformity of exosome distribution. When the degree of crosslinking reaches more than 85% and the exosome distribution deviation coefficient is less than 15%, the preparation is completed. Otherwise, adjust the concentration gradient distribution of the photoinitiator and repeat step S06 until the standard requirements are met.

[0034] Hypoxia pretreatment refers to culturing cells in a hypoxic environment to activate intracellular hypoxia response pathways, thereby promoting the secretion of exosomes with enhanced biological activity. Hypoxia-enhanced exosomes are exosomes secreted by stem cells cultured in a hypoxic environment, which exhibit stronger abilities to promote matrix synthesis and resist apoptosis compared to exosomes cultured under normoxic conditions.

[0035] Collagen methacrylamide precursor refers to a polymer precursor obtained by modifying type I collagen with methacrylic anhydride. It possesses the characteristic of undergoing free radical polymerization under the action of a photoinitiator to form a three-dimensional network structure. Electrospinning pretreatment technology refers to the technique of stretching polymer solutions into nanofibers using a high-voltage electrostatic field and collecting them to form a fiber scaffold. By adjusting process parameters, positively charged scaffolds are prepared for immobilizing exosomes.

[0036] Ultrasonic dispersion technology refers to a method that utilizes the cavitation effect and mechanical vibration of ultrasound to disrupt exosome aggregation and promote their uniform dispersion in solution. Magnetic field-assisted mixing technology refers to a technique that uses a magnetic stirrer to generate eddy currents under the influence of a magnetic field to promote thorough mixing of exosomes with the pregelation solution.

[0037] Layered photocrosslinking technology refers to a method of dividing a thick hydrogel into multiple thin layers and sequentially performing photocrosslinking, ensuring overall crosslinking uniformity by controlling the thickness of each layer and the irradiation time. Fiber optic transmission systems utilize optical fibers to guide a light source to different depths within the hydrogel for irradiation, solving the problem of limited light penetration depth.

[0038] Crosslinking degree refers to the percentage of monomers that have undergone crosslinking reactions in a hydrogel, and is used to characterize the completeness of crosslinking in the hydrogel. Exosome distribution deviation coefficient is the ratio of the standard deviation to the average distribution density of exosomes in the hydrogel, and is used to evaluate the uniformity of exosome distribution. Photoinitiator concentration gradient distribution refers to a method of setting different concentrations of photoinitiator at different depths of the hydrogel to compensate for light intensity attenuation and ensure uniform crosslinking.

[0039] Step S05 also includes a step of optimizing the mixing process parameters using a game theory model. Specifically, the optimal combination of ultrasonic power, magnetic field strength, and mixing time parameters is determined through a game process between the upper-level model and the lower-level model. The game theory model includes an upper-level model that aims to maximize the exosome dispersion efficiency and a lower-level model that aims to minimize the loss of exosome activity.

[0040] The objective function of the upper-level model is used to calculate the maximum exosome dispersion efficiency. The inputs include ultrasound power, magnetic field strength, and mixing time, and the output is the standardized dispersion efficiency value. The objective function is expressed as follows: The constraint condition is ultrasonic power. magnetic field strength Mixing time .

[0041] The objective function of the lower-level model is used to calculate the minimum value of exosome activity loss. The inputs include ultrasound power, magnetic field strength, and mixing time, and the output is the standardized activity retention rate. The objective function is expressed as follows: The constraint condition is ultrasonic power. magnetic field strength Mixing time .

[0042] The standardized dispersion efficiency value refers to the ratio of exosome dispersion efficiency to the baseline dispersion efficiency, used to evaluate the effectiveness of the dispersion process. The standardized activity retention rate refers to the ratio of exosome activity retention rate to initial activity, used to evaluate the degree to which exosome biological activity is maintained during mixing. The coupling term... and Used to establish the mutual influence relationship between the upper-level model and the lower-level model, where and is the coupling coefficient.

[0043] The ultrasonic power refers to the power output of the ultrasonic dispersion device, which originates from the power control system of the ultrasonic device and is used to control the degree of exosome dispersion. The magnetic field strength refers to the magnetic induction intensity generated by the magnetic field-assisted mixing device, which originates from the intensity adjustment system of the magnetic field generator and is used to adjust the flow state of the mixture. The mixing time refers to the duration of ultrasonic dispersion and magnetic field-assisted mixing, which originates from the process time control system and is used to determine the processing cycle of the mixing process.

[0044] The specific implementation methods of the above steps are described in detail below.

[0045] The specific implementation of step S01 is as follows: First, bone marrow mesenchymal stem cells are seeded into a cell culture dish and cultured under normoxic conditions until the confluence reaches 70% to 80%. The confluence is judged by observing the percentage of the area of ​​cells covering the bottom of the culture dish under a microscope. Then, the culture dish is transferred to a hypoxic incubator and the gas composition is adjusted to maintain the oxygen concentration at 2% and the carbon dioxide concentration at 5%. At the same time, the temperature control system is set to 37°C and cultured continuously for 24 hours to activate the intracellular hypoxia-inducible factor signaling pathway. The activation of this pathway can promote the secretion of exosomes rich in pro-angiogenic factors and anti-apoptotic factors. After the culture is completed, the supernatant in the culture dish is carefully collected with a sterile pipette and transferred to a sterile centrifuge tube for temporary storage at 4°C for later use. The purpose of this step is to stimulate stem cells to produce exosomes with enhanced biological activity through hypoxia stress, thereby improving the tissue repair capacity of the final hydrogel.

[0046] The specific implementation of step S02 involves purifying exosomes from cell culture supernatant using a fractional separation technique combining differential centrifugation and ultracentrifugation. First, the collected supernatant is centrifuged at 300g for 10 minutes to remove suspended intact cells. After centrifugation, the supernatant is carefully transferred to a new centrifuge tube, avoiding the bottom precipitate. Then, it is centrifuged at 2000g for 10 minutes to remove large cell debris and apoptotic bodies. After transferring the supernatant again, it is centrifuged at 10000g for 30 minutes to remove microvesicles and other large particulate impurities. The obtained supernatant... The liquid needs to be filtered through a 0.22 μm pore size filter membrane to further remove residual particulate matter. Finally, the filtrate is centrifuged at 100,000 g for 70 minutes to precipitate the exosomes at the bottom of the centrifuge tube. The supernatant is discarded, and the precipitate is resuspended in phosphate buffer solution and washed again at 100,000 g for 70 minutes to remove residual protein contaminants. The precipitate collected after two ultracentrifugations is the purified hypoxia-pretreated enhanced exosomes. This fractionation separation technology achieves high-purity separation of exosomes based on the difference in sedimentation rate of particles of different sizes in the centrifugal force field.

[0047] The specific implementation of step S03 involves converting type I collagen into a methacrylamide derivative with photocrosslinking capabilities through chemical modification. First, type I collagen powder is weighed and dissolved in a phosphate buffer solution with a pH of 7.4 to prepare a 10% (w / w) collagen solution. The solution is continuously stirred at 4°C for 12 hours using a magnetic stirrer to ensure complete dissolution and a homogeneous solution. The solution is then transferred to a constant-temperature water bath, and the temperature is slowly increased to 70°C to enhance the activity of the collagen molecular chains. Under continuous stirring, a methacrylic anhydride solution is slowly added dropwise to achieve a molar ratio of 1.5:1 between the total amino and hydroxyl groups of the collagen and the methacrylic anhydride. The ester groups in the methacrylic anhydride can nucleophilically react with the amino and hydroxyl groups of the collagen side chains. The substitution reaction generates stable amide and ester bonds, thereby introducing methacrylamide groups into collagen molecules. The reaction process needs to be maintained for 4 hours, and the progress of the reaction is judged by monitoring the change of pH value of the solution. When the pH value stabilizes, it indicates that the reaction is basically completed. The reaction product is dialyzed in deionized water for 48 hours through a dialysis bag to remove unreacted methacrylic anhydride and small molecule byproducts. During the dialysis process, the dialysate is changed every 8 hours to improve the purification efficiency. Finally, the dialyzed solution is freeze-dried. After pre-freezing at -80°C for 12 hours, it is transferred to a freeze dryer and dried under a vacuum of less than 10 Pa for 36 hours to obtain a fluffy collagen methacrylamide precursor powder. This modification process endows collagen with the ability to undergo free radical polymerization under the action of photoinitiators, providing reactive groups for subsequent photocrosslinking.

