A gel microsphere carrying amniotic mesenchymal stem cells, its preparation and application

Abstract

This invention provides a gel microsphere carrying amniotic mesenchymal stem cells, its preparation, and its application. Based on polyethylene glycol diacrylate (PEGDA) and powdered amniotic membrane, it incorporates various osteogenic differentiation factors and anti-inflammatory modulators, and carries amniotic mesenchymal stem cells (hA-MSCs). During the preparation of the gel microspheres, microfluidic technology is used to achieve high precision and consistency, ensuring that the average size of non-spherical microspheres is precisely controlled between 80 and 200 micrometers, facilitating transplantation or injection and ensuring uniform distribution and effective action of cells in vivo. Through photopolymerization, the gel microspheres rapidly solidify under mild conditions, maintaining cell activity and forming a cross-linked network structure. The gel microspheres not only possess good biocompatibility and degradability but also mimic the in vivo microenvironment, promoting cell survival, proliferation, and differentiation, regulating inflammatory responses, and creating a favorable microenvironment for bone defect repair.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering and tissue engineering, specifically a gel microsphere carrying amniotic mesenchymal stem cells and its preparation and application. Background Technology

[0002] Bone defects are a major challenge in clinical medicine. Traditional treatments such as surgery and medication, while providing some relief for patients, have significant limitations. Surgical treatment not only has a long recovery period but may also be accompanied by complications such as bleeding, infection, and nerve or vascular damage, and is particularly difficult to repair for large or complex-shaped bone defects. Drug therapy mainly accelerates bone regeneration through chemicals or growth factors that promote bone healing, but its efficacy is often unstable and may cause systemic side effects.

[0003] In recent years, with the rapid development of regenerative medicine and tissue engineering technologies, biomaterials combined with cell therapy have provided a new perspective for bone defect repair. Gel materials, due to their advantages such as high plasticity, ease of manipulation, and ability to provide three-dimensional growth space for cells, have shown great potential in bone defect repair. For example, some gels based on materials such as hydroxyapatite, collagen, or polylactic acid have been used to fill bone defects and promote bone regeneration. However, existing gel materials still have shortcomings in terms of biocompatibility, the matching degree between degradation rate and bone regeneration rate, and the ability to promote bone differentiation. Some gel materials may induce inflammatory responses after implantation, affecting cell survival and differentiation; at the same time, too rapid a degradation rate may lead to incomplete bone regeneration, while too slow a rate may hinder the formation of new bone tissue. Summary of the Invention

[0004] This invention provides a gel microsphere carrying amniotic mesenchymal stem cells to address the technical problems of donor limitation, immune rejection, inflammatory response, and insufficient biocompatibility in the treatment of bone defects.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a gel microsphere carrying amniotic mesenchymal stem cells, comprising the following components:

[0006] Excipients: 2 wt%-10 wt% polyethylene glycol diacrylate and 1 wt%-3 wt% powdered amnion;

[0007] Components that promote bone differentiation: sodium sapolixalate (0.1wt%-0.5wt%), mannitol (0.5wt%-2wt%), and L-arginine (0.1wt%-0.5wt%).

[0008] Anti-inflammatory modulators: 0.1wt%-0.5wt% meglumine, 0.05wt%-0.5wt% o-phenanthroline, and 0.05wt%-0.5wt% acesulfame potassium;

[0009] Cellular components: Cell concentration of 1×10 7 Amniotic mesenchymal stem cells (cells / mL);

[0010] Photopolymerization initiator: 0.1wt%-2wt% of 1-hydroxycyclohexylphenyl ketone.

[0011] Preferably, the polyethylene glycol diacrylate has a molecular weight greater than 3000 Daltons, and the powdered amniotic membrane has a particle size of less than 50 micrometers. Both, as excipients, give the gel good biocompatibility and mechanical stability, and the pore size of the cross-linked hydrogel is suitable for cell growth. The osteogenic component can promote the differentiation of amniotic mesenchymal stem cells into osteoblasts, thereby accelerating the bone defect repair process. The anti-inflammatory modulator can inhibit the inflammatory response, improve the repair effect, and reduce the risk of immune rejection. The amniotic mesenchymal stem cells can self-renew and differentiate into various cell types, and have broad application potential.

[0012] This invention also discloses the preparation of gel microspheres carrying amniotic mesenchymal stem cells. The gel microspheres are the same as those mentioned above, prepared by mixing with the photopolymerization initiator 1-hydroxycyclohexylphenyl ketone and using microfluidics technology to precisely control the flow and interaction of fluids in microchannels, achieving high-precision and high-consistency preparation of gel microspheres. Ultraviolet light irradiation is used to initiate a free radical polymerization reaction, forming a cross-linked network structure hydrogel inside the droplets, thereby encapsulating the amniotic mesenchymal stem cells in the formed novel gel material. The novel gel microspheres are obtained by screen screening.

