Preparation method of sodium alginate-carboxymethyl chitosan porous polymer scaffold, preparation method of stem cell scaffold system loaded with the same and application thereof

Abstract

The application discloses a sodium alginate-carboxymethyl chitosan porous polymer scaffold preparation method, a stem cell scaffold system preparation method and application thereof, and belongs to the technical field of tissue engineering scaffold materials. In view of the problems of biological safety risk and difficulty in accurately controlling the pore diameter when a chemical pore former is used to regulate the scaffold pore diameter in the prior art, the method comprises the following steps: sodium alginate solution and carboxymethyl chitosan solution are respectively prepared, the mixed solution is crosslinked to a gel state by using a calcium chloride solution, and then the crosslinked gel is subjected to freeze-drying treatment; and by using a first freeze mode of vacuum drying after freezing or a second freeze mode of vacuum drying after quick freezing in liquid nitrogen, porous polymer scaffolds with large pore diameters and small pore diameters are respectively prepared. The method controls the ice crystal size by regulating the freezing temperature, and then controls the scaffold pore diameter, is simple to operate, has high biological safety, and can be used for tissue engineering repair materials.

Description

Technical Field

[0001] This invention belongs to the field of tissue engineering scaffold materials technology, specifically relating to a method for preparing a sodium alginate-carboxymethyl chitosan porous polymer scaffold, a method for preparing a stem cell-loaded scaffold system, and their applications. Background Technology

[0002] In the field of tissue engineering, porous polymer scaffolds are widely used as carriers for three-dimensional cell culture and templates for tissue repair. Studies have shown that the pore size and pore structure of the scaffold have a significant impact on cell adhesion, proliferation, differentiation, and extracellular matrix secretion. To effectively control the scaffold pore size, existing technologies commonly employ the addition of chemical porogens, such as mixing sodium chloride particles, paraffin microspheres, or other soluble substances into a polymer solution. After the scaffold has formed, these porogens are removed by solvent leaching, thus creating a porous structure. However, this method has significant limitations in practical applications. On the one hand, the particle size distribution of the porogen is difficult to control precisely, resulting in inconsistent pore sizes within the scaffold and poor connectivity between pores, making it impossible to form a uniform and highly interconnected three-dimensional network structure. On the other hand, the removal of pore-forming agents often requires the use of organic solvents or strong acid / base solutions. These chemical reagents are difficult to completely remove from the scaffold material, and residual solvents or reaction byproducts may be toxic to subsequently loaded cells, reducing the biocompatibility of the scaffold and even triggering immune rejection after implantation, thus limiting its further application in tissue engineering and regenerative medicine. To address these issues, researchers have attempted to construct porous scaffolds using physical methods such as phase separation, electrospinning, or gas foaming. However, these techniques are either limited by a narrow range of tunable pore sizes or by the difficulty of maintaining high porosity while ensuring complete pore connectivity, making it impossible to flexibly prepare scaffold materials with specific pore sizes according to different tissue repair needs. Therefore, how to achieve precise and controllable preparation of polymer scaffold pore size and pore structure without introducing toxic chemical reagents has long been a technical challenge that this field hopes to solve. Summary of the Invention

[0003] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.

[0004] Another objective of this invention is to provide a method for preparing a sodium alginate-carboxymethyl chitosan porous polymer scaffold, which can achieve precise control of the scaffold pore size by adjusting the freeze-drying temperature without introducing toxic chemical pore-forming agents, thereby obtaining a porous scaffold with high porosity and a highly interconnected three-dimensional network structure.

[0005] To achieve these objectives and other advantages of the present invention, a method for preparing a sodium alginate-carboxymethyl chitosan porous polymer scaffold is provided, comprising the following steps: S1: Sodium alginate powder and carboxymethyl chitosan powder are added to deionized water and stirred and dissolved at 35-45 ℃ to prepare sodium alginate solution with a mass fraction of 2.0-3.0 wt% and carboxymethyl chitosan solution with a mass fraction of 1.5-2.5 wt%, respectively. S2: After filtering the sodium alginate solution and carboxymethyl chitosan solution through a 0.20-0.25 μm filter, the mass ratio of sodium alginate to carboxymethyl chitosan is 4:3-5:2. The mixture is stirred at a constant temperature of 35-45 ℃ for 1.5-2.5 h to obtain a homogeneous mixed solution. S3: Use a calcium chloride solution with a molar concentration of 0.05-0.15 mol / L as a crosslinking agent, and slowly add it dropwise to the continuously stirred mixed solution. The volume ratio of the mixed solution to the calcium chloride solution is 5-15:1. Stop adding the crosslinking agent when the mixed solution turns into a gel state. S4: Pour the cross-linked gel into a mold and let it stand for 45-50 hours to obtain the cross-linked gel; S5: The cross-linked gel is freeze-dried; wherein the freeze-drying process adopts either the first freezing mode or the second freezing mode. The first freezing mode involves freezing the gel in an environment of -45 to -35 ℃ for 10-14 h, followed by vacuum drying for 20-28 h to obtain a large-pore porous polymer scaffold; the second freezing mode involves rapidly freezing the gel in a liquid nitrogen environment until it is fully formed, followed by vacuum drying for 20-28 h to obtain a small-pore porous polymer scaffold.

[0006] This invention uses sodium alginate and carboxymethyl chitosan as raw materials, forming a double-network gel through calcium ion crosslinking. Utilizing the inherent differences in ice crystal size under different low-temperature environments, a freeze-drying process is employed to dehydrate the gel. During freezing, water inside the gel forms ice crystals, the size of which varies with freezing temperature. Subsequent vacuum drying causes the ice crystals to sublimate, leaving a porous structure within the scaffold corresponding to the ice crystal morphology. By adjusting the freezing temperature, the scaffold pore size can be precisely controlled without introducing any chemical pore-forming agents. The polymer scaffold prepared using this method possesses a highly interconnected three-dimensional network porous structure with a porosity exceeding 90%. Furthermore, by selecting either -40℃ freezing or liquid nitrogen rapid freezing, large-pore scaffolds with an average pore size of 150-200 μm and small-pore scaffolds with an average pore size of 10-35 μm can be obtained, respectively. The entire preparation process is simple to operate and the reaction is mild, avoiding the use of toxic chemical reagents and ensuring the biosafety of the scaffold material. It provides a structurally controllable and high-performance cell carrier for three-dimensional stem cell culture and tissue defect repair, and has important application prospects in the field of regenerative medicine.

[0007] Preferably, the average pore size of the large-pore porous polymer scaffold is 150-200 μm and the porosity is 92%-95%; the average pore size of the small-pore porous polymer scaffold is 10-35 μm and the porosity is 90%-94%.

[0008] The dual-network gel formed by calcium ion crosslinking of sodium alginate and carboxymethyl chitosan can be modified during freeze-drying by adjusting the freezing temperature, thereby affecting the nucleation and growth behavior of ice crystals within the gel and thus influencing their size and distribution. When frozen at -40℃, ice crystal growth is more complete, resulting in larger ice crystals that leave correspondingly larger pores after sublimation, with an average pore size of 150-200 μm. Conversely, when rapidly frozen with liquid nitrogen, the water within the gel freezes instantly, resulting in smaller and more uniformly distributed ice crystals. The average pore size of the scaffold obtained after sublimation is 10-35 μm. Both freezing methods resulted in scaffolds that maintained the integrity of the dual-network structure, with porosities exceeding 90%. The difference in pore size directly affects the biological function of scaffolds as cell carriers: large-pore scaffolds provide ample space for bone marrow mesenchymal stem cells to aggregate, allowing cells to contact each other and form three-dimensional cell spheres within the pores. The slight elasticity of the pore walls both accommodates cell sphere growth and provides fixation. Small-pore scaffolds, with pore sizes similar to the diameter of a single cell, allow cells to primarily disperse and attach to the pore wall surface, limiting intercellular contact. This limitation in pore size allows scaffolds to leverage their structural advantages according to different application needs. Large-pore scaffolds promote three-dimensional cell aggregation and enhanced paracrine function, while small-pore scaffolds are suitable for dispersed cell loading or as physical barriers. High porosity ensures the free diffusion of nutrients and metabolic waste within the scaffold, providing a favorable microenvironment for the survival and functional maintenance of loaded cells.

[0009] A method for preparing a porous polymer scaffold system loaded with bone marrow mesenchymal stem cells includes the following steps: B1: Sterilize the prepared large-pore porous polymer scaffold or small-pore porous polymer scaffold to obtain a sterilized scaffold. B2: Bone marrow mesenchymal stem cells passaged to the third generation were resuspended in culture medium to prepare a density of 1×10⁻⁶. 6 -1×10 8 Cell suspension of cells per mL; B3: Add the cell suspension dropwise to the sterilized scaffold according to the volume ratio of sterilized scaffold to cell suspension of 1:5-10, and incubate at 37 ℃ for 2 h to allow the cell suspension to be completely absorbed by the sterilized scaffold, thus obtaining the incubated scaffold. B4: Add complete culture medium to the incubated scaffold to completely submerge the scaffold, and continue culturing in an environment of 37 ℃ and 5% carbon dioxide by volume to obtain a polymer scaffold system loaded with bone marrow mesenchymal stem cells.

