Gel microspheres carrying amniotic mesenchymal stem cells as well as preparation and application of gel microspheres

Abstract

According to the gel microspheres loaded with amniotic mesenchymal stem cells as well as the preparation and the application of the gel microspheres loaded with the amniotic mesenchymal stem cells, polyethylene glycol diacrylate (PEGDA) and powdery amniotic membranes are taken as the basis, various osteogenic differentiation promoting factors and anti-inflammatory regulators are combined, and the amniotic mesenchymal stem cells (hA-MSC) are loaded; when the gel microspheres are prepared, high precision and high consistency of the gel microspheres are realized by utilizing a microfluid technology, so that the average size of the non-spherical microspheres is accurately controlled between 80 microns and 200 microns, the non-spherical microspheres are convenient to transplant or inject, and uniform distribution and effective action of cells in vivo are ensured; through a photopolymerization reaction, the gel microspheres are rapidly cured under mild conditions, keep cell activity and form a cross-linked network structure, and the gel microspheres not only have good biocompatibility and degradability, but also can simulate an in-vivo microenvironment, promote cell survival, proliferation and differentiation, regulate inflammatory response and create a favorable microenvironment for bone defect repair.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical engineering and tissue engineering, and specifically relates to a gel microsphere carrying amniotic mesenchymal stem cells and the preparation and application thereof. Background Art

[0002] Bone defects are a major challenge in clinical medicine. Traditional treatments such as surgery and medication, although they can relieve patients' symptoms to a certain extent, have obvious limitations. Surgical treatment not only has a long recovery period, but may also be accompanied by complications such as bleeding, infection, nerve or vascular damage, and is particularly difficult to repair for large or complex 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 technology, biomaterials combined with cell therapy have provided a new perspective for bone defect repair. Gel materials, due to their strong plasticity, easy operation, 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 deficiencies in biocompatibility, matching of degradation rate with bone regeneration rate, and ability to promote bone differentiation. Some gel materials may induce inflammatory responses after implantation, affecting cell survival and differentiation; at the same time, too fast a degradation rate may lead to incomplete bone regeneration, while too slow a degradation rate may hinder the formation of new bone tissue. Summary of the invention

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

[0005] To achieve the above object, the present invention provides the following technical solution: a gel microsphere carrying amniotic mesenchymal stem cells, comprising the following components: Excipients: 2wt%-10wt% of polyethylene glycol diacrylate and 1wt%-3wt% of powdered amniotic membrane; Osteodifferentiation promoting ingredients: 0.1wt%-0.5wt% of sapolisan sodium, 0.5wt%-2wt% of mannitol, and 0.1wt%-0.5wt% of L-arginine; Anti-inflammatory regulators: 0.1wt%-0.5wt% of meglumine, 0.05wt%-0.5wt% of o-phenanthroline, and 0.05wt%-0.5wt% of acesulfame potassium; Cell content: Cell concentration is 1×10 7 cells / mL of amniotic mesenchymal stem cells; Photopolymerization initiator: 0.1wt%-2wt% of 1-hydroxycyclohexyl phenyl ketone.

[0006] Preferably, the molecular weight of the polyethylene glycol diacrylate is greater than 3000 Daltons, and the particle size of the powdered amniotic membrane is less than 50 microns. The two serve as excipients to give the gel good biocompatibility and mechanical stability, and the pore size of the hydrogel formed by cross-linking is suitable for cell growth. The osteogenic differentiation 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 a variety of cell types, and have wide application potential.

[0007] The present invention also discloses a method for preparing gel microspheres carrying amniotic mesenchymal stem cells. The gel microspheres are the above-mentioned gel microspheres carrying amniotic mesenchymal stem cells. By mixing the photopolymerization initiator 1-hydroxycyclohexyl phenyl ketone and adopting microfluidic technology, the flow and interaction of the fluid in the microchannel are precisely controlled to achieve high-precision and high-consistency preparation of the gel microspheres. The free radical polymerization reaction is initiated by ultraviolet light irradiation to form a hydrogel with a cross-linked network structure inside the droplets, and the amniotic mesenchymal stem cells are then wrapped in the formed new gel material, and the new gel microspheres are obtained through screen screening.