[0013] Preferably, the specific steps include:

[0014] S1. Dissolve polyethylene glycol diacrylate in deionized water, add powdered amniotic membrane to form a solution of a certain concentration; the mass-volume concentration range of the polyethylene glycol diacrylate is 2%-10%, and the mass-volume concentration range of the amniotic membrane is 1%-3%, forming a uniform excipient solution.

[0015] S2. Add the osteogenic differentiation-promoting components, which include sodium sapoxetine, mannitol, and L-arginine, to the excipient solution at a mass ratio of 5%-20% of the total mass, and stir thoroughly until completely dissolved.

[0016] S3. Add the anti-inflammatory regulator at a mass ratio of 1%-5% of the total mass to the solution and continue stirring until the mixture is homogeneous. The anti-inflammatory regulator includes meglumine, o-phenanthroline, and acesulfame potassium.

[0017] S4. Add amniotic mesenchymal stem cell suspension and stir well;

[0018] S5. Using 1-hydroxycyclohexylphenyl ketone as a photopolymerization initiator, the mixture is mixed with the solution obtained in S4 and injected into the fluid channel of the microfluidic device. By precisely controlling the flow rate and volume, the fluid is ensured to flow stably in the microchannel. An ultraviolet light source is set at the outlet of the microfluidic device. When the mixed solution flows through the outlet, it is irradiated by ultraviolet light, which initiates a free radical polymerization reaction to form a colloidal solution. The colloidal solution reaches the screen to prepare a hydrogel.

[0019] S6. The hydrogel is ejected from the screen to obtain novel gel microspheres.

[0020] Preferably, the temperature of the excipient solution in step S1 does not exceed 40°C and is prepared for subsequent processes.

[0021] Preferably, after adding the amniotic mesenchymal stem cell suspension in step S5, it serves as the inner phase of the microfluidic, with a flow rate range of 8 μL / min. The intensity and irradiation time of the ultraviolet light at the outlet of the microfluidic device are adjusted according to the photopolymerization initiator 1-hydroxycyclohexylphenyl ketone and the precursor, and the polymerization time does not exceed 8 minutes to ensure efficient polymerization while avoiding damage to stem cells.

[0022] Preferably, in step S6, the screen is a wire sieve with a pore size of 100 micrometers.

[0023] The present invention also discloses the application of a gel microsphere carrying amniotic mesenchymal stem cells. The gel microsphere is the aforementioned gel microsphere carrying amniotic mesenchymal stem cells. The gel microsphere is implanted into the bone defect site by injection or surgery, has good biocompatibility, and will not cause severe immune or rejection reactions.

[0024] Preferably, the gel microspheres are suitable for repairing various types of bone defects, including those in the skull, spine, and limbs.

[0025] Preferably, the proportions of the components in the gel material are adjusted according to specific application requirements. The preparation is simple and quick, enabling the efficient production of large quantities of gel microspheres. Furthermore, these materials are easy to store and transport, facilitating clinical use.

[0026] The present invention has the following advantages over the prior art:

[0027] This invention provides a gel microsphere carrying amniotic mesenchymal stem cells (hA-MSCs), based on polyethylene glycol diacrylate (PEGDA) and powdered amniotic membrane, combined with various osteogenic differentiation factors and anti-inflammatory modulators. The gel microspheres are prepared using microfluidic technology to achieve high precision and uniformity, ensuring the average size of non-spherical microspheres is precisely controlled between 80 and 200 micrometers, facilitating transplantation or injection and ensuring uniform cell distribution and effective action in vivo. Through photopolymerization, the gel microspheres rapidly solidify under mild conditions, maintaining cell activity and forming a cross-linked network structure. These gel microspheres not only possess good biocompatibility and biodegradability but also mimic the in vivo microenvironment, promoting cell survival, proliferation, and differentiation, regulating inflammatory responses, and creating a favorable microenvironment for bone defect repair.

[0028] The following technical problems existing in the prior art have been solved:

[0029] (1) Problem of low cell survival rate: In traditional cell transplantation therapy, cells often have difficulty adapting to the new environment after transplantation, resulting in low survival rates. This invention solves the problem of low cell survival rate by using innovative materials combined with amniotic mesenchymal stem cells. Specifically: 1) Innovative materials: By scientifically proportioning polyethylene glycol diacrylate (PEGDA) and powdered amniotic membrane as excipients, hydrogel microspheres with a cross-linked network structure are prepared using photopolymerization. This microsphere structure provides cells with a microenvironment similar to the extracellular matrix, which is conducive to cell attachment, growth, and differentiation. 2) Application of amniotic mesenchymal stem cells: Amniotic mesenchymal stem cells are an ideal cell source due to their low immunogenicity, high proliferative capacity, and multi-lineage differentiation potential. In the gel microspheres, amniotic mesenchymal stem cells are effectively encapsulated, protecting the cells from direct impact from the external environment and continuously releasing growth factors through the sustained-release effect of the microspheres, promoting cell survival and proliferation. Experimental results showed that in animal experiments using amniotic mesenchymal stem cells carried by the gel microspheres of this invention for bone defect repair, the cell survival rate was increased by about 30% compared with the control group, and the cells had good morphology, showing good proliferative activity.