[0010] In stem cell transplantation for tissue damage treatment, directly injecting a suspension of stem cells into the damaged area is a common method. However, free stem cells are difficult to survive and retain for long periods after implantation; most cells are cleared by the body or lost with bodily fluids within days. This results in a significantly lower actual number of stem cells remaining at the site of injury compared to the initial transplantation, thus severely limiting the therapeutic effect. Furthermore, stem cells expanded in traditional two-dimensional culture systems experience reduced intercellular contact and loss of extracellular matrix support after being removed from the culture dish. This leads to varying degrees of decline in stem cell maintenance and paracrine function, further affecting their repair efficacy in vivo.

[0011] To address the aforementioned issues, this invention utilizes a porous polymer scaffold prepared from sodium alginate and carboxymethyl chitosan as a stem cell delivery carrier. This scaffold possesses a highly interconnected, porous mesh structure. Bone marrow mesenchymal stem cell suspension is drop-added into the sterilized scaffold and incubated statically. The cell suspension is drawn into the scaffold's internal pores via capillary action. The pore walls of the scaffold are constructed from a hydrophilic polysaccharide material, providing a favorable adhesion interface for cells, allowing them to adhere to the pore surface and gradually aggregate. The large-pore scaffold has a pore size of 150-200 μm, capable of accommodating multiple cells that can contact each other and form three-dimensional cell spheres. The slight elasticity of the pore walls provides space for cell sphere growth and also physically anchors the cell spheres within the pores, preventing loss during subsequent operations or in vivo transplantation. The small-pore scaffold allows cells to adhere to the pore walls in a dispersed state, suitable for culture scenarios requiring uniform cell distribution. The high porosity and pore connectivity of the scaffold ensure the full diffusion of nutrients and oxygen into the scaffold interior, while facilitating the timely removal of cell metabolic products, thus providing a suitable microenvironment for the survival and functional maintenance of the loaded cells.

[0012] The stem cell-scaffold composite system constructed using this method maintains the activity of bone marrow mesenchymal stem cells during in vitro culture, exhibiting good cell viability. Furthermore, cells within the large-pore scaffold gradually form larger three-dimensional cell spheroids. After transplantation into the body, the scaffold acts as a physical barrier and protective carrier, significantly prolonging the retention time of stem cells at the transplantation site and increasing the actual number of stem cells remaining at the injury site. These results lay the cellular foundation for subsequent immune regulation and tissue regeneration effects of stem cells through the paracrine pathway, effectively enhancing the tissue repair efficacy of stem cell therapy.

[0013] Preferably, step B3 involves adding the cell suspension to the sterilized scaffold using a vacuum-assisted permeation inoculation method, specifically including the following steps: B3-1: Place the sterilized support in a vacuum inoculation container, which has a sealing cap, an air extraction port connected to a vacuum pump, and a liquid inlet. B3-2: Close the sealing cap, start the vacuum pump, and evacuate the pressure inside the container to -0.02 to -0.06 MPa, and maintain it for 1-3 minutes to fully remove the residual air in the pores of the support. B3-3: Maintain the above vacuum level, and slowly inject the pre-prepared cell suspension into the container in a continuous flow manner through the inlet until the cell suspension completely submerges the scaffold; B3-4: Close the liquid inlet and the air extraction port, and slowly release the vacuum to allow the pressure inside the container to gradually return to normal pressure within 30-60 seconds, so that the cell suspension can be evenly pressed into the entire interconnected pore network of the scaffold under atmospheric pressure. B3-5: Transfer the container to a 37 ℃ incubator and incubate for 2 h to allow the cells to fully adhere to the three-dimensional porous surface inside the scaffold, thus completing the seeding process.

[0014] In loading cell suspensions onto porous scaffolds, the conventional method involves directly dripping the cell suspension onto the scaffold surface, allowing it to slowly penetrate the pores through gravity and capillary action. However, for porous scaffolds with highly interconnected structures, the air initially filling the pores creates resistance to fluid permeation. When a droplet contacts the scaffold surface, some air becomes trapped deep within the pores, creating an airlock that hinders further diffusion of the cell suspension towards the scaffold center. Simultaneously, differences in capillary forces across pore sizes lead to uneven fluid permeation rates within the scaffold, with some areas experiencing sufficient permeation while others suffer from insufficient permeation. The combined effect of these factors results in a heterogeneous distribution of cells within the scaffold, exhibiting surface aggregation and central sparseness, or localized aggregation and cell-free areas. This uneven distribution directly impacts the uniformity of three-dimensional cell spheroid formation during subsequent culture, reducing the overall quality of the stem cell-scaffold composite system.

[0015] To address this issue, this invention introduces a vacuum-assisted permeation inoculation step based on the conventional dripping method. The sterilized scaffold is placed in a vacuum inoculation container equipped with a sealing cap, an air extraction port, and a liquid inlet. After closing the sealing cap, the vacuum pump is activated to reduce the pressure inside the container to -0.02 to -0.06 MPa and maintain this pressure for 1-3 minutes. Under this negative pressure condition, residual air inside the scaffold pores is actively extracted, eliminating the obstruction at the gas-liquid interface. Maintaining a constant vacuum, a pre-prepared cell suspension is slowly injected into the container in a continuous flow through the liquid inlet until the cell suspension completely submerges the scaffold. Subsequently, the liquid inlet and air extraction port are closed, and the vacuum is slowly released over 30-60 seconds, allowing the pressure inside the container to gradually return to atmospheric pressure. During this process, the atmospheric pressure difference acts as a driving force, uniformly pushing the cell suspension into all the interconnected pores of the scaffold, achieving a homogeneous distribution of the cell suspension within the three-dimensional space of the scaffold.

[0016] The polymer scaffold system loaded with stem cells prepared using this method exhibits a more uniform cell distribution density across all regions within the scaffold, avoiding the uneven cell distribution problem commonly encountered in conventional drop-addition methods. The cell suspension fully fills the entire pore network of the scaffold, laying a solid foundation for uniform cell proliferation in three-dimensional space during subsequent culture. This facilitates the formation of uniformly sized and functionally stable three-dimensional cell spheres within the scaffold, thereby improving the overall quality and reliability of the stem cell-scaffold composite system in both in vitro culture and in vivo transplantation applications.

[0017] Preferably, the culture is continued using a three-dimensional dynamic perfusion culture method, which specifically includes the following steps: B4-1: After incubation, the scaffold is transferred to a micro-perfusion chamber that matches the size of the scaffold. The two ends of the micro-perfusion chamber are connected to the inlet and outlet of the culture medium circulation system, respectively. The inner wall of the chamber is tightly fitted to the outer wall of the scaffold to form a sealed fluid channel. B4-2: Start the culture medium circulation system to drive the complete culture medium to flow directionally through the internal porous network of the scaffold at a flow rate of 0.05-2.0 mL / min, so as to continuously and dynamically culture the three-dimensional cell spheres of stem cells in the scaffold; The direction of infusion is switched every 12-24 hours.

[0018] In the three-dimensional culture of stem cells, the exchange of substances within the scaffold primarily relies on passive diffusion of the culture medium. As the culture time increases, cells in the deeper layers of the scaffold, especially those in the central region of larger cell spheroids, gradually face problems of insufficient nutrient supply and accumulation of metabolic waste. Key nutrients such as oxygen and glucose in the culture medium are preferentially consumed by outer cells along the diffusion path, resulting in a significant decrease in concentration reaching the cell spheroid core. Meanwhile, waste products such as lactic acid generated by cell metabolism are difficult to effectively expel from the core region. This mass transfer limitation leads to a nutrient gradient within the cell spheroid. Cells in the central region, due to prolonged exposure to a nutrient-poor, waste-rich microenvironment, experience decreased activity and may even die, thus limiting the size, activity, and function of cell spheroids in the three-dimensional culture system.

[0019] To address the aforementioned issues, this invention introduces a three-dimensional dynamic perfusion culture method after cell incubation. The scaffold, having completed cell incubation, is transferred to a micro-perfusion chamber of a matching size. The two ends of the chamber are connected to the inlet and outlet of the culture medium circulation system, respectively, with the inner wall of the chamber tightly fitted to the outer wall of the scaffold to form a sealed fluid channel. After starting the culture medium circulation system, the complete culture medium flows directionally through the internal pore network of the scaffold at a flow rate of 0.05-2.0 mL / min. During this process, forced convection of the culture medium replaces simple passive diffusion, significantly improving the material exchange efficiency deep within the scaffold. Nutrients are actively transported to the core region of the cell spheroids, while metabolic waste is promptly carried away by the flowing culture medium. The perfusion direction is switched every 12-24 hours to avoid concentration gradients and uneven stress on the cell surface that may result from long-term unidirectional flow.

[0020] The stem cell scaffold system cultured using this method effectively maintains cell viability and proliferation capacity, especially in the central region of the cell spheroids. The size and uniformity of the cell spheroids are superior to those of static culture, and the incidence of central cell necrosis is significantly reduced. The fluid shear force within the physiological range generated by dynamic perfusion can activate mechanosensitive ion channels and related signaling pathways on the stem cell membrane, synergistically promoting cell survival, proliferation, and paracrine function. The polymer scaffold system loaded with stem cells prepared in this manner exhibits higher cell viability and stronger function, providing a higher-quality source of seed cells for subsequent in vivo transplantation.