[0008] As preferably, the method specifically includes the following steps: S1. dissolving polyethylene glycol diacrylate in deionized water, adding powdered amniotic membrane to form a solution of a certain concentration; the mass volume ratio of the polyethylene glycol diacrylate is in the range of 2%-10%, and the mass volume ratio of the amniotic membrane is in the range of 1%-3%, to form a uniform excipient solution; S2. Adding the osteogenic differentiation component to the excipient solution in an amount of 5% to 20% of the total mass, wherein the osteogenic differentiation component includes sodium sapolisan, mannitol, and L-arginine, and stirring until completely dissolved; S3. adding an anti-inflammatory regulator to the solution in an amount of 1% to 5% of the total mass, and continuing to stir until uniformly mixed, wherein the anti-inflammatory regulator includes meglumine, o-phenanthroline, and acesulfame potassium; S4. Add amnion mesenchymal stem cell suspension and stir evenly; S5. 1-hydroxycyclohexyl phenyl ketone is used as a photopolymerization initiator to mix with the mixed solution obtained in S4, and then injected into the fluid channel of the microfluidic device, and the flow rate and flow rate are precisely controlled to ensure the stable flow of the fluid in the microchannel; an ultraviolet light source is set at the outlet of the microfluidic device, and when the mixed solution flows through the outlet, it is irradiated by ultraviolet light to initiate a free radical polymerization reaction to form a colloidal solution, and the colloidal solution reaches the screen to prepare a hydrogel; S6. Pushing the hydrogel out from the screen, thereby preparing novel gel microspheres.

[0009] Preferably, the temperature of the excipient solution in step S1 does not exceed 40° C. for subsequent steps.

[0010] Preferably, after adding the amniotic membrane mesenchymal stem cell suspension in step S5, as the inner phase of the microfluid, the flow rate range is 8 μL / min, and the intensity and irradiation time of the ultraviolet light at the outlet of the microfluidic device are adjusted according to the photopolymerization initiator 1-hydroxycyclohexyl phenyl ketone and the precursor, and the polymerization time does not exceed 8 minutes to ensure efficient polymerization while avoiding damage to the stem cells.

[0011] Preferably, in step S6, the screen is a wire sieve with a pore size of 100 microns.

[0012] The present invention also discloses an application of gel microspheres carrying amniotic mesenchymal stem cells. The gel microspheres are the gel microspheres carrying amniotic mesenchymal stem cells mentioned above. The gel microspheres are implanted into bone defect sites through injection or surgery, have good compatibility, and will not cause severe immune response or rejection reaction.

[0013] Preferably, the gel microspheres are suitable for repairing various types of bone defects including skull, spine, and limb bones.

[0014] Preferably, the gel material adjusts the proportion of each component according to specific application requirements. The preparation is simple and quick, and a large amount of gel microsphere materials can be efficiently prepared. At the same time, these materials are also easy to store and transport, and are convenient for clinical use.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a gel microsphere carrying amniotic mesenchymal stem cells, which is based on polyethylene glycol diacrylate (PEGDA) and powdered amniotic membrane, combined with a variety of osteogenic differentiation factors and anti-inflammatory regulators, and carries amniotic mesenchymal stem cells (hA-MSC); when preparing the gel microspheres, microfluidic technology is used to achieve high precision and high consistency of the gel microspheres, so that the average size of the non-spherical microspheres is accurately controlled between 80 and 200 microns, which is convenient for transplantation or injection, ensuring that the cells are evenly distributed and effectively act in the body; through photopolymerization reaction, the gel microspheres are quickly solidified under mild conditions, maintaining cell activity and forming a cross-linked network structure. The gel microspheres not only have good biocompatibility and degradability, but also can simulate the in vivo microenvironment, promote cell survival, proliferation and differentiation, regulate inflammatory response, and create a favorable microenvironment for bone defect repair.

[0016] The following technical problems existing in the prior art are solved: (1) Low cell survival rate: In traditional cell transplantation therapy, cells often find it difficult to adapt to the new environment after transplantation, resulting in low survival rate. The present 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 and other excipients, hydrogel microspheres with a cross-linked network structure are prepared by 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 proliferation ability and multidirectional differentiation potential. In the gel microspheres, amniotic mesenchymal stem cells are effectively encapsulated, which not only protects the cells from direct impact of the external environment, but also continuously releases growth factors through the sustained release effect of the microspheres to promote cell survival and proliferation. The experimental results show that in an animal experiment using the gel microspheres of the present invention to carry amniotic mesenchymal stem cells for bone defect repair, the cell survival rate was increased by about 30% compared with the control group, and the cell morphology was good, showing good proliferation activity.