[0030] (2) Problem of poor treatment effect: Traditional treatment materials cannot effectively promote cell proliferation and differentiation, or lack the necessary bioactive factors to guide the direction of tissue repair, resulting in unsatisfactory treatment effect. To solve this problem, the present invention takes the following measures: 1) Interaction of components: Osteogenesis-promoting factors (such as sodium sapolixalate, mannitol, L-arginine) and anti-inflammatory regulators (glucamine, o-phenanthroline, acesulfame potassium) are integrated in the gel microspheres. These components can work synergistically to promote the proliferation, differentiation and matrix synthesis of chondrocytes, while inhibiting the inflammatory response and accelerating the tissue repair process. 2) Design of non-spherical microsphere structure: By designing a non-spherical microsphere structure, the stability and operability of the material at the defect site are improved, the release kinetics of bioactive factors are optimized, and their uniform distribution and continuous effect in the defect area are ensured, thereby improving the treatment effect. Animal model experiments show that, in the bone defect area treated with the gel microspheres of the present invention, histological examination during the 8-week observation period showed that the quality of cartilage regeneration was significantly improved, the defect area was effectively filled by newly formed cartilage tissue, and no obvious inflammatory response was observed. Compared with the control group, the repaired area showed better repair effects in terms of cartilage thickness and collagen fiber arrangement.

[0031] (3) Issues of Precision and Consistency in Preparation: Traditional gel preparation methods often struggle to guarantee the precision and consistency of materials, leading to unstable therapeutic effects. To address this issue, this invention employs the following measures: This preparation technique incorporates microfluidics, enabling high precision and consistency in the preparation of gel microspheres. By precisely controlling the geometry, flow rate, and fluid pressure of the microfluidic microchannels, gel microspheres with uniform size and stable morphology can be prepared. These highly consistent microspheres not only facilitate uniform cell distribution and effective action but also improve the filling effect and stability of the therapeutic material at the defect site, thereby enhancing the predictability and reliability of the therapeutic effect.

[0032] (4) Issues with cell distribution and uniformity: In traditional cell transplantation therapy, cell distribution is often difficult to control, leading to uneven therapeutic effects. The gel microspheres prepared by this technology achieve uniform cell distribution. This uniform cell distribution not only improves cell survival and proliferation activity but also promotes intercellular interactions and synergistic effects, thereby enhancing the repair effect of the therapeutic material at the defect site.

[0033] (5) Issues regarding preparation efficiency and cost: Traditional gel preparation often requires complex processes and a significant amount of time, resulting in high preparation costs. This preparation method, through automated and continuous production, greatly improves the preparation efficiency of gel microspheres and reduces preparation costs. This efficient and low-cost preparation not only facilitates large-scale production but also reduces the cost of therapeutic materials, enabling more patients to benefit from this innovative bone defect repair solution.

[0034] It has the following beneficial effects:

[0035] (1) Innovative material preparation: This invention uses polyethylene glycol diacrylate (PEGDA) combined with amniotic membrane excipients to prepare hydrogel microspheres through photopolymerization reaction, providing a suitable growth environment for cells.

[0036] (2) Highly efficient cell encapsulation: hA-MSCs with low immunogenicity, high proliferation and multi-lineage differentiation potential are used to enhance their osteogenic differentiation potential and anti-inflammatory ability through pretreatment and are effectively encapsulated in gel microspheres.

[0037] (3) Unique morphology design: It adopts a non-spherical microbead morphology, which is convenient for transplantation or injection, effectively preventing the movement of cells, blood and other substances, and ensuring the precision and efficiency of the repair process.

[0038] (4) Improve cell survival rate: The novel gel microsphere material significantly improves cell survival rate through encapsulation and protection, solving the problem of low cell survival rate in traditional cell transplantation.

[0039] (5) Synergistic therapeutic effect: The gel integrates components that promote osteogenic differentiation and anti-inflammatory regulation, which work synergistically with hA-MSCs to promote chondrocyte proliferation, differentiation and matrix synthesis, and have good anti-inflammatory regulatory function.

[0040] (6) Local administration reduces side effects: The novel gel microsphere material uses a local administration method to directly deliver drugs or cells to the defect site, reducing drug loss and lowering the risk of systemic side effects.

[0041] (7) Improved preparation efficiency and precision: Microfluidic technology enables precise control and manipulation of fluids at the micro- and nanoscale, ensuring that the generated gel microspheres have high monodispersity and size uniformity. By optimizing the design of microfluidic channels, the size, shape, and composition of gel microspheres can be precisely controlled to achieve personalized customization and meet the treatment needs of different patients.

[0042] (8) Enhanced cell encapsulation efficiency: This preparation technology can efficiently encapsulate hA-MSCs into gel microspheres while maintaining cell activity and function.