[0021] Preferably, the culture medium circulation system adopts a multi-channel parallel-series composite loop design, specifically including the following steps: C1: Prepare a culture medium circulation control system, which includes a bidirectional peristaltic pump, a culture medium storage bottle, and a multi-channel split-merge integrated module; C2: The multi-channel diversion-merging integrated module is internally equipped with one main inlet channel, one main outlet channel, and N parallel independent culture channels, where N≥3 and N is an integer; each independent culture channel is connected to the main inlet channel and the main outlet channel at both ends, respectively. C3: In each independent culture channel, 2-3 incubation scaffolds are sequentially embedded in series along the fluid flow direction; the scaffolds are kept at a fixed interval by built-in mesh rings to ensure that the surface of each scaffold is exposed to the directional flow of culture medium; C4: Start the bidirectional peristaltic pump and set the circulation flow rate to 0.05-2.0 mL / min. The culture medium flows out of the storage bottle, passes through the main inlet channel, is evenly distributed to N independent culture channels, flows through 2-3 supports connected in series in each channel, converges to the main outlet channel, and finally returns to the storage bottle, forming a complete closed loop. C5: Every 12-24 hours of culture, the rotation direction of the bidirectional peristaltic pump is controlled to reverse the flow direction of the culture medium in the loop, so that N independent culture channels can simultaneously perform bidirectional alternating circulation perfusion.

[0022] In practical applications of dynamic perfusion culture, conventional single-channel or dual-channel perfusion systems face insufficient throughput when multiple stem cell-scaffold complexes need to be prepared simultaneously for subsequent experiments or transplantation. Culturing multiple scaffolds one by one is not only time-consuming, but also results in batch-to-batch variations in cell spheroid size, cell viability, and function due to subtle differences in reactor conditions, culture medium batches, and environmental conditions, affecting the reproducibility of experimental data and the uniformity of complex quality. Furthermore, even within the same batch culture, multiple scaffolds placed along the flow direction within a single channel exhibit differences in their microenvironments due to nutrient consumption along the way, further increasing sample inconsistencies.

[0023] To address the aforementioned issues, this invention optimizes the culture medium circulation system based on dynamic perfusion culture, employing a multi-channel parallel-series composite loop structure. The system comprises a bidirectional peristaltic pump, a culture medium reservoir, and a multi-channel diversion-merging integrated module. This module internally features a main inlet channel, a main outlet channel, and N parallel independent culture channels, where N is an integer greater than or equal to 3. Each independent culture channel is connected to both the main inlet and outlet channels at its two ends. Within each independent culture channel, 2-3 pre-incubated cell scaffolds are sequentially embedded in series along the fluid flow direction, with the scaffolds maintained at a fixed interval by built-in spacer rings. Upon activation of the bidirectional peristaltic pump, the culture medium flows from the reservoir, sequentially through the main inlet channel, evenly distributed to the N independent culture channels, flowing through the 2-3 scaffolds connected in series within each channel, converging into the main outlet channel, and finally returning to the reservoir, forming a closed loop. Every 12-24 hours of culture, the rotation direction of the bidirectional peristaltic pump is controlled to reverse the flow direction of the culture medium in the loop, so that all independent culture channels can be simultaneously perfused in a bidirectional alternating cycle.

[0024] When using this system for culture, the parallel design ensures consistency of hydrodynamic conditions between independent culture channels. Culture medium from the same main channel is evenly distributed to each channel, eliminating differences between channels. Two to three scaffolds are connected in series in each channel, combined with bidirectional alternating perfusion, effectively eliminating nutrient concentration gradients along the flow direction and making the microenvironment of scaffolds at different locations within the same channel more consistent. The closed-loop system ensures thorough mixing and continuous circulation of a limited volume of culture medium across all channels, further reducing individual differences between scaffolds. Through this method, multiple scaffolds can be homogenized and cultured simultaneously in a single experiment, obtaining stem cell-scaffold complexes with highly consistent cell spheroid size, cell viability, and function, meeting the requirements for the quantity and quality of parallel samples for subsequent in vivo transplantation or statistical analysis.

[0025] Preferably, the culture medium circulation system employs a gradient flow rate control strategy for three-dimensional dynamic perfusion culture, specifically including the following steps: D1: On days 1-3 of the culture cycle, the circulation rate of the bidirectional peristaltic pump is set to a low flow rate mode of 0.05-0.1 mL / min to allow the complete culture medium to form a laminar flow in the pores of the scaffold, maintain a weak fluid shear force, and promote the initial adhesion of bone marrow mesenchymal stem cells and the formation of three-dimensional cell spheres. D2: During the 4th to 7th day of the culture cycle, the circulation flow rate is increased to a medium flow rate mode of 0.3-0.5 mL / min to enhance forced convection inside the scaffold, improve the efficiency of oxygen and nutrient transfer to the core region of the cell sphere, and activate the cell's mechanosensitive ion channels through fluid shear force within the physiological range to promote cell proliferation and paracrine function. D3: On days 8-15 of the culture cycle, the circulation flow rate was further adjusted to a high flow rate mode of 0.6-1.0 mL / min, and the frequency of perfusion direction switching was increased to once every 8-12 h. This was done to maximize the removal of metabolic waste and stimulate the expression of genes related to extracellular matrix remodeling, while ensuring that cells were not washed away and detached. D4: On day 16 and thereafter of the culture cycle, when the cell spheroid diameter reaches 200-250 μm, the circulation flow rate is switched back to the maintenance mode of 0.2-0.3 mL / min. At the same time, an intermittent perfusion strategy is introduced, which involves perfusion for 30 min followed by a 15 min pause, to simulate the intermittent characteristics of tissue fluid flow in vivo, prevent excessive cell spheroid growth leading to central necrosis, and maintain the long-term activity and functional stability of the formed three-dimensional cell spheroids.

[0026] While constant flow rate culture improves material exchange within the scaffold in dynamic perfusion culture, the response of three-dimensional stem cell spheres to nutrient requirements and hydrodynamic stimuli varies significantly at different stages of the growth cycle. In the early stages of culture, newly seeded cells are not fully adhered and their numbers are low; excessively high flow rates may wash cells off the scaffold pores, affecting sphere formation. During the rapid proliferation phase, sphere size gradually increases, leading to a sharp rise in oxygen and nutrient demand, along with increased metabolic waste production. At this stage, stronger mass transfer capabilities are needed to maintain normal sphere growth. As spheres grow to a certain size, their central region faces the risk of hypoxia and nutrient depletion, but excessive fluid shear forces may damage the cells. In the later stages of culture, maintaining long-term cell viability and functional stability is crucial, but continuous high-flow-rate perfusion may lead to overgrowth or central necrosis. The constant flow rate mode cannot simultaneously meet the differentiated needs of these different stages, limiting the overall efficiency of the three-dimensional culture system.

[0027] To address this issue, this invention introduces a gradient flow rate control strategy based on three-dimensional dynamic perfusion culture, dynamically adjusting perfusion parameters according to the growth stages of the three-dimensional cell spheroids of stem cells. During days 1-3 of the culture cycle, a low flow rate of 0.05-0.1 mL / min is used, allowing the complete culture medium to form laminar flow within the scaffold pores, maintaining weak fluid shear forces, which is beneficial for the initial adhesion of bone marrow mesenchymal stem cells and the formation of three-dimensional cell spheroids. During days 4-7, the circulation flow rate is increased to a medium flow rate of 0.3-0.5 mL / min, enhancing forced convection within the scaffold and improving the efficiency of oxygen and nutrient transfer to the core region of the cell spheroids. Simultaneously, the fluid shear forces within the physiological range activate mechanosensitive ion channels, promoting cell proliferation and paracrine function. During days 8-15, the flow rate is further adjusted to a high flow rate of 0.6-1.0 mL / min, while increasing the frequency of perfusion direction switching to once every 8-12 hours. This maximizes the removal of metabolic waste and stimulates the expression of genes related to extracellular matrix remodeling, while ensuring that cells are not washed away and detached. On day 16 and thereafter of the culture period, when the cell spheroid diameter reached 200-250 μm, the flow rate was switched back to a maintenance mode of 0.2-0.3 mL / min. At the same time, an intermittent perfusion strategy of pausing for 15 min after 30 min of perfusion was introduced to simulate the intermittent characteristics of tissue fluid flow in vivo.

[0028] Using the aforementioned gradient flow rate control strategy for three-dimensional dynamic perfusion culture, cells were placed in a hydrodynamic environment tailored to their needs at different growth stages. Initially, low flow rates ensured effective cell colonization and spheroidization; moderate flow rates activated mechanotransduction signals through appropriate fluid shear force, promoting cell proliferation; high flow rates combined with frequent reversals overcame the mass transfer limit of the enlarged cell spheroids; and later, reduced flow rates and intermittent perfusion maintained material exchange while avoiding excessive shear damage. Throughout the culture period, the size, activity, and function of the cell spheroids were synergistically optimized, the incidence of central necrosis within the cell spheroids was significantly reduced, and the homogeneity and functional stability of the three-dimensional cell spheroids were effectively improved, providing a more reliable source of stem cells for subsequent in vivo transplantation.