[0048] The specific implementation of step S04 involves using electrospinning technology to prepare a nanofiber scaffold with positive charge properties for immobilizing loaded exosomes. First, the collagen methacrylamide precursor prepared in step S03 is dissolved in a mixed solvent of hexafluoroisopropanol and formic acid to prepare a spinning solution with a mass fraction of 8%. Chitosan is added to the spinning solution as a positively charged polymer additive, with its mass fraction controlled at 2%, so that the surface of the spun fibers carries a positive charge, facilitating the adsorption of negatively charged exosomes. The prepared spinning solution is loaded into a syringe and installed at the high-voltage electrode of the electrospinning equipment. Spinning parameters are set, including adjusting the voltage to 15kV to establish a sufficiently strong electric field between the needle and the receiving plate so that the spinning solution can overcome surface tension and form a Taylor cone. The distance between the needle and the receiving plate is set to 12cm to ensure that the fibers have sufficient time to propagate during flight. Solvent evaporation and stretching refinement are performed, with the propulsion speed controlled within the range of 0.8 mL / h to 1.2 mL / h to ensure the continuity and stability of the spinning process. The ambient humidity is controlled between 30% and 40% to prevent premature fiber solidification or adhesion. During the spinning process, the charged polymer jet flies at high speed towards the grounded receiving plate under the action of electric field force. During the flight, it undergoes stretching refinement and rapid solvent evaporation, and finally deposits on the receiving plate to form a uniform fiber network structure with a diameter of 500 nm to 800 nm. This structure has a high specific surface area and good pore connectivity, which is conducive to the penetration of the subsequent pre-gel solution and the fixation of exosomes. After spinning, the fiber scaffold is dried in a vacuum drying oven at 40°C for 2 hours to remove residual solvent. The positive charge density of the fiber surface is further enhanced by plasma surface treatment technology to improve the adsorption capacity of exosomes.

[0049] The specific implementation of step S05 involves uniformly mixing the collagen methacrylamide precursor with exosomes and optimizing the mixing process parameters using a game theory model. First, the collagen methacrylamide precursor powder prepared in step S03 is redissolved in a phosphate buffer solution to prepare a 5% (w / w) pregel solution. The photoinitiator 2-hydroxy-4-morpholinoacetophenone is added to the solution to achieve a final concentration of 0.05%, and the solution is stirred thoroughly for 30 minutes under light-protected conditions to ensure uniform dispersion of the photoinitiator. Then, the anoxic pretreatment-enhanced exosomes prepared in step S02 are added to a concentration of 200 μg and treated with ultrasonic dispersion technology. The game theory model is then used to further optimize the mixing process parameters. The optimization process is based on a two-layer optimization framework. The upper-layer model aims to maximize exosome dispersion efficiency, while the lower-layer model aims to minimize exosome activity loss. The objective function of the upper-layer model is obtained by calculating the combined effects of ultrasonic power, magnetic field strength, and mixing time on dispersion efficiency, resulting in a standardized dispersion efficiency value. The 0.6 power term of ultrasonic power reflects the breaking effect of ultrasonic cavitation on exosome aggregates, the 0.4 power term of magnetic field strength reflects the promoting effect of magnetic field-induced fluid eddy motion on dispersion, and the logarithmic term of mixing time indicates that dispersion efficiency exhibits a marginal decrease over time. A penalty term related to exosome activity loss is subtracted to couple the influence of the lower-layer model. The objective function of the layer model is obtained by calculating the standardized activity retention rate by evaluating the impact of process parameters on the integrity of exosome membrane structure and the activity of surface proteins. The exponential decay term in this function reflects the cumulative damage effect of the product of ultrasonic power and processing time on the exosome membrane structure. The negative correlation of the magnetic field strength term indicates that an appropriate magnetic field strength can protect exosome activity by reducing shear force. A reward term related to dispersion efficiency is added to achieve bidirectional coupling between the upper and lower layer models. The game-theoretic solution process uses an iterative algorithm. First, the parameters of the lower layer model are fixed to solve for the optimal solution of the upper layer model. Then, the optimal solution of the upper layer model is substituted into the lower layer model to solve for its optimal solution. This iterative process is repeated until the objective function values ​​of the upper and lower layer models converge. Iteration stops when the change is less than 1%. The optimal parameter combination is usually 120W to 150W ultrasonic power, 0.4T to 0.6T magnetic field strength, and 180s to 240s mixing time. Under the optimized parameter conditions, actual mixing operation is carried out. At the same time, magnetic field-assisted mixing technology is used to generate three-dimensional eddy motion under the magnetic field drive of a magnetic stir bar to further promote the thorough mixing of exosomes and pregel solution. The entire mixing process is carried out under 4℃ ice bath conditions to reduce the metabolic activity of exosomes and prevent the degradation of bioactive components. This step achieves a balance between efficient dispersion and activity protection of exosomes through game model optimization, ensuring the uniform distribution of exosomes in hydrogel and the effective preservation of biological functions.

[0050] The specific implementation of step S06 involves using a layered photocrosslinking technology combined with an optical fiber transmission system to achieve three-dimensional crosslinking and curing of the pregel. First, the pregel solution containing the exosomes prepared in step S05 is slowly injected into the electrospinning scaffold prepared in step S04. Capillary action is used to ensure the pregel solution fully penetrates the pore structure of the fiber network. Then, the filled scaffold is placed on the sample stage of a rotating irradiation device. This device allows for 360-degree continuous rotation of the sample to ensure uniform illumination from all directions. The photocrosslinking system uses a 405nm blue LED light source with an adjustable intensity range of 5. Up to 20 To address the limited penetration depth of light in hydrogels, an optical fiber transmission system was introduced. This system consists of a 200 μm diameter multimode fiber array, with the fibers arranged at 2 mm intervals and inserted into the pregel at different depths. Each fiber's end is specially treated to achieve lateral light scattering, creating a spherical irradiation area around it. A layered photocrosslinking process divides the hydrogel vertically into several layers, each with a thickness controlled between 1 mm and 2 mm. Photocrosslinking is performed sequentially from the bottom layer upwards, with each layer irradiated for 30 seconds. The photoinitiator decomposes under 405 nm blue light excitation, generating free radicals that initiate free radical polymerization between methacrylamide groups. During polymerization, collagen molecular chains are bridged by methacrylamide to form a covalently cross-linked three-dimensional network structure. The rate of the crosslinking reaction is influenced by light intensity. The concentration of photoinitiator and photoinitiator are controlled together. By adjusting these two parameters, the gelation time and crosslinking density can be precisely controlled. The rotating irradiation device rotates continuously at 10 rpm during each layer of photocrosslinking to ensure that all radial positions of the sample receive the same light dose. After the interlayer crosslinking is completed, the next layer of photocrosslinking process begins immediately to ensure that a continuous crosslinking network is formed between layers and to avoid delamination. The entire photocrosslinking process requires strict control of the ambient temperature to be maintained at 25°C to prevent temperature fluctuations from affecting the crosslinking reaction rate. After photocrosslinking is completed, the hydrogel is soaked in phosphate buffer solution for 30 minutes to remove unreacted photoinitiator and soluble components. This step solves the technical problem of uneven crosslinking of thick hydrogels through the synergistic effect of layered photocrosslinking and optical fiber transmission system, realizing the in-situ fixation of exosomes in a three-dimensional network.