[0043] (9) High-throughput preparation: This preparation technology features high throughput, enabling the preparation of large quantities of gel microspheres in a short time. This significantly improves production efficiency, reduces preparation costs, and provides more possibilities for clinical applications.

[0044] (10) Facilitates drug delivery and release control: Gel microspheres prepared using this technology enable localized delivery and controlled release of drugs. Drugs can be encapsulated within the gel microspheres, allowing for slow and sustained release. This release method helps reduce systemic side effects and improves therapeutic efficacy. Attached Figure Description

[0045] Figure 1 The flowchart shows the preparation process of gel microspheres carrying amniotic mesenchymal stem cells.

[0046] Figure 2 In Specific Embodiment 2, the survival of amniotic mesenchymal stem cells in the gel material is demonstrated using a cell counter.

[0047] Figure 3 In Specific Example 2, the ability of cells to differentiate into osteoblasts or cartilage is assessed by detecting the expression of the differentiation-related gene RUNX2.

[0048] Figure 4 In specific embodiment 3, the survival of amniotic mesenchymal stem cells in the gel material is demonstrated using a cell counter.

[0049] Figure 5 In Specific Example 3, the ability of cells to differentiate into osteoblasts or cartilage was assessed by detecting the expression of the differentiation-related gene RUNX2.

[0050] Figure 6 In Specific Example 4, the ability of cells to differentiate into osteoblasts or cartilage was assessed by detecting the expression of the differentiation-related gene RUNX2. Detailed Implementation

[0051] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. However, it is not limited to a specific instance, and this method can be applied to similar situations. Example 1

[0052] Please see Figure 1 This embodiment discloses a gel microsphere carrying amniotic mesenchymal stem cells and its preparation. Please refer to the relevant figures. The gel microsphere carrying amniotic mesenchymal stem cells includes:

[0053] ①Excipients:

[0054] Polyethylene glycol diacrylate (PEGDA): As the main component of hydrogels, it provides good mechanical properties and biocompatibility.

[0055] Powdered amniotic membrane: Rich in various growth factors and extracellular matrix components, which help cell growth and tissue repair.

[0056] ② Osteogenesis-promoting factors:

[0057] Sodium saprolide, mannitol, and L-arginine: These components can stimulate cells to differentiate into osteogenic structures, promoting the repair and regeneration of cartilage defects.

[0058] ③ Anti-inflammatory modulators:

[0059] Meglumine, o-phenanthroline, and acesulfame potassium: have anti-inflammatory and immunomodulatory effects, can reduce inflammatory responses, and provide a favorable microenvironment for cell growth.

[0060] ④ Amniotic mesenchymal stem cells (hA-MSC):

[0061] It has multi-directional differentiation potential and can differentiate into chondrocytes, participating in the repair and reconstruction of cartilage tissue.

[0062] ⑤ Photopolymerization initiator:

[0063] 1-Hydroxycyclohexylphenyl ketone: As an initiator of photopolymerization, it can rapidly initiate the polymerization of PEGDA under ultraviolet light irradiation to form a hydrogel with a cross-linked network structure.

[0064] The above-mentioned components, through scientific formulation and careful preparation, together constitute the novel gel microsphere material of this invention, providing an efficient, safe, and biodegradable material for bone defect repair.

[0065] This embodiment discloses a method for preparing the aforementioned gel microspheres carrying amniotic mesenchymal stem cells. The method first involves mixing polyethylene glycol diacrylate (PEGDA) with powdered amniotic membrane as the base excipient. Then, a variety of carefully selected osteogenic differentiation factors, such as sodium sapolixalate, mannitol, and L-arginine, as well as anti-inflammatory modulators, such as meglumine, o-phenanthroline, and acesulfame potassium, are added to ensure the material possesses excellent bioactivity and anti-inflammatory properties. Simultaneously, amniotic mesenchymal stem cells (hA-MSCs) with multi-lineage differentiation potential are uniformly dispersed in the mixture. Next, using advanced microfluidic technology, the mixture is precisely prepared into non-spherical microspheres by controlling the fluid flow rate and volume. The average size of these microspheres is precisely controlled between 80 and 200 micrometers to ensure that the microspheres are easy to transplant or inject while guaranteeing uniform distribution and effective action of the cells in vivo. Finally, under mild and cell-friendly conditions, the gel microspheres were rapidly solidified via photopolymerization to form hydrogel microspheres with a stable cross-linked network structure. These microspheres were then extruded from a 100 μm screen to prepare non-spherical microbeads. After washing, drying, and quality inspection, they were aseptically packaged and stored for later use. The gel microspheres prepared by this method exhibit excellent biocompatibility and osteogenic differentiation-promoting ability, providing a highly efficient treatment option for bone defect repair.

[0066] Specifically, the following steps are included:

[0067] I. Material Preparation

[0068] (1) Ethylene glycol diacrylate (PEGDA): PEGDA with a molecular weight greater than 3000 Daltons was selected as the main excipient. It has good water solubility to ensure the formation of a stable cross-linked network structure in the hydrogel, while maintaining good biocompatibility and mechanical properties. The concentration range is 2%-10% (w / v).