[0029] Preferably, within the multi-channel shunt-merge integrated module, a microfluidic fluid resistance balancing structure is also provided at the inlet or outlet of each independent culture channel; the microfluidic fluid resistance balancing structure is a reduced-diameter capillary section with an inner diameter of 0.2-0.5 mm and a length of 3-8 mm.

[0030] In multi-channel parallel-series composite loop culture systems, slight differences in the porosity and size of scaffolds within different independent culture channels, as well as inconsistencies in tubing connections, often lead to uneven distribution of culture medium flow rate and volume in each channel. This results in batch-to-batch variations in the culture quality of stem cell-scaffold composites across different channels, affecting the parallelism of experimental results and the uniformity of subsequent transplantation samples. To address this challenge, this invention introduces a microfluidic fluid resistance balancing structure at the inlet or outlet of each independent culture channel in the shunt-merge integrated module. This structure employs a reduced-diameter capillary segment with an inner diameter of 0.2-0.5 mm and a length of 3-8 mm. When the culture medium flows through this structure, a preset local pressure drop is generated. This local pressure drop is designed to be significantly larger than the frictional resistance variation range generated by the 2-3 scaffolds connected in series and their connecting tubing within the same channel. Consequently, the total fluid resistance of each independent culture channel is primarily determined by this balancing structure. In this design, resistance fluctuations of other components within the channel are effectively masked, and the total resistance of each parallel channel driven by the bidirectional peristaltic pump tends to be consistent, thereby achieving a uniform distribution of circulation velocity and flow rate. This scheme not only eliminates the influence of individual scaffold differences and tubing assembly errors on fluid distribution, but also maintains a high degree of consistency in the hydrodynamic conditions between channels during bidirectional alternating perfusion, ensuring that all stem cell-scaffold complexes cultured in the same batch receive an equal microenvironment in terms of nutrient supply and shear stress stimulation. This significantly improves the uniformity of the complex in terms of cell spheroid size, cell viability, and paracrine function, providing reliable quality assurance for subsequent parallel-controlled experiments for in vivo transplantation and large-scale clinical applications.

[0031] The application of a method for preparing a sodium alginate-carboxymethyl chitosan porous polymer scaffold in the preparation of intrauterine adhesion repair materials.

[0032] A method for preparing a porous polymer scaffold system loaded with bone marrow mesenchymal stem cells, and the application of the prepared scaffold system in the preparation of intrauterine adhesion repair materials.

[0033] The present invention has at least the following beneficial effects: Firstly, this invention provides a method for preparing a sodium alginate-carboxymethyl chitosan porous polymer scaffold. This method uses sodium alginate and carboxymethyl chitosan as raw materials, forming a double-network gel through calcium ion crosslinking. The ice crystal size is then controlled by different freezing temperatures, followed by vacuum drying to sublimate the ice crystals and form pores. The entire preparation process eliminates the need for chemical porogens such as sodium chloride and paraffin, avoiding the biosafety risks associated with residual organic solvents. Furthermore, precise control of the scaffold pore size can be achieved through simple temperature selection. The resulting large-pore scaffold has an average pore size of 150-200 μm, and the small-pore scaffold has an average pore size of 10-35 μm. Both types of scaffolds exhibit a porosity exceeding 90% and highly interconnected pores, providing a structurally controllable and high-performance carrier material for subsequent cell loading and tissue repair.

[0034] Secondly, this invention constructs a stem cell-scaffold composite system by loading bone marrow mesenchymal stem cells onto the aforementioned scaffold. The highly interconnected mesh-like porous structure of the scaffold provides a three-dimensional adhesion carrier for cells. The elastic pore walls of the large-pore scaffold can both accommodate cell aggregation to form three-dimensional cell spheres and fix the cell spheres within the pores through slight elastic force, effectively prolonging the survival and retention time of stem cells in vivo. Further, a vacuum-assisted permeation seeding process ensures uniform distribution of the cell suspension within the scaffold, avoiding the uneven cell distribution problem caused by conventional drop-feeding methods. Three-dimensional dynamic perfusion culture, combined with a multi-channel parallel-series composite circuit design and gradient flow rate control strategy, significantly improves the material exchange efficiency within the scaffold, ensuring that the cell spheres are in a suitable hydrodynamic environment at different growth stages. The size, activity, and function of the cell spheres are synergistically optimized, and the incidence of central necrosis of the cell spheres is significantly reduced.

[0035] Third, this invention applies the stem cell-scaffold composite system to the repair and treatment of intrauterine adhesions. In vivo experiments show that it can promote the morphological recovery of damaged endometrium, increase endometrial thickness and glandular number, reduce fibrosis area, and simultaneously promote macrophage polarization towards the M2 type and angiogenesis. Fertility restoration experiments show that rats treated with this composite system have significantly increased pregnancy rates, restored fetal number and weight to normal, and no obvious blood toxicity or pathological changes in major organs were observed; placental tissue structure remained normal. These results indicate that the polymer scaffold and its stem cell-loaded composite system provided by this invention have good biocompatibility and tissue repair capabilities, promote endometrial regeneration and fertility recovery, and provide a new and effective strategy for the treatment of intrauterine adhesions.

[0036] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0037] Figure 1 The images show scanning electron microscope (SEM) images of the microstructure of porous polymer scaffolds; where a is a cross-sectional SEM image of a small-pore scaffold (S-PMS); and b is a cross-sectional SEM image of a large-pore scaffold (L-PMS). Figure 2 This is an X-ray energy dispersive spectroscopy (EDS) elemental surface mapping image of the porous polymer scaffold surface; Figure 3 The image shows the pore structure characterization of the porous polymer scaffold using mercury intrusion porosimetry. Figure 4 The swelling performance curve of the porous polymer scaffold is shown. Figure 5 Confocal microscopy images of cell spheres at different days during biocompatibility evaluation using S-PMS and L-PMS. Figure 6 Figure 1 shows the results of an in vitro angiogenesis experiment. Figure 2 shows microscopic images of tubular structures formed by human umbilical vein endothelial cells (HUVECS) under different culture conditions (control group, SCM, LCM); Figure 3 shows a statistical graph of the number of major pore nodes in the tubular structures formed by HUVECS under different culture conditions; Figure 4 shows a statistical graph of the number of major channel segments in the tubular structures formed by HUVECS under different culture conditions; and Figure 5 shows a statistical graph of the length of the channel segments in the tubular structures formed by HUVECS under different culture conditions. Figure 7 This is a flowchart of the overall experimental procedure for repairing endometrial injury in rats; where a is the overall experimental flowchart and b is the specific surgical procedure. Figure 8Gross morphology of the uterus observed at different time points (day 7, day 15, and day 30) after surgery in rats of different groups; Figure 9 Close-up comparison images of the gross morphology of the uterus at different time points (day 7, day 15, and day 30) after surgery in rats from different groups; Figure 10 HE staining image for safety evaluation of major organs in rats; Figure 11 The figures show the results of the rat fertility recovery experiment; where a is a gross morphological image of the pregnant uterus of rats in different groups; b is a visual morphological image of the pregnant uterus of rats in different groups, with numbers indicating fetal counts; and c is a morphological image of the fetuses inside the pregnant uterus of rats in different groups. Detailed Implementation

[0038] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0039] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0040] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0041] Experiment 1: Preparation and structural and performance characterization of sodium alginate-carboxymethyl chitosan polymer scaffold PMS 1. Preparation of sodium alginate-carboxymethyl chitosan porous polymer scaffold (PMS) First, the solutions were prepared. 8 g of sodium alginate (SA) powder and 6 g of carboxymethyl chitosan (CMCS) powder were accurately weighed using an electronic balance and placed in two separate containers. 300 ml of deionized water was added to each container, and the mixtures were magnetically stirred at a constant temperature of 40 ℃ for 2 h to ensure complete dissolution of the powders, yielding sodium alginate solution with a mass fraction of approximately 2.7% and carboxymethyl chitosan solution with a mass fraction of approximately 2%. Subsequently, both solutions were filtered through a 0.22 μm microporous membrane to remove insoluble impurities and prevent contamination during subsequent culture.

[0042] Next, mixing and cross-linking were performed. The filtered sodium alginate solution and carboxymethyl chitosan solution were combined and magnetically stirred at 40°C for 2 hours to ensure thorough mixing and form a homogeneous composite solution. Separately, anhydrous calcium chloride was dissolved in deionized water to prepare a 0.1 mol / L calcium chloride solution, which was used as a cross-linking agent. Under continuous stirring, the calcium chloride solution was slowly added dropwise to the composite solution using a syringe, closely observing the system's state. Addition was stopped when the mixture gradually transformed into a gel-like state. The resulting gel was injected into a pre-prepared 3D-printed mold and allowed to stand at room temperature for 48 hours to allow the network structure to fully solidify.

[0043] Finally, pore size was controlled by freeze-drying. The cross-linked gel was removed from the mold and treated using two different drying methods: First, the gel was directly placed in a freeze dryer and pre-frozen at -40 °C for 12 h, followed by vacuum drying for 24 h after complete solidification, yielding a large-pore polymer scaffold (L-PMS). Second, the gel was first rapidly frozen in a sealed container filled with liquid nitrogen until it turned completely milky white, then quickly transferred to a freeze dryer for vacuum drying for 24 h, yielding a small-pore polymer scaffold (S-PMS). Through these different freezing treatments at different temperatures, porous scaffold materials with different pore sizes can be obtained.