[0051] The specific implementation of step S07 involves quality testing of the crosslinking degree and exosome distribution uniformity of the prepared hydrogel. The crosslinking degree is determined using a swelling-weighing method. The hydrogel sample is soaked in phosphate buffer solution for 24 hours until swelling equilibrium is reached, and its wet weight is weighed. Subsequently, it is dried in a vacuum drying oven at 60℃ to constant weight, and the dry weight is weighed. The crosslinking degree is calculated based on the ratio of the dry weight to the theoretical dry weight at complete crosslinking. When the crosslinking degree reaches 85% or higher, it indicates that the hydrogel has sufficient mechanical strength and stability. The exosome distribution uniformity is tested using fluorescent labeling combined with confocal microscopy. First, exosomes are labeled with the fluorescent dye PKH26 to emit red fluorescence under excitation light. The labeled exosomes are then loaded into the hydrogel according to the method in step S05. A confocal laser scanning microscope is used to perform three-dimensional scanning of different regions of the hydrogel to obtain spatial distribution images of the exosomes. The fluorescence intensity at each location is statistically analyzed using image analysis software, and the ratio of its standard deviation to the mean is calculated to obtain the exosome distribution deviation coefficient. When this coefficient is less than 15%, the exosome distribution is considered to meet the uniformity requirements. If the test results do not meet the standards, the reasons need to be analyzed and adjustments made. For cases of insufficient crosslinking, the crosslinking degree can be improved by increasing the concentration of photoinitiator or extending the illumination time. For cases of uneven exosome distribution, the mixing process parameters in step S05 need to be optimized or the concentration gradient distribution of photoinitiator needs to be adjusted. The method for adjusting the concentration gradient distribution of photoinitiator is to use microfluidic technology to set different concentrations of photoinitiator at different depths of the hydrogel during the preparation of the pregel solution. The concentration of photoinitiator at the surface position near the light source is set to 0.03% to 0.04%, the concentration at the middle layer is set to 0.05% to 0.06%, and the concentration at the deep layer is set to 0.07% to 0.08%. This gradient distribution can compensate for the effect of light intensity attenuation with increasing depth, so that each layer obtains a similar effective photoinitiator excitation amount, thereby achieving uniform crosslinking. After the adjustment is completed, the photocrosslinking process in step S06 is repeated and the quality test is performed again until all indicators meet the standard requirements. This step ensures the performance stability and repeatability of the final product through strict quality control and parameter feedback adjustment mechanism.

[0052] It should be noted that the key technical ideas of this invention include hypoxia pretreatment to enhance exosome bioactivity, game model optimization of exosome loading process, and three-dimensional molding technology combining layered photocrosslinking with optical fiber transmission. The synergistic effect of these technical ideas significantly improves the overall performance of tissue engineering scaffolds.

[0053] Hypoxia pretreatment enhances exosome bioactivity by simulating the ischemic and hypoxic microenvironment in vivo, activating the hypoxia response mechanism of stem cells and causing them to secrete exosomes enriched with hypoxia-inducible factors. Compared to exosomes cultured under normoxic conditions, exosomes pretreated with hypoxia exhibited stronger biological effects in promoting endothelial cell migration and tube formation and inhibiting apoptosis. This pretreatment strategy enhances the therapeutic potential of exosomes from the source, laying the foundation for the tissue repair function of subsequent hydrogel scaffolds. At the same time, the precise control of 2% oxygen concentration and 24-hour treatment time avoids cell damage caused by excessive hypoxia, maximizing the enrichment of bioactive components while ensuring the yield of exosomes.

[0054] The game-theoretic model for optimizing exosome loading technology innovatively introduces bilayer game theory into the biomaterial preparation process. By establishing an adversarial optimization framework that maximizes dispersion efficiency and minimizes activity loss, it achieves the optimal balance between physical dispersion effect and biological activity protection. Traditional exosome loading methods often simply pursue dispersion uniformity while ignoring the damage to the exosome membrane structure caused by ultrasound and shear force, or sacrifice dispersion effect to protect activity, leading to exosome aggregation in the matrix. The game-theoretic model of this invention establishes a mutual constraint relationship between two optimization objectives through the coupling terms of the upper and lower objective functions. The iterative solution process can automatically search for parameter combinations that allow both objectives to reach acceptable levels simultaneously. This intelligent optimization strategy significantly improves the effective loading capacity and spatial distribution uniformity of exosomes in hydrogels, creating ideal conditions for the continuous release of exosomes and the exertion of their biological functions.

[0055] The three-dimensional molding technology combining layered photocrosslinking and fiber optic transmission overcomes the limitations of traditional photocrosslinking methods in the preparation of thick-layer hydrogels. By decomposing the overall crosslinking process into the gradual solidification of multiple thin layers, each layer can receive sufficient light to achieve complete crosslinking. The introduction of the fiber optic transmission system further solves the problem of limited light penetration depth, enabling light to be directly transmitted to the deep internal regions of the hydrogel. The array arrangement of multiple optical fibers and the lateral scattering design ensure the uniformity of light intensity distribution throughout the entire volume. This combination of technologies not only improves the crosslinking uniformity and mechanical properties of the hydrogel, but also avoids the problem of excessive crosslinking of the surface layer and insufficient crosslinking of the inner layer. At the same time, the temperature control and rotation irradiation mechanism in the layered crosslinking process ensure that the loaded exosomes will not be deactivated due to local overheating or uneven light irradiation, providing a feasible technical route for the preparation of large-size tissue engineering scaffolds with uniform performance.

[0056] The synergistic effect of these three key technological approaches is reflected in their joint construction of a complete technological chain from the preparation of bioactive substances to loading optimization and scaffold molding. The hypoxia pretreatment technology provides high-quality functional exosomes as a source of bioactivity, the game theory model optimization technology ensures that these exosomes can be uniformly dispersed in the matrix material in the best state, and the layered photocrosslinking technology transforms the liquid precursor loaded with exosomes into a solid scaffold with a stable three-dimensional structure. The three are organically connected in the temporal and spatial dimensions, and each link specifically solves the key scientific problems in the tissue engineering scaffold preparation process. Compared with the defects of low exosome loading rate, uneven distribution and unstable scaffold performance in the existing technology, this invention significantly improves the biological function and structural integrity of the scaffold through multi-technology synergy, and provides a novel biomaterial platform with excellent performance and controllable preparation for tissue repair and regenerative medicine applications.

[0057] It should be noted that this invention also solves the following technical problem: the poor structural stability caused by incomplete internal crosslinking of thick materials in traditional photocrosslinked hydrogel preparation. In existing photocrosslinked collagen hydrogel preparation processes, due to the limited penetration ability of light in biomaterials, when the hydrogel thickness exceeds a certain range, the light intensity received by the internal region decreases sharply, leading to incomplete polymerization of the collagen methacrylamide precursor, resulting in a gradient distribution of crosslinking density and uneven structural strength. This insufficient internal crosslinking not only affects the mechanical properties and long-term stability of the hydrogel, but may also cause exosomes to escape from uncrosslinked regions, disrupting its distribution uniformity. This invention innovatively employs layered photocrosslinking technology to decompose thick hydrogels into multiple thin layers for sequential processing. Combined with an optical fiber transmission system, the light source is directly introduced into the interior of the material for irradiation. Simultaneously, a photoinitiator concentration gradient distribution strategy is used to compensate for the attenuation differences of light intensity at different depths, achieving uniform crosslinking of the entire hydrogel system and ensuring that exosomes can obtain a stable fixation environment and long-lasting distribution stability at all depth levels.