[0069] (2) Amniotic membrane: The amniotic membrane powder formed by freeze drying is selected and strictly processed and sterilized. It is used as a component of the gel and is rich in various growth factors and extracellular matrix components, which helps cell growth and differentiation and provides a suitable environment for the adhesion, proliferation and differentiation of stem cells. The concentration range is 1 wt%-3 wt%.

[0070] (3) Sarpagin: A naturally occurring plant extract that promotes cell differentiation toward osteogenic direction through its specific bioactive effects, thereby helping to repair and regenerate bone tissue. The concentration range is 0.05 wt%-0.1 wt%.

[0071] (4) Mannitol: Here it plays an auxiliary role in promoting osteogenic differentiation, indirectly promoting osteogenic differentiation by influencing the cellular environment or metabolic pathways. The concentration range is 0.1wt%-0.5wt%.

[0072] (5) L-Arginine: An essential amino acid involved in protein synthesis, it plays a positive role in cartilage repair. It can improve arthritis and bone erosion through metabolic reprogramming and disrupting the purine metabolism of osteoclasts, thereby indirectly promoting the repair and regeneration of bone tissue. The concentration range is 0.05wt%-0.2wt%.

[0073] (6) Meglumine: A basic substance that can inhibit the production of pro-inflammatory factors such as interleukin-1β, reduce joint inflammation, and help provide a favorable microenvironment for cell growth, thereby promoting tissue repair and regeneration. The concentration range is 0.05wt%-0.1wt%.

[0074] (7) Phenanthroline: As a metal ion chelating agent, it can reduce the expression of MMP3 and MMP13 mRNA. These two matrix metalloproteinases are related to inflammatory responses and tissue damage, and it plays a role in inhibiting inflammatory responses. The concentration range is 0.01wt%-0.05wt%.

[0075] (8) Acesulfame potassium: As a chemically synthesized substance, it has a variety of biological activities. Acesulfame potassium has been found to have certain anti-inflammatory effects. By inhibiting the inflammatory response, acesulfame potassium helps to provide a good microenvironment for cell growth, with a concentration range of 0.01wt%-0.06wt%.

[0076] (9) Amniotic mesenchymal stem cells (hA-MSCs): hA-MSCs are isolated and cultured from the amnion of healthy donors and undergo rigorous testing and screening to ensure that they have good multi-lineage differentiation potential and biological activity.

[0077] (10) 1-Hydroxycyclohexylphenyl ketone: As an initiator of photopolymerization, it should be weighed accurately and stored properly to avoid its failure or degradation.

[0078] II. Preparation Process

[0079] Step 1: Solution Preparation

[0080] 1) Dissolve PEGDA in an appropriate amount of deionized water to form a PEGDA solution.

[0081] 2) Mix the powdered amniotic membrane with an appropriate amount of solvent, and disperse it evenly by ultrasound or stirring to form an amniotic membrane suspension.

[0082] 3) Dissolve sodium sapoxetine, mannitol, and L-arginine in a solvent to form a solution of osteogenic differentiation factors.

[0083] 4) Dissolve meglumine, o-phenanthroline, and potassium acesulfame in a solvent to form an anti-inflammatory modulatory solution.

[0084] Step 2: Mixing and Homogenizing

[0085] 1) Mix the PEGDA solution, amniotic membrane suspension, osteogenic differentiation factor solution and anti-inflammatory regulator solution in a predetermined ratio, and add an appropriate amount of hA-MSC.

[0086] 2) Use a magnetic stirrer or ultrasonic processor to disperse the mixture evenly and ensure that all components are fully mixed.

[0087] Step 3: Add photopolymerization initiator

[0088] 1) Dissolve 1-hydroxycyclohexylphenyl ketone in a small amount of solvent, then add it to the above mixture and stir until homogeneous.

[0089] Step 4: Preparation of gel droplets using microfluidics

[0090] 1) Using a microfluidic device, the mixture from step 3 is injected into a microchannel at a certain flow rate to form stable droplets.

[0091] 2) By precisely controlling the geometry and flow rate of the microchannels, gel droplets with uniform size and shape are prepared.

[0092] Step 5: Photopolymerization reaction

[0093] 1) Place the microfluidic device containing gel droplets under an ultraviolet light source to initiate a photopolymerization reaction.

[0094] 2) Control the irradiation time and intensity of ultraviolet light to ensure that PEGDA is fully polymerized to form a hydrogel with a cross-linked network structure.

[0095] Step Six: Preparation of Gel Microspheres

[0096] 1) Collect the polymerized hydrogel microspheres from the microfluidic device.

[0097] 2) Use screen screening or other methods to process gel microspheres with a non-spherical microbead morphology and ensure that their average size is in the range of 80 to 200 micrometers.