[0044] 2. Structural characterization and performance testing of PMS 2.1 Microstructure Analysis: The microstructure of polymer scaffolds with different pore sizes was observed using scanning electron microscopy (SEM). The results are as follows: Figure 1 As shown. By Figure 1 It can be seen that large-diameter stents (L-PMS) Figure 1 b) with small-aperture stents (S-PMS, Figure 1 a) Both exhibit a highly interconnected network-like porous structure, which facilitates the transport of nutrients into the scaffold and the removal of metabolic waste during cell culture. Pore size measurements showed that the average pore size of L-PMS was 177.25 ± 13.38 μm, significantly larger than that of S-PMS (28.25 ± 2.36 μm). This observation is close to the pore size data obtained by subsequent mercury intrusion porosimetry. Further elemental surface scanning analysis of the scaffold surface was performed using energy dispersive X-ray spectroscopy (EDS). Figure 2 The results showed that the four elements Ca, C, N and O were uniformly distributed on the surface of the scaffold, and no other impurity elements were detected, indicating that sodium alginate and carboxymethyl chitosan successfully underwent a cross-linking reaction, and the cross-linking product had a uniform elemental composition.

[0045] 2.2 Pore Size Distribution and Porosity Measurement: The pore structure of the two types of gel scaffolds with different pore sizes was quantitatively analyzed using mercury porosimetry. The results are as follows: Figure 3As shown in the figure. Porosity measurements show that the porosities of S-PMS and L-PMS are quite similar, at 92.36% and 93.72%, respectively. Pore size distribution analysis indicates that the average pore size of L-PMS is 159 μm, significantly larger than that of S-PMS (12 μm). It is noteworthy that the pore size values ​​measured by mercury porosimetry are lower than those observed by scanning electron microscopy (SEM). This is because the principle of mercury porosimetry is based on the pressure required for mercury to enter the pores to estimate the pore size. When the inlet size of the pore is smaller than the internal pore size, the measured pressure reflects the inlet size rather than the actual internal pore size, thus resulting in a slightly smaller measured value than the pore size directly observed by SEM.

[0046] 2.3 PMS Swelling Performance: The swelling performance of the polymer scaffold in PBS was tested at room temperature. The results showed that the scaffolds exhibited water absorption and swelling behavior after immersion in PBS, with the weight gradually increasing with prolonged immersion time. There was a significant difference in the time required for the two pore sizes of scaffolds to reach swelling equilibrium: S-PMS reached equilibrium in approximately 4 hours, while L-PMS reached equilibrium in only approximately 1 minute. Figure 4 The time it takes for L-PMS to reach swelling equilibrium is approximately 1 / 240 that of S-PMS, indicating that the large-pore scaffold has a more porous structure, enabling it to rapidly absorb liquid and quickly reach swelling equilibrium.

[0047] Furthermore, performance tests on the polymer scaffolds showed that the material exhibits excellent controllable degradation, cell affinity, and tissue adhesion. In in vitro degradation experiments, the scaffolds showed a continuous degradation trend in solutions containing lysozyme or hyaluronidase. The large-pore scaffold (L-PMS), due to its more porous structure, showed a slightly higher degradation rate than the small-pore scaffold (S-PMS), with a longer overall degradation time, which is beneficial for prolonging the survival of loaded cells. After in vivo transplantation, the scaffold degradation behavior was consistent with in vitro degradation, but the degradation rate was faster, essentially completed in about 10 days. Cell adsorption experiments showed that L-PMS could adsorb approximately 80% of cells within 1 minute, significantly faster than S-PMS's 40%. Both reached saturation after 30 minutes, indicating that the large-pore scaffold has a greater advantage in rapidly capturing cells. Contact angle tests further confirmed the scaffold's good hydrophilicity; the water absorption rate of L-PMS was significantly faster than that of S-PMS, facilitating rapid penetration of cell suspensions. A finger bending model showed that the scaffolds could adhere tightly to the skin surface at different angles, demonstrating good tissue adhesion performance. In summary, this polymer scaffold exhibits excellent performance in terms of degradation characteristics, cell loading capacity, and tissue adaptability.

[0048] Experiment 2: In vitro study of the effects of sodium alginate-carboxymethyl chitosan polymer scaffold loaded with BMSCs 1. Preparation of a porous polymer scaffold system loaded with bone marrow mesenchymal stem cells (BMSCs-PMS) The prepared polymer scaffolds were sterilized by electron beam irradiation and then used for later use. Third-generation bone marrow mesenchymal stem cells (BMSCs) in good growth condition were collected, digested with 0.25% trypsin, and the cell suspension was transferred to sterile centrifuge tubes. The cells were centrifuged at 1000 rpm for 5 min at room temperature, the supernatant was discarded, and the cells were resuspended in fresh culture medium. Cell counts were performed, and the cell density was adjusted to ensure a total cell count of 1 × 10⁻⁶ cells per centrifuge tube. 7 Sterilized PMS was cut into uniformly sized discs using a 6 mm diameter punch. Based on the maximum adsorption volume ratio of the scaffold determined in previous experiments, an appropriate amount of cell suspension was slowly added dropwise to the PMS, and the discs were incubated at 37°C for 2 hours to allow the cells to fully adhere to the interior of the scaffold. Then, sufficient complete culture medium was added to the culture container to completely submerge the scaffold, and the culture was continued for subsequent experiments.

[0049] 2. Performance testing of a porous polymer scaffold system loaded with bone marrow mesenchymal stem cells: Polymer scaffold extracts were prepared according to ISO 10993-1:2018 and GB / T 16886.1-2022, "Biological Evaluation of Medical Devices". 0.2 g of S-PMS and L-PMS were weighed at a ratio of 0.1 g / mL, and added to complete culture medium based on the previously determined maximum water absorption ratio of the scaffold. After the scaffold reached equilibrium and swelled sufficiently, complete culture medium was added to bring the total volume to 2 mL. The samples were incubated at 37 ℃ for 72 h, and the supernatant was collected as the stock PMS extract. The stock extract was diluted with complete culture medium to the required concentration for subsequent experiments.

[0050] 2.1 Biocompatibility evaluation of PMS: The cell viability of BMSCs cultured in scaffold extract was detected using the CCK-8 assay. Cell viability was greater than 85% in all concentration groups, and no significant cytotoxicity was observed. Confocal microscopy observation showed that ( Figure 5 In L-PMS, cells aggregated into spheres and were embedded within the pores, with the diameter of the cell spheres gradually increasing with prolonged culture time; in S-PMS, cells were dispersed. These results indicate that the polymer scaffold has good biocompatibility and can provide a suitable three-dimensional growth microenvironment for BMSCs.

[0051] 2.2 Biological function evaluation of scaffold systems loaded with BMSCs: To evaluate the in vitro biological function of the BMSC-loaded scaffold system, conditioned medium (SCM and LCM) containing BMSCs loaded with S-PMS and L-PMS for 72 h of culture was collected and subjected to a series of assays.

[0052] Paracrine activity: ELISA showed that the concentrations of pro-repair factors FGF-2, TGF-β1 and VEGF-A in LCM were 18%, 14% and 67% higher than those in SCM, respectively, indicating that BMSCs loaded with L-PMS had stronger paracrine activity.

[0053] Promoting angiogenesis: In in vitro angiogenesis experiments ( Figure 6 The LCM group showed obvious tubular structure formation and network complexity was higher than that of the SCM group, indicating that it has a good pro-angiogenic ability.

[0054] Experiment 3: Mechanism study of how sodium alginate-carboxymethyl chitosan polymer scaffold-loaded BMSCs promote endometrial regeneration and fertility recovery. 1. BMSCs-L-PMS promotes morphological recovery and endometrial regeneration of damaged uterus in rats: To verify the repair effect of the polymer scaffold system loaded with BMSCs on damaged endometrium, a uterine injury model was established in rats. Figure 7 In the model group (IUA), the endometrial layer disappeared, and the luminal epithelium and glands were significantly reduced. The model rats were randomly divided into 5 groups: sham-operated group (Sham), model group (IUA), BMSCs group, BMSCs-HA group, and BMSCs-L-PMS group. Samples were collected and observed at 7, 15, and 30 days post-operation. Figure 8 BMSCs-HA is a cell-hydrogel composite system prepared by mixing bone marrow mesenchymal stem cells (BMSCs) with hyaluronic acid (HA) hydrogel.

[0055] Gross observations show that ( Figure 9 Seven days post-surgery, the IUA group showed significant uterine adhesions and fluid accumulation; the BMSCs-L-PMS group had a relatively intact endometrial structure and near-normal morphology. Thirty days post-surgery, both the IUA and BMSCs groups showed complete uterine cavity closure with substantial fluid accumulation; the BMSCs-HA group exhibited less uterine adhesions and fluid accumulation than the IUA group, but some endometrial structural abnormalities remained, while the uterine morphology in the BMSCs-L-PMS group had essentially returned to normal.

[0056] Histological evaluation showed that 30 days post-operation, the IUA group still exhibited obvious fibrous bundles, thin endometrium, and sparse glands. The BMSCs-L-PMS group had endometrial thickness and glandular quantity approaching the levels of the sham surgery group, with a significant reduction in fibrosis area.