[0017] (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. In order to solve this problem, the present invention adopts the following measures: 1) Interaction of components: In the gel microspheres, osteogenic differentiation factors (such as sodium sapolis, mannitol, L-arginine) and anti-inflammatory regulators (methylglucamine, o-phenanthroline, acesulfame potassium) are integrated. These components can work synergistically to promote the proliferation, differentiation and matrix synthesis of chondrocytes, while inhibiting inflammatory reactions and accelerating the tissue repair process. 2) Design of non-spherical microbead structure: By designing a non-spherical microbead 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 action 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, during the 8-week observation period, histological examination showed that the quality of cartilage regeneration was significantly improved, the defect area was effectively filled with new cartilage tissue, and no obvious inflammatory reaction was observed. Compared with the control group, the cartilage thickness and collagen fiber arrangement in the repair area showed better repair effects.

[0018] (3) Problems with preparation accuracy and consistency: Traditional gel preparation often has difficulty in ensuring the accuracy and consistency of materials, resulting in unstable treatment effects. In order to solve this problem, the present invention has taken the following measures: The preparation technology introduces microfluidic technology, so that the preparation process of gel microspheres can achieve high accuracy and high consistency. By precisely controlling the geometric dimensions, flow rate and fluid pressure of the microfluidic microchannel, gel microspheres with uniform size and stable morphology can be prepared. Such highly consistent microspheres are not only conducive to the uniform distribution and effective action of cells, but also improve the filling effect and stability of the treatment material at the defect site, thereby enhancing the predictability and reliability of the treatment effect.

[0019] (4) Problems of cell distribution and uniformity: In traditional cell transplantation therapy, the distribution of cells is often difficult to control, resulting in uneven treatment effects. The gel microspheres prepared by this technology achieve uniform distribution of cells. This uniform cell distribution not only improves the survival rate and proliferation activity of cells, but also promotes the interaction and synergy between cells, thereby enhancing the repair effect of the therapeutic material at the defect site.

[0020] (5) The problem of preparation efficiency and cost: Traditional gel preparation often requires complex processes and a lot of time, and the preparation cost is high. This preparation greatly improves the preparation efficiency of gel microspheres and reduces the preparation cost through automated and continuous production methods. This efficient and low-cost preparation is not only conducive to large-scale production, but also reduces the cost of treatment materials, allowing more patients to benefit from this innovative bone defect repair solution.

[0021] It has the following beneficial effects: (1) Innovative material preparation: The present invention combines polyethylene glycol diacrylate (PEGDA) with amniotic membrane excipients to prepare hydrogel microspheres through photopolymerization reaction to provide a suitable growth environment for cells.

[0022] (2) Efficient cell encapsulation: hA-MSCs with low immunogenicity, high proliferation and multidirectional differentiation potential are used to enhance their osteogenic differentiation potential and anti-inflammatory ability through pretreatment, and are effectively encapsulated in gel microspheres.

[0023] (3) Unique morphological design: The non-spherical microbeads are easy to transplant or inject, effectively preventing the movement of cells, blood and other substances, ensuring the accuracy and efficiency of the repair process.

[0024] (4) Improve cell survival rate: The new gel microsphere material significantly improves cell survival rate through encapsulation and protection, solving the problem of low cell survival rate in traditional cell transplantation.

[0025] (5) Synergistic therapeutic effect: The gel contains 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 functions.

[0026] (6) Local administration reduces side effects: The new gel microsphere material uses local administration to directly deliver drugs or cells to the defect site, reduce drug loss, and reduce the risk of systemic side effects.

[0027] (7) Improve preparation efficiency and precision: Microfluidics technology can precisely control and manipulate fluids at the micro-nanoscale, ensuring that the generated gel microspheres are highly monodisperse and uniform in size. By optimizing the design of microfluidic channels, the size, shape, and composition of the gel microspheres can be precisely controlled to achieve personalized customization to meet the treatment needs of different patients.