[0058] Furthermore, this invention addresses the technical problems of poor controllability of bioactivity and low batch-to-batch consistency in exosome preparation. Traditional exosome preparation typically employs the natural secretion process of cells under normoxic culture conditions. Due to the random fluctuations in cell metabolic state caused by the culture environment, the obtained exosomes exhibit significant batch-to-batch variability in key quality indicators such as protein content, growth factor composition, and membrane structure integrity, making it difficult to meet the requirements of product standardization and functional consistency for biomedical applications. This invention establishes a standardized culture protocol for hypoxia pretreatment, precisely controlling a hypoxic environment with an oxygen concentration of 2% to simulate physiological hypoxia in vivo. This activates the expression regulation of key transcription factors such as HIF-1α within cells, significantly improving the standardization of bioactivity and batch-to-batch consistency of exosome products. This provides crucial assurance for the accuracy and reproducibility of exosome distribution control in subsequent hydrogel preparation.

[0059] Specifically, the principle of this invention is as follows: The fundamental principle behind solving the core technical problem of uneven distribution of exosomes in collagen hydrogels lies in its construction of a multi-dimensional synergistic control technology system to overcome the physicochemical barriers to exosome distribution. First, ultrasonic dispersion technology utilizes the cavitation effect generated by ultrasound in a liquid to create a localized high-temperature, high-pressure microenvironment. Through acoustic flow and microjets, it effectively disrupts the electrostatic attraction and van der Waals interactions between exosomes, dissociating aggregated exosome clumps into a monodisperse state, laying the foundation for subsequent uniform distribution. Second, magnetic field-assisted mixing technology uses a magnetic stirrer to generate complex three-dimensional eddy currents under the action of a magnetic field. These eddies promote thorough mixing of exosomes and collagen precursor solutions at both macroscopic and microscopic scales, overcoming the dead-angle effect and shear unevenness problems of traditional stirring methods, and achieving uniform dispersion of exosomes in three-dimensional space. Furthermore, the introduction of a game theory model resolves the multi-objective conflict in process parameter optimization. By striking a balance between maximizing dispersion efficiency in the upper-level model and optimizing activity maintenance in the lower-level model, the optimal parameter combination that achieves both sufficient dispersion and maintains exosome bioactivity is automatically searched, avoiding local optima that might result from optimizing a single parameter. In addition, the positively charged fiber scaffold prepared by electrospinning utilizes electrostatic interactions to form stable electrostatic adsorption with the negative charges on the exosome surface. This adsorption force not only prevents re-aggregation and sedimentation of exosomes during crosslinking but also guides exosomes to achieve an orderly distribution along the fiber network structure, forming a stable spatially fixed pattern. Finally, layered photocrosslinking technology combined with an optical fiber transmission system ensures uniform crosslinking of the collagen matrix at different depths. By controlling the consistency of crosslinking rate and density, exosome migration and redistribution caused by uneven crosslinking are prevented, maintaining the long-term stability of exosome distribution.

[0060] The following provides a specific embodiment 1 of the present invention, and the specific implementation of each step in this embodiment 1 is described in detail below.

[0061] The specific implementation of step S01 involves culturing bone marrow mesenchymal stem cells to 70% to 80% confluence and then placing them in a solution containing 2% oxygen and 5%... Cells were cultured in a hypoxic environment at 37°C for 24 hours as a hypoxic pretreatment, and the cell culture supernatant was collected. This step mainly activates the hypoxia-inducible factor signaling pathway through hypoxia stress, prompting cells to secrete exosomes with stronger biological activity. The hypoxic environment was established using a three-gas incubator for precise control. The concentration is 2%. The concentration is 5%, and the equilibrium gas is The temperature was strictly maintained at 37℃, and the culture time was set to 24 hours to ensure that hypoxia-inducible factors were fully activated without causing excessive stress damage to cells. After the culture was completed, the supernatant was collected using aseptic techniques and immediately transferred to a 4℃ environment for storage until use.

[0062] The specific implementation of step S02 involves centrifuging the collected cell culture supernatant sequentially at 300g for 10 minutes, 2000g for 10 minutes, and 10000g for 30 minutes to remove cells and cell debris. Then, the supernatant is ultracentrifuged at 100000g for 70 minutes twice to collect the precipitate and obtain hypoxia-pretreated enhanced exosomes. This differential centrifugation process achieves stepwise purification of exosomes based on the difference in sedimentation rate of particles of different sizes in the centrifugal force field. The centrifugal force is set according to Stokes' law to ensure the effectiveness of each separation stage. After each centrifugation, the supernatant should be carefully aspirated to avoid disturbing the precipitate. Finally, two 100000g ultracentrifugations can obtain high-purity exosomes.

[0063] The specific implementation of step S03 involves dissolving type I collagen in a phosphate buffer solution to prepare a 10% (w / w) collagen solution. Methacrylic anhydride is then slowly added at 70°C to react with the amino and hydroxyl groups of collagen. After the reaction is complete, the solution is dialyzed and freeze-dried to obtain a collagen methacrylamide precursor. This chemical modification process introduces photocrosslinkable methacryloyl groups through a nucleophilic substitution reaction between the ester groups of methacrylic anhydride and the side chain functional groups of collagen. The reaction temperature of 70°C increases the reaction rate without damaging the triple helix structure of collagen. The molar ratio of methacrylic anhydride to the total amount of amino and hydroxyl groups is controlled at 1.5:1 to ensure adequate modification. After a reaction time of 4 hours, unreacted reagents are removed by dialyzed, and the solution is freeze-dried to obtain a powdered precursor material that is easy to store.

[0064] The specific implementation of step S04 involves preparing a positively charged collagen fiber scaffold using electrospinning pretreatment technology. By adjusting the spinning voltage to 15kV and the collection distance to 12cm, a uniform fiber network structure with a fiber diameter of 500nm to 800nm ​​is formed. During the electrospinning process, the formation and stretching of the polymer jet follow the principles of electrohydrodynamics. A strong electric field gradient is established between the needle and the receiving plate to overcome the surface tension of the solution and form a Taylor cone. Under the action of the electric field, the jet is accelerated towards the receiving plate and undergoes solvent evaporation and stretching during its flight. Adding 2% chitosan to the spinning solution imparts positive charge characteristics to the fiber surface. Precise control of the spinning parameters ensures that the fiber diameter is uniformly distributed in the range of 500nm to 800nm.

[0065] The specific implementation of step S05 involves redissolving the collagen methacrylamide precursor prepared in step S03 and uniformly mixing it with the photoinitiator to form a pregel solution. 200 μg of the hypoxia-pretreated enhanced exosomes prepared in step S02 are then added, and ultrasonic dispersion and magnetic field-assisted mixing techniques are used to ensure uniform dispersion of the exosomes. This step employs a game theory model to optimize the mixing process parameters to achieve a balance between maximizing exosome dispersion efficiency and minimizing activity loss. The game theory model includes a two-layer optimization framework consisting of an upper-level model and a lower-level model, which is solved iteratively to obtain the optimal parameter combination.