[0098] Step 7: Washing and Drying

[0099] 1) Wash the gel microspheres with sterile saline or distilled water to remove unreacted residues.

[0100] 2) Dry the washed gel microspheres to remove excess moisture.

[0101] Step 8: Quality Inspection and Packaging

[0102] 1) Perform quality checks on the prepared gel microspheres, including testing for indicators such as appearance, size, and biocompatibility.

[0103] 2) Aseptically package the qualified gel microspheres and store them under appropriate conditions for later use.

[0104] Through the above preparation method, this invention successfully prepared a gel microsphere carrying amniotic mesenchymal stem cells. This gel microsphere exhibits excellent biocompatibility, osteogenic differentiation promotion, and anti-inflammatory regulatory capabilities, and can be used as a filler for soft tissue reconstruction, effectively promoting the rapid repair of cartilage defects. Furthermore, the preparation method is simple and controllable, suitable for large-scale production, providing an innovative and efficient treatment solution for bone defect repair. Example 2

[0105] This embodiment discloses the preparation and application experiments of gel microspheres carrying amniotic mesenchymal stem cells.

[0106] I. Material Preparation

[0107] PEGDA: PEGDA with a molecular weight of 3000 Daltons was selected at a concentration of 5% (w / v).

[0108] Amniotic membrane powder: concentration 2 wt%.

[0109] Sodium sapolixacin: Concentration 0.075 wt%.

[0110] Mannitol: concentration 0.3 wt%.

[0111] L-arginine: concentration 0.1 wt%.

[0112] Meglumine: Concentration 0.075 wt%.

[0113] o-phenanthroline: concentration 0.03 wt%.

[0114] Acetylsulfamate potassium: concentration 0.03 wt%.

[0115] Amniotic mesenchymal stem cells (hA-MSCs): 1 × 10⁻⁶ per milliliter of solution 6 The proportion of cells added.

[0116] Photopolymerization initiator: 1-hydroxycyclohexylphenyl ketone, added at a ratio of 0.5%.

[0117] II. Preparation

[0118] 1) Dissolve PEGDA in deionized water and heat to 40°C to accelerate dissolution and form a homogeneous solution.

[0119] 2) Disperse the amniotic membrane powder in the above PEGDA solution and stir with a magnetic stirrer for 2 hours to ensure that the amniotic membrane powder is evenly dispersed.

[0120] 3) Dissolve sodium sapoxetine, mannitol, L-arginine, meglumine, o-phenanthroline and potassium acesulfame in deionized water to form their respective solutions.

[0121] 4) Mix all the above solutions together and add hA-MSC. Stir gently to avoid damaging the cells.

[0122] 5) Add the photopolymerization initiator and sonicate for 5 minutes to achieve uniform dispersion of each component.

[0123] 6) Use the above liquid as the internal phase of the microfluidic system, and control the flow rate at 8 μL / min. Adjust the intensity and irradiation time of ultraviolet light at the outlet using the microfluidic device.

[0124] 7) Irradiate the microfluidic under ultraviolet light for 5 minutes to initiate a photopolymerization reaction and form a hydrogel.

[0125] 8) Gel microspheres of non-spherical shape were obtained by screening with a sieve, washed three times with deionized water, and then freeze-dried.

[0126] 9) After quality inspection, aseptically package and store at -20°C for later use.

[0127] III. Experimental Section

[0128] 1) Cell viability detection: The prepared gel microspheres are placed in cell culture medium and cultured for a period of time. The cells are then stained with trypan blue dye to distinguish between live and dead cells and to count them.

[0129] 2) Assessment of osteogenic differentiation capacity: Gel microspheres were implanted into a rat animal model (bone defect site), and samples were collected at predetermined time points (e.g., 4 weeks, 8 weeks, 12 weeks) for histological, biochemical and molecular biological analysis to assess osteogenic differentiation capacity and bone repair effect.

[0130] IV. Experimental Results:

[0131] 1) Please refer to Figure 2 Cell viability testing results showed that the prepared gel microspheres had good cell compatibility in the culture medium, with a cell viability rate of over 90%.

[0132] 2) Please refer to Figure 3 In vitro cell detection experiments showed that the gel microspheres could significantly promote the repair of bone defects and had a strong osteogenic differentiation capacity. Their RUNX2 index was better than that of the untreated control group. Example 3

[0133] This embodiment discloses the preparation and application experiments of gel microspheres carrying amniotic mesenchymal stem cells.

[0134] I. Material Preparation

[0135] PEGDA: PEGDA with a molecular weight of 5000 Daltons was selected at a concentration of 8% (w / v).

[0136] Amniotic membrane powder: concentration 1.5 wt%.

[0137] Sodium sapolixacin: Concentration 0.1 wt%.

[0138] Mannitol: concentration 0.4 wt%.

[0139] L-Arginine: Concentration 0.15 wt%.

[0140] Meglumine: Concentration 0.05 wt%.

[0141] o-phenanthroline: concentration 0.02 wt%.

[0142] Acetylsulfamate potassium: concentration 0.05 wt%.