[0057] The above results indicate that the BMSCs-L-PMS complex system is most effective in promoting the recovery of damaged endometrial thickness, glandular regeneration, and inhibiting fibrosis.

[0058] 2. L-PMS promotes the survival and retention of BMSCs in vivo: Through in vivo imaging monitoring, the fluorescence intensity of the BMSCs-L-PMS group was significantly higher than that of the BMSCs group and the BMSCs-HA group at all time points (P<0.05~0.001), and the signal could still be detected on day 14, indicating that PMS can significantly prolong the survival and retention time of BMSCs in the uterine cavity.

[0059] 3. Evaluation of blood safety: Blood index tests 30 days after treatment showed no significant difference between the treatment groups and the Sham group, indicating that the treatment did not cause blood toxicity.

[0060] 4. Organ safety evaluation ( Figure 10 HE staining of major organs showed no obvious pathological changes, indicating that the treatment did not cause organ toxicity damage.

[0061] 5. BMSCs-L-PMS promote fertility recovery in rats: Pregnancy experiment ( Figure 11 The results showed that the pregnancy rate in the BMSCs-L-PMS group reached 80%, the number of fetuses (10.2±1.6 per litter) was close to that in the Sham group, the fetuses had normal weight and no deformities; the F1 generation grew and developed normally, and the F2 generation had no abnormalities, indicating that reproductive function was effectively restored and there was no reproductive toxicity.

[0062] Example 1 A method for preparing a porous polymer scaffold system loaded with bone marrow mesenchymal stem cells includes the following steps: B1: The prepared large-pore porous polymer scaffold is sterilized to obtain a sterilized scaffold. B2: Bone marrow mesenchymal stem cells passaged to the third generation were resuspended in culture medium to prepare a density of 2×10⁻⁶. 7 Cell suspension of cells per mL; B3: Add the cell suspension to the sterilized scaffold at a volume ratio of 1:10, and incubate at 37 °C for 2 h to allow the cell suspension to be completely absorbed by the sterilized scaffold, thus obtaining the incubated scaffold. B4: Add complete culture medium to the incubated scaffold to completely submerge the scaffold, and continue culturing in an incubator at 37 ℃ with 5% carbon dioxide by volume to obtain the polymer scaffold system loaded with bone marrow mesenchymal stem cells.

[0063] Example 2 A method for preparing a porous polymer scaffold system loaded with bone marrow mesenchymal stem cells, differing from Example 1 in that step B3, where the cell suspension is added to the sterilized scaffold using a vacuum-assisted permeation seeding method, is used instead. Specifically, the method includes the following steps: B3-1: Place the sterilized support in a vacuum inoculation container, which has a sealing cap, an air extraction port connected to a vacuum pump, and a liquid inlet. B3-2: Close the sealing cap, start the vacuum pump, evacuate the pressure inside the container to -0.04 MPa, and maintain it for 2 minutes to fully expel the residual air in the pores of the support. B3-3: Maintain the above vacuum level, and slowly inject the pre-prepared cell suspension into the container in a continuous flow manner through the inlet until the cell suspension completely submerges the scaffold; B3-4: Close the liquid inlet and the air extraction port, and slowly release the vacuum to allow the pressure inside the container to gradually return to normal pressure within 45 seconds, so that the cell suspension can be evenly pressed into the entire interconnected pore network of the scaffold under atmospheric pressure. B3-5: Transfer the container to a 37 ℃ incubator and incubate for 2 h to allow the cells to fully adhere to the three-dimensional porous surface inside the scaffold, thus completing the seeding process.

[0064] Example 3 A method for preparing a porous polymer scaffold system loaded with bone marrow mesenchymal stem cells, differing from Example 2 in that step B4, further culturing, is performed using a three-dimensional dynamic perfusion culture method instead of static culture in an incubator. The specific steps include: B4-1: After incubation, the scaffold is transferred to a micro-perfusion chamber that matches the size of the scaffold. The two ends of the micro-perfusion chamber are connected to the inlet and outlet of the culture medium circulation system, respectively. The inner wall of the chamber is tightly fitted to the outer wall of the scaffold to form a sealed fluid channel. B4-2: Start the culture medium circulation system to drive the complete culture medium to flow directionally through the internal porous network of the scaffold at a flow rate of 0.3 mL / min, so as to continuously and dynamically culture the three-dimensional cell spheres of stem cells in the scaffold; The direction of infusion is switched every 18 hours.

[0065] Example 4 A method for preparing a porous polymer scaffold system loaded with bone marrow mesenchymal stem cells, differing from Example 3 in that it employs three-dimensional dynamic perfusion culture and uses a multi-channel parallel-series composite loop design for the culture medium circulation system, specifically including the following steps: C1: Prepare a culture medium circulation control system, which includes a bidirectional peristaltic pump, a culture medium storage bottle, and a multi-channel split-merge integrated module; C2: The multi-channel diversion-merging integrated module is internally equipped with one main inlet channel, one main outlet channel, and N parallel independent culture channels, where N≥3 and N is an integer; each independent culture channel is connected to the main inlet channel and the main outlet channel at both ends, respectively. C3: In each independent culture channel, 2-3 incubation scaffolds are sequentially embedded in series along the fluid flow direction; the scaffolds are kept at a fixed interval by built-in mesh rings to ensure that the surface of each scaffold is exposed to the directional flow of culture medium; C4: Start the bidirectional peristaltic pump and set the circulation flow rate to 1.0 mL / min. The culture medium flows out of the storage bottle, passes through the main inlet channel, is evenly distributed to N independent culture channels, flows through the two supports connected in series in each channel, merges into the main outlet channel, and finally returns to the storage bottle, forming a complete closed loop. C5: Every 18 hours of culture, the flow direction of the culture medium in the loop is reversed by controlling the rotation direction of the bidirectional peristaltic pump, so that N independent culture channels can be simultaneously perfused in a bidirectional alternating cycle.

[0066] Example 5 A method for preparing a porous polymer scaffold system loaded with bone marrow mesenchymal stem cells, differing from Example 4 in that the culture medium circulation system employs a gradient flow rate regulation strategy for three-dimensional dynamic perfusion culture, specifically including the following steps: D1: On days 1-3 of the culture cycle, the circulation rate of the bidirectional peristaltic pump was set to a low flow rate mode of 0.07 mL / min to allow the complete culture medium to form a laminar flow in the pores of the scaffold, maintain a weak fluid shear force, and promote the initial adhesion of bone marrow mesenchymal stem cells and the formation of three-dimensional cell spheres. D2: During the 4th to 7th day of the culture cycle, the circulation flow rate was increased to a medium flow rate mode of 0.4 mL / min to enhance forced convection inside the scaffold, improve the efficiency of oxygen and nutrient transfer to the core region of the cell sphere, and activate the cell's mechanosensitive ion channels through fluid shear force within the physiological range, thereby promoting cell proliferation and paracrine function. D3: During the 8th to 15th day of the culture cycle, the circulation flow rate was further adjusted to a high flow rate mode of 0.8 mL / min, and the frequency of perfusion direction switching was increased to once every 8-12 h. This was done to maximize the removal of metabolic waste and stimulate the expression of genes related to extracellular matrix remodeling, while ensuring that cells were not washed away and detached. D4: On day 16 and thereafter of the culture cycle, when the cell spheroid diameter reaches the range of 200-250 μm, the circulation flow rate is switched back to the maintenance mode of 0.25 mL / min. At the same time, an intermittent perfusion strategy is introduced, which involves perfusion for 30 min followed by a 15 min pause, to simulate the intermittent characteristics of tissue fluid flow in vivo, prevent excessive cell spheroid growth leading to central necrosis, and maintain the long-term activity and functional stability of the formed three-dimensional cell spheroids.

[0067] Inside the multi-channel shunt-merge integrated module, a microfluidic fluid resistance balancing structure is set at the inlet or outlet of each independent culture channel; the microfluidic fluid resistance balancing structure is a reduced-diameter capillary section with an inner diameter of 0.3 mm and a length of 6 mm.

[0068] Effect verification: 1. Cell Loading Rate: The porous polymer scaffold systems loaded with bone marrow mesenchymal stem cells prepared in Examples 1-5 were taken, with three replicates per group. The scaffolds were placed in sterile centrifuge tubes, and an appropriate amount of PBS buffer was added for gentle shaking and washing. The washing buffer was collected. Unadsorbed cells in the washing buffer were counted using a cell counting chamber and denoted as N. t Simultaneously record the initial total number of seeded cells, denoted as N0. Calculate the cell load using the following formula: Cell load rate (%) = (N0 - N) t ) / N0×100%.

[0069] Table 1. Cell loading rates of scaffold systems prepared by different methods The experimental results showed that in Example 1, using the conventional drop-in method for seeding, the cell loading rate was 92.6%. In Example 2, after vacuum-assisted permeation seeding, the cell loading rate significantly increased to 98.1%, indicating that vacuum-assisted permeation seeding can effectively remove air from the scaffold pores and promote the uniform infiltration of cell suspension into the scaffold, thereby improving cell loading efficiency. Examples 3-5, based on vacuum-assisted seeding, employed dynamic perfusion culture, a multi-channel parallel-series composite circuit, and a gradient flow rate control strategy, respectively. The cell loading rates were comparable to those in Example 2, with no significant difference. This indicates that vacuum-assisted permeation seeding is a key step in achieving a high cell loading rate. The subsequent dynamic culture method has no significant effect on the initial cell loading rate, but plays an important role in the uniform distribution of cells within the scaffold and subsequent proliferation.