[0066] The objective function of the upper-level model is used to calculate the maximum value of exosome dispersion efficiency, and the formula is expressed as follows:

[0067] ;

[0068] In the formula, The exosome dispersion efficiency under the current process conditions is expressed in units of... ; The baseline dispersion efficiency is expressed in units of Its value was obtained through experimental determination under standard process conditions; Ultrasonic power, measured in W; The reference ultrasonic power is expressed in W, with a value of 100W. This refers to the magnetic field strength, measured in tons (T). The reference magnetic field strength is expressed in tons (T) and is 0.5T. This is the mixing time, measured in seconds. The baseline mixing time is in seconds and is set to 180 seconds. The coupling coefficient of the upper-level model is dimensionless and ranges from 0.1 to 0.3. The exosome activity loss rate is dimensionless. The baseline activity loss rate is dimensionless and its value is obtained through bioactivity detection experiments under baseline process conditions.

[0069] The parameter acquisition method is as follows: The polydispersity index of exosome particle size distribution was obtained by measuring dynamic light scattering technology. The smaller the polydispersity index, the higher the dispersion efficiency. The measurement steps include: Step 1, diluting the mixed sample to an appropriate concentration; Step 2, measuring the particle size distribution using a dynamic light scattering instrument at 25°C; Step 3, recording the polydispersity index and converting it into a dispersion efficiency value through a calibration curve. The power control system of the ultrasonic equipment directly reads the output power, which is equipped with a power sensor that monitors the output power in real time and displays the current value on a digital display screen. The intensity is set through a magnetic field generator intensity adjustment system, which uses a Hall sensor to measure the magnetic induction intensity and maintains the stability of the set value through a feedback control loop. The mixing time is the cumulative time recorded by the timer of the process time control system from the start of mixing to the stop. The exosome membrane protein markers were obtained using an experimental assay. The measurement steps included: Step 1, extracting exosome samples before and after mixing; Step 2, detecting the expression levels of membrane proteins such as CD63 and CD81 using Western blotting; Step 3, calculating the percentage decrease in protein expression levels after mixing relative to before mixing, which is the activity loss rate.

[0070] The objective function of the lower-level model is used to calculate the maximum value of exosome activity retention, and the formula is expressed as follows:

[0071] ;

[0072] In the formula, Exosome activity under current process conditions, expressed in relative fluorescence units (RFU). Initial exosome activity, expressed in relative fluorescence units (RFU); It is a natural exponential function; The coupling coefficient of the lower-level model is dimensionless and ranges from 0.05 to 0.15; the meanings of the other parameters are the same as those of the upper-level model; it should be noted that this formula is an empirical fitting model, where the magnetic field strength term... The dimensions are eliminated through normalization, i.e., in actual calculations... ,in The reference magnetic field strength is set to 1T.

[0073] The parameter acquisition method is as follows: Exosome functional activity assay was performed, and the measurement steps included: Step 1, co-culturing the mixed exosomes with target cells for 24 hours; Step 2, measuring cell viability using a cell proliferation assay kit; Step 3, calculating the fold increase in cell viability relative to the negative control group to characterize exosome activity, and the results were expressed as relative fluorescence units (RFU). The initial bioactivity of the mixed pre-exosomes was determined using the same cell function assays, and their ability to promote cell proliferation was characterized by relative fluorescence units.

[0074] The game theory model is solved using an iterative optimization algorithm. The specific calculation process is as follows: Initialize parameters. , , Set the number of iterations for the median of each constraint range. superscript Indicates the first The next iteration; Step 1, fix the parameters of the lower-level model and solve for the optimal solution of the upper-level model, i.e., under the constraints... W, T, Maximize under s ,get , , Step 2: Substitute the optimal solution from the upper layer into the lower layer model to solve for the optimal solution. The largest parameter combination; Step 3, determine the convergence condition. and If the condition is met, output the optimal parameters and terminate the iteration; otherwise, let... Return to step 1; in step 4, obtain the optimal parameters. , , Applied to actual mixing processes, where superscript This represents the optimal value after game optimization.

[0075] The specific implementation of step S06 involves transferring the pre-gel solution loaded with exosomes onto the electrospinning scaffold prepared in step S04. Layered photocrosslinking technology combined with an optical fiber transmission system is used to irradiate each layer with 405nm blue light for 30 seconds. A rotating irradiation device is employed to ensure uniform illumination in all directions. During the photocrosslinking process, the photoinitiator decomposes under blue light excitation, generating free radicals that initiate the free radical polymerization reaction of methacrylamide groups to form a three-dimensional crosslinked network. To ensure the uniformity of crosslinking in the thick hydrogel, an optical fiber transmission system is used to guide the light source to different depths. The crosslinking thickness of each layer is controlled between 1mm and 2mm, and the irradiation intensity is set to 10 nm. Up to 15 The rotating irradiation device operates at a speed of 10 rpm to ensure that each radial position receives the same light dose.

[0076] The conversion rate of the crosslinking reaction can be characterized by the following formula:

[0077] ;

[0078] In the formula, The cross-linking reaction conversion rate is dimensionless. This represents the initial concentration of methacrylamide groups, in units of... ; Irradiation time The remaining concentration of methacrylamide groups, in units of ; The apparent reaction rate constant is expressed in units of 1000 m / s. ; Light intensity, unit: ; The duration of illumination is measured in seconds (s).

[0079] The parameter acquisition method is as follows: The degree of substitution of the collagen methacrylamide precursor and the concentration of the pregel solution were calculated. The degree of substitution was obtained by measuring the integral ratio of the methacrylamide characteristic peak to the collagen characteristic peak using 1H NMR spectroscopy. The concentration of remaining double bonds was determined by ultraviolet spectrophotometry. The measurement steps included: step 1, taking samples at regular intervals during the crosslinking process; step 2, dissolving the sample in tetrahydrofuran; step 3, measuring the absorbance value at 280 nm; and step 4, converting the absorbance to double bond concentration using a standard curve. This is an empirical parameter, obtained by fitting conversion rate data under different light intensities and time conditions through preliminary experiments; a typical value is 0.015. Up to 0.025 . The light intensity distribution of the irradiated area is directly measured using an optical power meter, and the average value is taken.

[0080] The specific implementation of step S07 is to detect the degree of crosslinking of the hydrogel and the uniformity of exosome distribution. When the degree of crosslinking reaches more than 85% and the exosome distribution deviation coefficient is less than 15%, the preparation is completed. Otherwise, the concentration gradient distribution of the photoinitiator is adjusted and step S06 is repeated until the standard requirements are met.

[0081] The formula for calculating the degree of crosslinking is as follows:

[0082] ;

[0083] In the formula, The degree of crosslinking is expressed as %; The mass of the hydrogel after it has completely dried is expressed in grams. The dry weight at which complete cross-linking is theoretically achieved is in grams. The initial mass of the precursor material used to prepare the hydrogel, in grams; The mass fraction of the crosslinkable component of the collagen methacrylamide precursor is dimensionless.

[0084] The parameter acquisition method is as follows: The hydrogel sample was determined by the swelling and weighing method. The measurement steps included: Step 1, immersing the hydrogel sample in phosphate buffer solution for 24 hours until swelling equilibrium was reached; Step 2, removing the sample and blotting off surface moisture with filter paper; Step 3, drying in a vacuum drying oven at 60℃ until constant weight; Step 4, weighing the dry weight using a precision balance. . The mass of collagen methacrylamide precursor powder weighed during the preparation process was accurately measured using an analytical balance. Based on the degree of substitution and molecular structure of the precursor material, the mass fraction of the crosslinkable component for methacrylamide-modified collagen with a degree of substitution of 40% to 60% is approximately 0.85 to 0.92.