[0143] Amniotic mesenchymal stem cells (hA-MSCs): at a rate of 2 × 10⁻⁶ per milliliter of solution. 6 The proportion of cells added.

[0144] Photopolymerization initiator: 1-hydroxycyclohexylphenyl ketone, added at a ratio of 1%.

[0145] II. Preparation

[0146] 1) Dissolve PEGDA and add amniotic membrane powder, then stir magnetically for 4 hours.

[0147] 2) Dissolve the other components separately, mix them, and then add hA-MSC and photopolymerization initiator.

[0148] 3) Sonicate for 10 minutes to ensure uniform dispersion.

[0149] 4) The liquid is used as the internal phase of the microfluidic, and the flow rate is controlled at 8 μL / min.

[0150] 5) Irradiate with ultraviolet light for 10 minutes to form a hydrogel.

[0151] 6) Screening, washing, drying, quality inspection, aseptic packaging and storage.

[0152] III. Experimental Section

[0153] 1) Cell viability detection: The prepared gel microspheres are placed in cell culture medium and cultured for a period of time. The cells are then stained with trypan blue dye to distinguish between live and dead cells and to count them.

[0154] 2) Animal model experiments: Gel microspheres were implanted into bone defects in animal models to evaluate their repair effect.

[0155] IV. Experimental Results

[0156] 1) Please refer to Figure 4 The prepared gel microspheres exhibit good cell compatibility in the culture medium, with a cell survival rate exceeding 90%, and even reaching over 95%.

[0157] 2) Please refer to Figure 5 In vitro cell experiments revealed that the modified gel microspheres exhibited better performance in bone defect repair, with improvements in histological scores and biochemical indicators at the animal level. Example 4

[0158] This embodiment discloses the preparation and application experiments of gel microspheres carrying amniotic mesenchymal stem cells.

[0159] I. Material Preparation

[0160] PEGDA: PEGDA with a molecular weight of 8000 Dalton was selected at a concentration of 2% (w / v).

[0161] Amniotic membrane powder: concentration 3 wt%.

[0162] Sodium sapoxetine: Concentration 0.05 wt%.

[0163] Mannitol: concentration 0.1 wt%.

[0164] L-arginine: concentration 0.05 wt%.

[0165] Meglumine: Concentration 0.1 wt%.

[0166] o-phenanthroline: concentration 0.01 wt%.

[0167] Acetylsulfamate potassium: concentration 0.06 wt%.

[0168] Amniotic mesenchymal stem cells (hA-MSCs): at a concentration of 0.5 × 10⁻⁶ per milliliter of solution. 6 The proportion of cells added.

[0169] Photopolymerization initiator: 1-hydroxycyclohexylphenyl ketone, added at a ratio of 0.25%.

[0170] II. Preparation:

[0171] 1) Dissolve PEGDA and add amniotic membrane powder, then stir magnetically for 3 hours.

[0172] 2) Dissolve the other components separately, mix them, and then add hA-MSC and photopolymerization initiator.

[0173] 3) Sonicate for 8 minutes to ensure uniform dispersion.

[0174] 4) The liquid is used as the internal phase of the microfluidic, and the flow rate is controlled at 8 μL / min.

[0175] 5) Irradiate with ultraviolet light for 8 minutes to form a hydrogel.

[0176] 6) Screening, washing, drying, quality inspection, aseptic packaging and storage.

[0177] III. Experimental Section

[0178] 1) Cell differentiation direction assessment: Using a specific differentiation medium, gel microspheres were cultured together with hA-MSCs, and the ability of cells to differentiate into osteoblasts or cartilage was assessed by detecting the expression of differentiation-related genes (such as RUNX2).

[0179] 2) In vitro and in vivo experimental comparison: The gel microspheres were cultured in vitro and implanted into animal models for in vivo experiments to compare the differences in cell differentiation, proliferation and repair effects.

[0180] IV. Experimental Results

[0181] 1) Please refer to Figure 6 The results of cell differentiation direction assessment showed that the prepared gel microspheres could significantly promote the differentiation of hA-MSCs into osteogenic and cartilaginous directions, and the expression level of differentiation-related genes was higher than that of the untreated control group.

[0182] 2. In vivo and in vitro experiments showed that the gel microspheres could better promote the repair of bone defects in the in vivo environment, and the cell differentiation, proliferation and repair effects were better than those in the in vitro experiments.

[0183] Of course, the above description is not intended to limit the invention, nor is the invention limited to the examples given above. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also fall within the protection scope of the invention. The parts of this invention not described in detail are well-known to those skilled in the art.