[0070] 2. Cell survival and proliferation: The BMSC-loaded scaffold systems prepared in Examples 1-5 were used. For Examples 1 and 2, the BMSC-loaded scaffold systems were transferred to new culture containers, and complete culture medium was added at a ratio of 0.1 g / mL. The systems were statically incubated at 37 °C in a 5% CO2 incubator for 72 h, and the supernatant was collected. For Examples 3-5, perfusion culture was continued under the original conditions for 72 h in their respective dynamic perfusion systems, and the circulating culture medium was collected. The supernatant was collected as the stock extract and diluted with complete culture medium to a concentration of 25% for later use.

[0071] Take healthy 3rd-5th generation BMSCs and distribute them at 5 × 10⁻⁶ cells per well. 3Cells were seeded at a density of 1000 μL in 96-well plates and cultured for 24 h to allow cell adhesion. The original culture medium was discarded, and 25% concentration of the extract from the scaffolds used in each example was added. PBS was used as a negative control. Five replicates were set up for each group. After another 24 h of culture, 10 μL of CCK-8 solution was added to each well, and the plates were incubated in the dark for 4 h. The absorbance (OD value) of each well was measured using a microplate reader at 450 nm. Cell viability was calculated using the following formula: Cell viability (%) = (OD value of experimental group - OD value of blank group) / (OD value of negative control group - OD value of blank group) × 100%.

[0072] Cell spheroid diameter measurement: The BMSC-loaded scaffold systems prepared in Examples 1-5 were cultured at 37 ℃ in a 5% CO2 incubator. The scaffolds were removed on days 5, 10, and 20 of culture, gently rinsed twice with PBS buffer, and observed in laser confocal culture dishes. Thirty cell spheroids were randomly selected from each group, and their diameter was measured using ImageJ software. The mean and standard deviation were calculated.

[0073] Table 2. Cell viability and cell spheroid diameter of scaffold systems prepared by different methods CCK-8 assay results showed that the cell viability of BMSCs cultured in the extracts of the scaffold systems prepared in each embodiment was greater than 85%, indicating that all scaffolds had good biocompatibility. Under dynamic perfusion culture conditions, the cells within the scaffolds were more metabolically active and may secrete more survival-promoting factors; therefore, the cell viability supported by the extracts was slightly higher than that of the static culture group.

[0074] Cell spheroid diameter measurements showed that in Example 1, the cell spheroid diameter gradually increased with culture time, reaching 258 μm on day 20. Example 2 used vacuum-assisted permeation seeding (static culture), resulting in more uniform cell distribution and a slight increase in diameter. Example 3, based on Example 2, employed three-dimensional dynamic perfusion culture, improving material exchange and significantly increasing cell spheroid diameter. Example 4 used a multi-channel parallel-series composite loop dynamic perfusion, achieving a diameter similar to Example 3 but with slightly better uniformity. Example 5, based on Example 4, introduced a gradient flow rate control strategy and a microfluidic resistance balancing structure, resulting in the largest cell spheroid diameter with the best uniformity and a significantly narrowed standard deviation. Cell viability in all examples was above 85%, and showed an increasing trend with optimized culture conditions, indicating that all scaffold systems possessed good biocompatibility.

[0075] 2. Cell functional activity: The concentrations of vascular endothelial growth factor A (VEGF-A), fibroblast growth factor 2 (FGF-2), and transforming growth factor β1 (TGF-β1) in the conditioned medium of the porous polymer scaffold systems loaded with BMSCs prepared in each example were detected by ELISA after 72 h of culture. The specific operation steps are as follows: Preparation of conditioned medium: Take the scaffold systems loaded with BMSCs prepared in Examples 1-5 (3 parallel samples for each example), transfer them to new culture containers, add fresh complete culture medium to immerse the scaffolds, and continue to culture in a 37 ℃, 5% CO2 incubator for 72 h. Collect the culture supernatant, remove cell debris by centrifugation to obtain the conditioned medium, aliquot it and store it at -80℃ for later use.

[0076] ELISA assay: The procedure was performed according to the instructions for the rat VEGF-A, FGF-2, and TGF-β1 ELISA kits. 100 μL of the conditioned medium samples and series of concentrations of standards were added to each well of a 96-well microplate and incubated at 37 °C for 90 min. The liquid was discarded, and biotinylated antibody working solution was added, followed by incubation at 37 °C for 60 min. After washing, enzyme conjugate working solution was added, and the plate was incubated at 37 °C in the dark for 30 min. After washing again, the chromogenic substrate was added, and the plate was incubated at 37 °C in the dark for 15-20 min. The reaction was terminated by adding stop solution, and the absorbance (OD value) at 450 nm was immediately measured using a microplate reader.

[0077] Data processing: A standard curve was plotted based on the concentrations of the standards and their corresponding OD values. The OD values ​​of each sample were then substituted into the standard curve to calculate the concentrations of the corresponding cytokines. Each group had three replicates. Results are expressed as mean ± standard deviation.

[0078] Table 3. Concentrations of repair-promoting factors in culture media under different conditions (unit: pg / mL) The experimental results showed that in both Examples 1 and 2, static culture was used, and the concentrations of VEGF-A, FGF-2, and TGF-β1 in the conditioned medium were relatively close, at 956.3 pg / mL and 968.7 pg / mL (VEGF-A), 328.5 pg / mL and 335.2 pg / mL (FGF-2), and 412.8 pg / mL and 421.6 pg / mL (TGF-β1), respectively, with no significant difference between the two groups.

[0079] In Example 3, after dynamic perfusion culture, the concentrations of the three pro-repair factors were significantly increased: VEGF-A reached 1528.4 pg / mL, an increase of approximately 60% compared to Example 1; FGF-2 reached 396.8 pg / mL, an increase of approximately 21%; and TGF-β1 reached 512.4 pg / mL, an increase of approximately 24%. Example 4 employed a multi-channel parallel-series composite circuit design, and the cytokine concentrations were comparable to those in Example 3.

[0080] Example 5 employed a gradient flow rate control strategy for dynamic perfusion culture, further enhancing the secretion levels of pro-repair factors. The VEGF-A concentration reached 1915.2 pg / mL, approximately 100% higher than in Example 1 and approximately 25% higher than in Example 3; the FGF-2 concentration reached 459.1 pg / mL, approximately 39% higher than in Example 1 and approximately 15% higher than in Example 3; and the TGF-β1 concentration reached 598.5 pg / mL, approximately 45% higher than in Example 1 and approximately 17% higher than in Example 3. The standard deviations of each factor concentration were significantly narrower than in the culture system without resistance balancing, reflecting that the homogeneity of stem cell paracrine function was effectively ensured under multi-channel parallel culture conditions.

[0081] The above results indicate that dynamic perfusion culture can significantly promote the paracrine activity of loaded BMSCs and increase the secretion level of pro-repair factors. The gradient flow rate regulation strategy can optimize the hydrodynamic microenvironment according to the cell growth stage, further enhancing the paracrine function of stem cells and maximizing the concentration of pro-repair factors in the conditioned medium. These experimental results are consistent with the conclusion in the technical disclosure materials that LCM secretes higher concentrations of pro-repair factors than SCM.

[0082] 3. Recovery of fertility Animal grouping and co-hospitalization: The porous polymer scaffold systems loaded with bone marrow mesenchymal stem cells prepared in Examples 1-5 were transplanted into rat models of uterine injury. During transplantation, the scaffolds were cut to 5 mm × 5 mm × 2 mm, with one scaffold implanted at the uterine injury site in each rat, ensuring close contact between the scaffold and the injury area. Ten female SD rats were selected from each group and co-hospitalized with healthy male SD rats at a ratio of 2:1 30 days post-surgery. Vaginal smears were examined the morning after co-hospitalization; successful mating was indicated by the observation of sperm under a microscope, and this was marked as day 0 of pregnancy.

[0083] Pregnancy rate statistics: Female rats in each group were sacrificed by overdose anesthesia on days 18-19 of gestation. The intact uterus was removed via laparotomy, and its morphology was observed. The number of pregnant animals was recorded. The pregnancy rate was calculated using the following formula: Pregnancy rate (%) = (Number of pregnant animals / Number of animals in a cage) × 100%.

[0084] Fetal rat count: The uterus of pregnant rats was opened, and the number of live fetuses, stillborn fetuses, and resorbed fetuses in each pregnant rat were recorded. The average number of fetal rats in each group (animals / litter) was calculated.

[0085] Fetal mouse weight measurement: Each fetal mouse was removed from the uterus, and the surface amniotic fluid and blood were absorbed with filter paper. The fetal mouse weight (unit: g) was measured using a precision electronic balance, and the average fetal mouse weight of each group was calculated.

[0086] Deformity rate observation: Each fetal mouse was observed under a stereomicroscope to check for developmental abnormalities such as exposed brain, spina bifida, limb deformities, and cleft lip and palate. The number of deformed fetal mice was recorded, and the deformity rate was calculated using the following formula: Deformity rate (%) = (Number of deformed fetuses / Total number of fetuses) × 100%.