[0085] The formula for calculating the exosome distribution deviation coefficient is expressed as follows:

[0086] ;

[0087] In the formula, The exosome distribution deviation coefficient is expressed in % (%). The standard deviation of exosome distribution density is given in units of 1000 m / s. ; This represents the average exosome distribution density, in units of... ; The quantity of the measurement area is dimensionless. For the first Exosome distribution density in each measurement region, in units of ; This is the index of the measurement area, with values ​​ranging from 1 to... .

[0088] The parameter acquisition method is as follows: The determination was performed using fluorescent labeling combined with confocal microscopy. The measurement steps included: Step 1, labeling exosomes with the fluorescent dye PKH26 to make them emit red fluorescence; Step 2, loading the labeled exosomes into a hydrogel using conventional methods; Step 3, performing a three-dimensional scan of the hydrogel using a confocal laser scanning microscope, dividing the scanned area into... Step 4: Use image analysis software to calculate the integral value of fluorescence intensity within each measurement unit; Step 5: Convert the fluorescence intensity into exosome mass concentration using a standard curve. . The value is determined based on the size of the hydrogel. For a cylindrical hydrogel with a diameter of 10 mm and a height of 5 mm, it is usually divided into 100 to 200 measurement units to obtain statistical significance.

[0089] It should be explained that the objective function of the upper-level model is based on a power-law model established by the synergistic effect of ultrasonic cavitation and magnetic field-induced flow on the influence of exosome dispersion efficiency. This function is expressed through a 0.6 power term of the ultrasonic power. This demonstrates the nonlinear relationship between ultrasonic energy input and the intensity of cavitation bubble collapse. The microjets and shock waves generated by the violent collapse of cavitation bubbles can effectively break up exosome aggregates, but their effectiveness decreases marginally with increasing power. The 0.4 power term of the magnetic field strength... This reflects the intensity of the eddy current motion driven by the Lorentz force generated by the magnetic field on the mixture. This eddy current motion promotes the uniform distribution of exosomes in three-dimensional space, but its contribution is relatively small compared to ultrasound due to the limitation of the magnetic field penetration depth. The logarithm of the mixing time... This reveals the dynamic characteristics of the dispersion process: a rapid initial dispersion rate followed by a gradual convergence to an equilibrium state, with coupling terms... The constraint of exosome activity loss on dispersion efficiency optimization is introduced to avoid excessive damage to the exosome membrane structure in pursuit of high dispersion. The advantage of this objective function over traditional single-objective optimization methods lies in its unified consideration of physical dispersion effects and bioactivity protection. Normalization eliminates the influence of parameters with different dimensions, allowing direct comparison of the contributions of each factor. The selection of the exponent term, based on nonlinear fitting of a large amount of experimental data, ensures the model's predictive accuracy. This multi-factor coupled model can quickly locate the optimal operating point in a complex process parameter space, significantly improving the stability and repeatability of the exosome loading process.

[0090] The objective function of the lower-level model is based on a quantitative description of the damage mechanism of exosomal membrane protein structure caused by ultrasonic shear force and thermal effects. The exponential decay term in this function is expressed as follows:

[0091] ;

[0092] This characterizes the ultrasonic power. and duration of action The product of these terms represents the cumulative destructive effect on exosome activity. The mathematical form of this term originates from a first-order reaction model in chemical kinetics, reflecting the exponential decay of membrane protein denaturation and lipid bilayer damage with increasing energy input. The coefficient 0.001 is an empirical parameter with dimensions of [missing value]. To ensure the exponent is dimensionless, the magnetic field strength term in the denominator is expressed as follows:

[0093] ;

[0094] This demonstrates the protective effect of a moderate magnetic field on exosomes by reducing local shear stress. Magnetic field-induced directional flow can reduce random collisions caused by turbulence, thereby maintaining the integrity of the exosome membrane structure. Coupling terms... A positive incentive relationship between activity retention and dispersion efficiency was established to encourage improved dispersion levels while preserving activity. The innovation of this objective function compared to existing technologies lies in its first-time integration of the thermodynamic stability and mechanical stress response of exosomal membrane proteins into a unified mathematical framework. An exponential decay model accurately describes the nonlinear cumulative characteristics of activity loss during ultrasonic treatment. The introduction of a magnetic field term reveals the physical mechanism of electromagnetic field protection of biomacromolecules, providing a new regulatory dimension for process optimization. The bidirectional coupling design allows the upper and lower layer models to reach a Nash equilibrium state through iterative solutions, achieving Pareto optimality for both dispersion efficiency and activity preservation. This game-theoretic framework, compared to traditional weighted multi-objective optimization methods, better handles conflicting relationships between objectives and avoids getting trapped in local optima.

[0095] The formula for crosslinking reaction conversion rate is based on the quasi-steady-state assumption in free radical polymerization kinetics. This formula simplifies the complex chain initiation, chain growth, and chain termination processes into an apparent first-order reaction model. The conversion rate is calculated as follows:

[0096] ;

[0097] Among them, light intensity The apparent rate constant is directly proportional to the decomposition rate of the photoinitiator, which in turn determines the rate of free radical generation in the system. By integrating microscopic dynamic parameters such as the quantum yield of the photoinitiator, the chain growth rate constant, and the chain termination rate constant, and through the light energy accumulation term... The product of these factors reflects the physical essence that the total photon flux determines the reaction process, while the exponential decay form reflects the kinetic characteristic that the reaction rate gradually decreases as the concentration of reactive groups decreases during the reaction. The advantage of this formula over traditional photocrosslinking processes lies in providing a theoretical tool for quantitatively predicting the degree of crosslinking under different illumination conditions. By controlling the combination of light intensity and time, the crosslinking density of the hydrogel can be precisely controlled, thereby adjusting its mechanical properties and degradation rate. The introduction of the apparent rate constant simplifies the complex reaction mechanism, allowing process engineers to optimize parameters without in-depth knowledge of free radical chemistry. This model, combined with layered photocrosslinking technology, enables independent control of the crosslinking degree of each layer of a thick hydrogel, providing a theoretical basis for the preparation of gradient structure scaffolds. This precise kinetic control ensures the in-situ fixation of exosomes during the crosslinking process, avoiding exosome leakage or aggregation due to uneven crosslinking.

[0098] The principle behind the formula for calculating the degree of crosslinking is based on the law of conservation of mass. It characterizes the completeness of crosslinking by measuring the mass percentage of insoluble crosslinked networks in the hydrogel. The formula is expressed as follows:

[0099] ;

[0100] The actual measured dry weight Dry weight when fully cross-linked with theory The comparison is based on the quality of the precursor material. and crosslinkable component ratio Calculations show that small molecules and solvents that did not participate in the crosslinking reaction are removed during the swelling and drying process, leaving only the crosslinked network that forms covalent bonds. Therefore, the dry weight ratio directly reflects the reaction conversion rate of the methacrylamide groups. The advantage of this formula over the traditional swelling method for determining the degree of crosslinking is that it eliminates the need for complex theoretical assumptions and model fitting; accurate crosslinking degree values ​​can be obtained directly through mass measurement, showing the mass fraction of crosslinked components. The introduction of this method takes into account the presence of non-reactive components in the precursor material, making the calculation results more consistent with reality. This method is applicable to various photocrosslinked hydrogel systems and has good versatility and repeatability. The 85% crosslinking degree threshold is set based on a comprehensive consideration of mechanical properties and biocompatibility, ensuring that the hydrogel has sufficient strength to support cell growth while maintaining moderate degradability to facilitate tissue remodeling. This simple and reliable characterization method provides a standardized means for hydrogel quality control, ensuring the consistency of different batches of products.