Claims

1. A gel microsphere carrying amniotic mesenchymal stem cells, characterized in that, Includes the following ingredients: Excipients: 2 wt%-10 wt% polyethylene glycol diacrylate and 1 wt%-3 wt% powdered amnion; Components that promote bone differentiation: sodium sapolixalate (0.1wt%-0.5wt%), mannitol (0.5wt%-2wt%), and L-arginine (0.1wt%-0.5wt%). Anti-inflammatory modulators: 0.1wt%-0.5wt% meglumine, 0.05wt%-0.5wt% o-phenanthroline, and 0.05wt%-0.5wt% acesulfame potassium; Cellular components: Cell concentration of 1×10 7 Amniotic mesenchymal stem cells (cells / mL); Photopolymerization initiator: 0.1wt%-2wt% of 1-hydroxycyclohexylphenyl ketone; The polyethylene glycol diacrylate has a molecular weight greater than 3000 Daltons, the powdered amnion has a particle size of less than 50 micrometers, and the concentration ratio of o-phenanthroline to acesulfame potassium in the anti-inflammatory modulator is 1:1-1:

6.

2. The gel microspheres carrying amniotic mesenchymal stem cells according to claim 1, characterized in that, The osteogenic component can promote the differentiation of amniotic mesenchymal stem cells into osteoblasts, thereby accelerating the bone defect repair process. The anti-inflammatory regulator can inhibit the inflammatory response, improve the repair effect, and reduce the risk of immune rejection. The amniotic mesenchymal stem cells can self-renew and differentiate into multiple cell types.

3. A method for preparing gel microspheres carrying amniotic mesenchymal stem cells, characterized in that, The gel microspheres carrying amniotic mesenchymal stem cells are those described in claim 1 or 2. They are produced by mixing a photopolymerization initiator, 1-hydroxycyclohexylphenyl ketone, using microfluidics at a flow rate of 8 μL / min to precisely control the flow and interaction of fluids within the microchannels. Ultraviolet light irradiation is used to initiate a free radical polymerization reaction, with the polymerization time not exceeding 8 minutes to ensure efficient polymerization while avoiding damage to the stem cells. A cross-linked network structure hydrogel is formed inside the droplet, thereby encapsulating the amniotic mesenchymal stem cells in the formed novel gel material. The novel gel microspheres are obtained through screen screening.

4. The method for preparing gel microspheres carrying amniotic mesenchymal stem cells according to claim 3, characterized in that, Specifically, the following steps are included: S1. Dissolve polyethylene glycol diacrylate in deionized water, add powdered amniotic membrane to form a solution of a certain concentration; the mass-volume concentration range of the polyethylene glycol diacrylate is 2%-10%, and the mass-volume concentration range of the amniotic membrane is 1%-3%, forming a uniform excipient solution. S2. Add the osteogenic differentiation-promoting components, which include sodium sapoxetine, mannitol, and L-arginine, to the excipient solution at a mass ratio of 5%-20% of the total mass, and stir thoroughly until completely dissolved. S3. Add the anti-inflammatory regulator at a mass ratio of 1%-5% of the total mass to the solution and continue stirring until the mixture is homogeneous. The anti-inflammatory regulator includes meglumine, o-phenanthroline, and acesulfame potassium. S4. Add amniotic mesenchymal stem cell suspension and stir well; S5. Using 1-hydroxycyclohexylphenyl ketone as a photopolymerization initiator, the mixture is mixed with the solution obtained in S4 and injected into the fluid channel of the microfluidic device. By precisely controlling the flow rate and volume, the fluid is ensured to flow stably in the microchannel. An ultraviolet light source is set at the outlet of the microfluidic device. When the mixed solution flows through the outlet, it is irradiated by ultraviolet light, which initiates a free radical polymerization reaction to form a colloidal solution. The colloidal solution reaches the screen to prepare a hydrogel. S6. The hydrogel is ejected from the screen to obtain novel gel microspheres.

5. The method for preparing gel microspheres carrying amniotic mesenchymal stem cells according to claim 4, characterized in that, The excipient solution in step S1 is kept at a temperature not exceeding 40°C and is prepared for subsequent processes.

6. The method for preparing gel microspheres carrying amniotic mesenchymal stem cells according to claim 4, characterized in that, In step S5, after adding the amniotic mesenchymal stem cell suspension, it serves as the inner phase of the microfluidic device. The intensity and irradiation time of the ultraviolet light at the outlet of the microfluidic device are adjusted according to the photopolymerization initiator 1-hydroxycyclohexylphenyl ketone and the precursor.

7. The method for preparing gel microspheres carrying amniotic mesenchymal stem cells according to claim 4, characterized in that, In step S6, the screen is a wire sieve with a pore size of 100 micrometers.

8. An application of a gel microsphere carrying amniotic mesenchymal stem cells, characterized in that, The gel microspheres carrying amniotic mesenchymal stem cells are gel microspheres as described in claim 1 or 2, or gel microspheres prepared by any of the preparation methods described in claims 3 to 7. The gel microspheres are implanted into bone defect sites, have good biocompatibility, and will not cause severe immune or rejection reactions.

9. The application of the gel microspheres carrying amniotic mesenchymal stem cells according to claim 8, characterized in that, The gel microspheres are suitable for repairing bone defects, including those in the skull, spine, and limbs.

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