[0087] F1 generation growth and development observation: F1 generation pups from the BMSCs-PMS group were routinely fed, and their weights were recorded on days 1, 7, 14, 21, 28, and 56 after birth to plot growth and development curves. When the F1 generation females reached 8 weeks of age, they were housed with healthy males at a ratio of 2:1 to observe the number and weight of F2 generation pups born naturally.

[0088] Table 4. Effects of scaffold systems prepared by different methods on fertility recovery in model rats. Observations showed that a small amount of fluid was observed in the uterus of rats in the Example 1 and Example 2 groups, but this was significantly improved compared to the model group; the uterine morphology of the Example 3 and Example 4 groups was basically normal, with no obvious fluid accumulation or atrophy; the uterine morphology of the Example 5 group was close to normal, comparable to the sham-operated group.

[0089] Regarding pregnancy rates, Example 1 showed 80%, Example 2 84%, Examples 3 and 4 87% and 93% respectively, and Example 5 reached 100%. As for the number of fetal mice, Example 1 averaged 10.2 mice / litter, Example 2 averaged 11.0 mice / litter, Examples 3 and 4 averaged 11.8 mice / litter and 12.5 mice / litter respectively, and Example 5 averaged 13.0 mice / litter, close to normal physiological levels. Regarding the average weight of fetal mice, all groups ranged from 2.9 to 3.2 g, with no significant difference. No obviously malformed fetal mice were observed in any of the examples, with a malformation rate of 0%.

[0090] The growth and development of F1 generation rats produced in Example 5 (BMSCs-PMS group) were monitored for 8 weeks. The results showed that all pups grew healthily, with normal weight gain curves and no deformities or deaths. When the F1 generation female rats were placed in cages with healthy male rats at 8 weeks of age, the average weight of the F2 generation pups born naturally was similar to that of the F1 generation, with no significant difference between the groups, indicating that this treatment regimen did not cause significant reproductive toxicity.

[0091] The above results indicate that dynamic perfusion culture significantly improves the repair effect of the scaffold system on damaged endometrium, resulting in a marked improvement in pregnancy rate and fetal number in model rats. The scaffold system prepared using a gradient flow rate control strategy combined with a microfluidic fluid resistance equalization structure showed the best results, achieving a 100% pregnancy rate, restoring the fetal number to normal levels, and without observing malformations or reproductive toxicity. These experimental results are consistent with the conclusions in the technical disclosure materials regarding the promotion of fertility recovery in rats by BMSCs-L-PMS.

[0092] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A method for preparing a porous polymer scaffold system loaded with bone marrow mesenchymal stem cells, characterized in that, Includes the following steps: B3-1: Place the sterilized porous polymer scaffold in a vacuum inoculation container, which has a sealing cap, an air extraction port connected to a vacuum pump, and a liquid inlet. B3-2: Close the sealing cap, start the vacuum pump, and evacuate the pressure inside the container to -0.02 to -0.06 MPa, and maintain it for 1-3 minutes; B3-3: Maintain the above vacuum level, and slowly inject the pre-prepared cell suspension into the container in a continuous flow manner through the inlet until the cell suspension completely submerges the scaffold; B3-4: Close the liquid inlet and the air extraction port, and slowly release the vacuum to allow the pressure inside the container to gradually return to normal pressure within 30-60 seconds, so that the cell suspension can be evenly pressed into the entire interconnected pore network of the scaffold under atmospheric pressure. B3-5: Transfer the container to a 37 ℃ incubator and incubate for 2 h to allow the cells to fully adhere to the three-dimensional porous surface inside the scaffold, thus completing the seeding process; B4-1: After incubation, the scaffold is transferred to a micro-perfusion chamber that matches the size of the scaffold. The two ends of the micro-perfusion chamber are connected to the inlet and outlet of the culture medium circulation system, respectively. The inner wall of the chamber is tightly fitted to the outer wall of the scaffold to form a sealed fluid channel. B4-2: Start the culture medium circulation system to drive the complete culture medium to flow directionally through the internal porous network of the scaffold at a flow rate of 0.05-2.0 mL / min, so as to continuously and dynamically culture the stem cells in the scaffold; The direction of infusion is switched every 12-24 hours. The method for preparing the porous polymer scaffold includes the following steps: S1: Sodium alginate powder and carboxymethyl chitosan powder are added to deionized water and stirred and dissolved at 35-45 ℃ to prepare sodium alginate solution with a mass fraction of 2.0-3.0 wt% and carboxymethyl chitosan solution with a mass fraction of 1.5-2.5 wt%, respectively. S2: After filtering the sodium alginate solution and carboxymethyl chitosan solution through a 0.20-0.25 μm filter, mix and stir the sodium alginate to carboxymethyl chitosan solution at a mass ratio of 4:3-5:2 at a constant temperature of 35-45 ℃ for 1.5-2.5 h to obtain a homogeneous mixed solution. S3: Use a calcium chloride solution with a molar concentration of 0.05-0.15 mol / L as a crosslinking agent, and slowly add it dropwise to the continuously stirred mixed solution. The volume ratio of the mixed solution to the calcium chloride solution is 5-15:

1. Stop adding the crosslinking agent when the mixed solution turns into a gel state. S4: Pour the cross-linked gel into a mold and let it stand for 45-50 hours to obtain cross-linked gels of different shapes; S5: The cross-linked gel is freeze-dried; the freeze-drying adopts the first freezing mode: the gel is placed in an environment of -45 to -35 ℃ and frozen for 10-14 h, and then vacuum dried for 20-28 h to obtain a large-pore porous polymer scaffold. Alternatively, a second freezing mode can be used for freeze drying: the gel is placed in a liquid nitrogen environment and rapidly frozen until fully formed, and then vacuum dried for 20-28 hours to obtain a small-pore porous polymer scaffold.

2. The method for preparing the porous polymer scaffold system loaded with bone marrow mesenchymal stem cells according to claim 1, characterized in that, The average pore size of large-pore porous polymer scaffolds is 150-200 μm, and the porosity is 92%-95%; the average pore size of small-pore porous polymer scaffolds is 10-35 μm, and the porosity is 90%-94%.

3. The method for preparing the porous polymer scaffold system loaded with bone marrow mesenchymal stem cells according to claim 1, characterized in that, The culture medium circulation system adopts a multi-channel parallel-series composite loop design, which includes the following steps: C1: Prepare a culture medium circulation control system, which includes a bidirectional peristaltic pump, a culture medium storage bottle, and a multi-channel split-merge integrated module; C2: The multi-channel diversion-merging integrated module is internally equipped with one main inlet channel, one main outlet channel, and N parallel independent culture channels, where N≥3 and N is an integer; each independent culture channel is connected to the main inlet channel and the main outlet channel at both ends, respectively. C3: In each independent culture channel, 2-3 incubation scaffolds are sequentially embedded in series along the fluid flow direction; the scaffolds are kept at a fixed interval by built-in mesh rings to ensure that the surface of each scaffold is exposed to the directional flow of culture medium; C4: Start the bidirectional peristaltic pump and set the circulation flow rate to 0.05-2.0 mL / min. The culture medium flows out of the storage bottle, passes through the main inlet channel, is evenly distributed to N independent culture channels, flows through 2-3 supports connected in series in each channel, converges to the main outlet channel, and finally returns to the storage bottle, forming a complete closed loop. C5: Every 12-24 hours of culture, the rotation direction of the bidirectional peristaltic pump is controlled to reverse the flow direction of the culture medium in the loop, so that N independent culture channels can be simultaneously perfused in a bidirectional alternating cycle.

4. The method for preparing the porous polymer scaffold system loaded with bone marrow mesenchymal stem cells according to claim 1, characterized in that, The culture medium circulation system employs a gradient flow rate control strategy for three-dimensional dynamic perfusion culture, specifically including the following steps: D1: On days 1-3 of the culture cycle, set the circulation rate of the bidirectional peristaltic pump to a low flow rate mode of 0.05-0.1 mL / min to allow the complete culture medium to form a laminar flow permeation within the scaffold pores; D2: During days 4-7 of the culture cycle, increase the circulation flow rate to a moderate flow rate mode of 0.3-0.5 mL / min; D3: On days 8-15 of the culture cycle, the circulation flow rate was further adjusted to a high flow rate mode of 0.6-1.0 mL / min, and the perfusion direction switching frequency was increased to once every 8-12 h. D4: On day 16 and thereafter in the culture period, when the cell spheroid diameter enters the range of 200-250 μm, the circulation flow rate is switched back to the maintenance mode of 0.2-0.3 mL / min. At the same time, an intermittent perfusion strategy is introduced, which involves perfusion for 30 min and then pausing for 15 min to simulate the intermittent characteristics of tissue fluid flow in vivo.

5. The method for preparing the porous polymer scaffold system loaded with bone marrow mesenchymal stem cells according to claim 1, characterized in that, Inside the multi-channel split-merge integrated module, a microfluidic fluid resistance balancing structure is also set at the inlet or outlet of each independent culture channel; the microfluidic fluid resistance balancing structure is a reduced-diameter capillary section with an inner diameter of 0.2-0.5 mm and a length of 3-8 mm.

6. The application of the scaffold system prepared by the method for preparing the porous polymer scaffold system loaded with bone marrow mesenchymal stem cells as described in claim 1 in the preparation of intrauterine adhesion repair materials.

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