[0101] The principle behind the exosome distribution deviation coefficient formula is to apply the statistical concept of coefficient of variation to quantify the uniformity of exosome distribution in the three-dimensional space of a hydrogel. The formula is expressed as follows:

[0102] ;

[0103] This formula derives a normalized dispersion index by calculating the ratio of the standard deviation to the mean of exosome density in each measurement region. It reflects the absolute fluctuation range of the data, while the coefficient of variation eliminates the influence of dimensions and average levels, allowing for direct comparison of homogeneity between different samples. The summation term... Iterate through all measurement units, calculate the density of each unit and the overall average. The degree of deviation is amplified by squaring, which increases the weight of larger deviations, making the indicator more sensitive to uneven distribution. Compared with traditional qualitative observation methods, this formula provides an objective and quantitative evaluation standard. The three-dimensional distribution data obtained by fluorescence imaging technology can comprehensively reflect the spatial location information of exosomes, avoiding the one-sidedness of two-dimensional slice observation. The 15% deviation coefficient threshold is set based on the biological requirements of continuous release and function of exosomes, ensuring that all parts of the hydrogel can provide similar bioactive signals. The high sensitivity of this method can identify small distribution differences and guide the fine control of the mixing process. The application of statistical methods makes the evaluation results reliable and reproducible, providing a scientific basis for the establishment of product quality standards. This quantitative uniformity evaluation system significantly improves the functional stability and clinical application potential of exosome-loaded hydrogels.

[0104] To better understand and implement this invention, the following is a specific application scenario of this invention, Example 2:

[0105] A technical team needed to prepare highly bioactive tissue engineering scaffold materials for spinal cord injury repair research. The team decided to use a hypoxia-pretreated exosome-loaded photocrosslinked collagen hydrogel technique to address the technical challenges of insufficient bioactivity and uneven exosome distribution in traditional scaffold materials.

[0106] The technical team first isolated mesenchymal stem cells from the bone marrow of experimental animals, and then adjusted the cell density to [a specific density] under standard culture conditions. Cells were cultured at a density of 10% fetal bovine serum (FBS) in DMEM medium using standard methods. When cell confluence reached 75%, the culture dishes were transferred to a tri-gas incubator with an oxygen concentration of 2%. The cells were pretreated with a 5% concentration of nitrogen at 37°C for 24 hours under hypoxic conditions. After pretreatment, 600 mL of cell culture supernatant was collected for subsequent exosome isolation.

[0107] Exosome isolation was strictly performed using differential centrifugation. First, cells were removed by centrifugation at 300g for 10 minutes at 4°C. Then, large cell debris was removed by centrifugation at 2000g for 10 minutes, followed by small cell debris and organelles removal by centrifugation at 10000g for 30 minutes. The pretreated supernatant was transferred to an ultracentrifuge tube and centrifuged at 100000g for 70 minutes at 4°C. The supernatant was discarded, and the precipitate was retained. The precipitate was resuspended in 50 mL of phosphate buffer and ultracentrifuged once more to improve exosome purity. The final exosome precipitate was resuspended in 200 μL of sterile phosphate buffer, and the exosome concentration was determined to be 2.8 mg / mL using a protein quantification method.

[0108] The collagen methacrylamide precursor was prepared using a chemical modification method. 50g of type I collagen was dissolved in 500mL of phosphate buffer to prepare a 10% (w / w) collagen solution. Under constant temperature water bath conditions of 70℃, 12.5mL of methacrylic anhydride was slowly added dropwise at a rate of 0.5mL / min, while simultaneously stirring continuously at 200rpm using a magnetic stirrer. The pH was maintained between 8.0 and 8.5 during the reaction, and the reaction time was 4 hours. After the reaction was complete, the product was transferred to a dialysis bag and dialyzed against deionized water at 4℃ for 48 hours, with the dialysate changed every 8 hours. After dialysis, the product was freeze-dried to obtain 32g of collagen methacrylamide precursor powder.

[0109] In the preparation of the electrospun scaffold, collagen methacrylamide precursor was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol to prepare a 12% (w / w) spinning solution. The electrospinning equipment parameters were set as follows: spinning voltage 15 kV, needle inner diameter 0.5 mm, dispensing speed 1.2 mL / h, and collection distance 12 cm. The ambient temperature was controlled at 25℃ and the relative humidity maintained at 35% during the spinning process. After 6 hours of continuous spinning, a fiber scaffold with a thickness of 150 μm was obtained, with fiber diameters mainly distributed in the range of 500-800 nm and an average fiber diameter of 650 nm.

[0110] The pregel solution was prepared using game-theoretic optimization of mixing parameters. 15g of collagen methacrylamide precursor was redissolved in 150mL of phosphate buffer, and 0.5% (w / w) of photoinitiator Irgacure 2959 was added to form the pregel solution. The optimal combination of process parameters, calculated through game-theoretic model optimization, was 120W ultrasonic power, 0.45T magnetic field strength, and 180s mixing time. Under these conditions, 200μg of hypoxia-pretreated exosomes were added to the pregel solution and dispersed using ultrasonic dispersion technology, while magnetic field-assisted mixing technology was used to ensure uniform distribution of the exosomes. The solution temperature was monitored during mixing and controlled below 25℃ to avoid loss of exosome activity.

[0111] like Figure 2 As shown, the dispersion efficiency of exosomes under different process parameters exhibits a significant parameter dependence. A pre-gel solution loaded with exosomes was uniformly coated onto the surface of an electrospinning scaffold, with a coating thickness controlled at 2 mm. Layered photocrosslinking technology was used for curing, dividing the entire thickness into four layers, each 500 μm thick. Irradiation was performed using a 405 nm blue LED light source equipped with an optical fiber transmission system, with a light power density set to 50. The first layer is irradiated for 30 seconds, the second layer for 35 seconds, the third layer for 40 seconds, and the fourth layer for 45 seconds. This incremental irradiation time is designed to compensate for the light intensity attenuation effect.

[0112] like Figure 3 As shown, the change in the degree of hydrogel crosslinking with irradiation time during the layered photocrosslinking process is clearly visible. Simultaneously, a rotating irradiation device was used, rotating the sample at 15 rpm to ensure uniform illumination in all directions. After each layer was irradiated, the degree of crosslinking and exosome distribution of that layer were immediately measured. When the crosslinking degree of the second layer was found to be only 82%, lower than the standard requirement of 85%, the technical team adjusted the photoinitiator concentration distribution strategy, increasing the photoinitiator concentration to 0.8% in that layer and repeating the photocrosslinking treatment until the degree of crosslinking reached 87%.

[0113] During the quality control phase, multiple testing methods were employed to verify product quality. The degree of crosslinking was assessed using the swelling ratio method, where hydrogel samples were immersed in phosphate buffer at 37°C for 24 hours, and the mass change was measured. The uniformity of exosome distribution was detected using a fluorescent labeling method; exosomes were labeled with DiO fluorescent dye, and their three-dimensional distribution within the hydrogel was observed using a confocal microscope. The final test results showed that the overall crosslinking degree of the hydrogel reached 89%, and the exosome distribution deviation coefficient was 12%, both meeting the technical requirements.

[0114] Table 1 shows the impact of different combinations of game theory model parameters on exosome dispersion:

[0115] Table 1. Effects of Game Theory Model Optimization Parameters on Exosome Dispersion Performance

[0116] The technical team evaluated the biocompatibility and bioactivity of the prepared hydrogels. In vitro cell culture experiments showed that the hydrogels loaded with hypoxic pretreated exosomes significantly promoted the proliferation and differentiation of nerve cells. Exosome release kinetics studies indicated that under physiological conditions, exosomes could be continuously released for 14 days, with the release curve exhibiting a biphasic characteristic of rapid initial release followed by slow release.

[0117] Mechanical property tests showed that the hydrogel had a compressive elastic modulus of 15.6 kPa, matching the mechanical properties of natural nerve tissue. The tensile strength reached 45.2 kPa, and the elongation at break was 180%, exhibiting good flexibility and deformability. Degradation performance tests indicated that in phosphate buffer containing collagenase, the hydrogel's mass loss rate reached 75% within 28 days, with the degradation rate coordinated with the tissue regeneration rate.

[0118] Table 2 shows the variation of hydrogel crosslinking uniformity under different irradiation parameters:

[0119] Table 2. Effect of layered photocrosslinking process parameters on crosslinking uniformity

[0120] Key technical parameters throughout the entire preparation process are controlled in real time through an online monitoring system to ensure the consistency and reproducibility of product quality. Temperature control accuracy is ±1℃, pH control accuracy is ±0.1, centrifugation speed control accuracy is ±50rpm, and light exposure time control accuracy is ±2s.

[0121] This invention represents a significant technological advancement over traditional methods for preparing exosome-loaded hydrogels. First, hypoxia pretreatment activates the HIF-1α signaling pathway within stem cells, promoting high expression of growth factors such as VEGF and PDGF, resulting in secreted exosomes with enhanced angiogenesis and tissue repair capabilities. Second, a game-theoretic model-optimized mixing process balances the trade-off between dispersion efficiency and activity retention, achieving efficient and uniform dispersion of exosomes while maximizing their bioactivity. Third, layered photocrosslinking technology combined with an optical fiber transmission system solves the technical challenge of uneven crosslinking within thick samples, ensuring overall crosslinking uniformity through layer-by-layer control and photoinitiator concentration gradient distribution. Fourth, the positively charged scaffold prepared through electrospinning pretreatment interacts electrostatically with the negatively charged exosomes, improving exosome immobilization efficiency and release controllability. The synergistic effect of these technological innovations results in a composite material that significantly outperforms traditional methods in terms of exosome loading, distribution uniformity, bioactivity retention, and mechanical properties.

[0122] It should be noted that the variables involved in this invention are explained in detail in Table 3.

[0123] Table 3. Variable Explanation Table

[0124] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a photocrosslinked collagen hydrogel loaded with exosomes after hypoxia pretreatment, characterized in that, Bone marrow mesenchymal stem cells were cultured to confluence and then placed in a hypoxic environment for hypoxia pretreatment. The cell culture supernatant was collected, and the collected cell culture supernatant was centrifuged to remove cells and cell debris. The precipitate was then collected by ultracentrifugation to obtain hypoxia pretreated and enhanced exosomes. Type I collagen was dissolved in phosphate buffer solution to prepare a collagen solution, which was then reacted with methacrylic anhydride, dialyzed, and freeze-dried to obtain collagen methacrylamide precursor. Positively charged collagen fiber scaffolds were prepared using electrospinning pretreatment technology. The collagen methacrylamide precursor was redissolved and uniformly mixed with a photoinitiator to form a pregel solution, which was then added to the hypoxia pretreatment. To enhance exosomes, ultrasonic dispersion and magnetic field-assisted mixing techniques were employed to ensure uniform exosome dispersion. The pre-gel solution loaded with exosomes was transferred to an electrospinning scaffold, and layered photocrosslinking technology combined with an optical fiber transmission system was used for layered irradiation. The degree of hydrogel crosslinking and the uniformity of exosome distribution were detected until the standard requirements were met to complete the preparation. Ultrasonic dispersion technology was used to break up exosome aggregates to achieve a monodisperse state. Magnetic field-assisted mixing technology generated three-dimensional eddy current motion to promote thorough mixing of exosomes and collagen precursor solution. The positively charged fiber scaffold prepared by electrospinning formed stable electrostatic adsorption with the negative charge on the surface of exosomes to prevent re-aggregation. The layered photocrosslinking technology refers to a method of dividing the hydrogel into multiple thin layers ranging from 1 mm to 2 mm and performing photocrosslinking sequentially, ensuring overall crosslinking uniformity by controlling the thickness of each layer and the irradiation time; Specifically, the positive charge is achieved by adding chitosan as a positively charged polymer additive to the spinning solution, with a mass fraction controlled at 2% so that the surface of the spun fiber carries a positive charge, which facilitates the adsorption of negatively charged exosomes.

2. The method for preparing exosome-loaded photocrosslinked collagen hydrogels under hypoxia pretreatment according to claim 1, characterized in that, The hypoxia pretreatment step specifically involves placing the cells in a hypoxic environment containing 2% oxygen and 5% carbon dioxide and culturing them at 37°C for 24 hours to activate the intracellular hypoxia response pathway and promote the secretion of exosomes with stronger biological activity.

3. The method for preparing exosome-loaded photocrosslinked collagen hydrogels under hypoxia pretreatment according to claim 2, characterized in that, The hypoxia pretreatment-enhanced exosomes refer to the exosomes secreted by stem cells cultured in a hypoxic environment.

4. The method for preparing exosome-loaded photocrosslinked collagen hydrogels under hypoxia pretreatment according to claim 3, characterized in that, The sequential centrifugation step specifically involves centrifuging the collected cell culture supernatant sequentially at 300g for 10 minutes, 2000g for 10 minutes, and 10000g for 30 minutes to remove cells and cell debris, and then repeating the process twice at 100000g for 70 minutes.

5. The method for preparing exosome-loaded photocrosslinked collagen hydrogels under hypoxia pretreatment according to claim 4, characterized in that, The collagen methacrylamide precursor refers to a polymer precursor obtained by modifying type I collagen with methacrylic anhydride.

6. The method for preparing exosome-loaded photocrosslinked collagen hydrogels according to claim 5, characterized in that, The electrospinning pretreatment process involves using a high-voltage electrostatic field to stretch the polymer solution into nanofibers and collect them to form a fiber scaffold. By adjusting the spinning voltage to 15kV and the collection distance to 12cm, a uniform fiber network structure with a fiber diameter of 500nm to 800nm ​​is formed.

7. The method for preparing exosome-loaded photocrosslinked collagen hydrogels under hypoxia pretreatment according to claim 6, characterized in that, The steps for detecting the degree of crosslinking of the hydrogel and the uniformity of exosome distribution are as follows: preparation is completed when the degree of crosslinking reaches 85% or more and the exosome distribution deviation coefficient is less than 15%; otherwise, the concentration gradient distribution of the photoinitiator is adjusted and the layered photocrosslinking steps are repeated until the standard requirements are met.

8. The method for preparing exosome-loaded photocrosslinked collagen hydrogels according to claim 7, characterized in that, The exosome distribution deviation coefficient refers to the ratio of the standard deviation of the exosome distribution density in the hydrogel to the average distribution density.

9. The method for preparing exosome-loaded photocrosslinked collagen hydrogels according to claim 8, characterized in that, The step of adding hypoxia pretreatment to enhance exosomes also includes a step of optimizing the mixing process parameters using a game model. The optimal combination of ultrasonic power, magnetic field strength, and mixing time parameters is determined through a game process between the upper and lower models.

10. The method for preparing exosome-loaded photocrosslinked collagen hydrogels according to claim 9, characterized in that, The game theory model includes an upper-level model that aims to maximize exosome dispersion efficiency and a lower-level model that aims to minimize exosome activity loss.

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