[0028] (8) Enhanced cell encapsulation efficiency: This preparation technology can efficiently encapsulate hA-MSCs into gel microspheres while maintaining cell activity and function.

[0029] (9) Achieve high-throughput preparation: This preparation technology has the characteristics of high throughput and can prepare a large number of gel microspheres in a short time. This greatly improves production efficiency, reduces preparation costs, and provides more possibilities for clinical applications.

[0030] (10) Convenient for drug delivery and release control: The gel microspheres prepared by this technology can achieve local delivery and controlled release of drugs. Drugs can be encapsulated inside the gel microspheres, thereby achieving slow and sustained release of drugs. This release method helps to reduce the systemic side effects of drugs and improve the therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The figure shows the flow chart of the preparation of gel microspheres carrying amniotic mesenchymal stem cells.

[0032] Figure 2 In the specific embodiment 2, the survival of amniotic mesenchymal stem cells in the gel material is demonstrated by a cell counter.

[0033] Figure 3 In specific example 2, the ability of cells to differentiate into osteoblasts or chondrocytes is evaluated by detecting the expression of the differentiation-related gene RUNX2.

[0034] Figure 4 In specific example 3, the survival of amniotic mesenchymal stem cells in the gel material is demonstrated by a cell counter.

[0035] Figure 5 In specific example 3, the ability of cells to differentiate into osteoblasts or chondrocytes is evaluated by detecting the expression of the differentiation-related gene RUNX2.

[0036] Figure 6 In specific example 4, the ability of cells to differentiate into osteoblasts or chondrocytes is evaluated by detecting the expression of the differentiation-related gene RUNX2. DETAILED DESCRIPTION

[0037] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below with reference to specific embodiments in conjunction with the accompanying drawings. However, the present invention is not limited to a specific example and this method can be applied to similar situations. Example 1

[0038] See also Figure 1 This embodiment discloses a gel microsphere carrying amniotic mesenchymal stem cells and its preparation. Please refer to the relevant drawings. The gel microsphere carrying amniotic mesenchymal stem cells includes: ① Excipients: Polyethylene glycol diacrylate (PEGDA): As the main component of hydrogel, it provides good mechanical properties and biocompatibility.

[0039] Powdered amniotic membrane: rich in various growth factors and extracellular matrix components, which helps cell growth and tissue repair.

[0040] ② Factors that promote osteogenic differentiation: Sodium sapolinate, mannitol, and L-arginine: These ingredients can stimulate cell differentiation towards osteoblastic direction and promote the repair and regeneration of cartilage defects.

[0041] ③ Anti-inflammatory regulators: Meglumine, 1,2-phenanthroline, and acesulfame potassium: have anti-inflammatory and immunomodulatory effects, can reduce inflammatory responses, and provide a good microenvironment for cell growth.

[0042] ④Amniotic membrane mesenchymal stem cells (hA-MSC): It has multidirectional differentiation potential and can differentiate into chondrocytes, participating in the repair and reconstruction of cartilage tissue.

[0043] ⑤ Photopolymerization initiator: 1-Hydroxycyclohexylphenyl ketone: As an initiator of photopolymerization reaction, it can quickly initiate the polymerization reaction of PEGDA under ultraviolet light to form a hydrogel with a cross-linked network structure.

[0044] The above ingredients are scientifically proportioned and carefully prepared to form the novel gel microsphere material of the present invention, which provides a highly efficient, safe and degradable material for bone defect repair.

[0045] This embodiment discloses a method for preparing the gel microspheres carrying amniotic mesenchymal stem cells, which first mixes polyethylene glycol diacrylate (PEGDA) with powdered amniotic membrane as a basic excipient, and then adds a variety of carefully selected osteogenic differentiation factors, such as sodium sapolis, mannitol, L-arginine, etc., and anti-inflammatory regulators, such as meglumine, o-phenanthroline, acesulfame potassium, etc., to ensure that the material has excellent biological activity and anti-inflammatory properties. At the same time, amniotic mesenchymal stem cells (hA-MSC) with multidirectional differentiation potential are evenly dispersed in the above mixture. Then, using advanced microfluidic technology, by precisely controlling the fluid flow rate and flow rate, the mixture is prepared into non-spherical microbeads, whose average size is precisely controlled between 80 and 200 microns, to ensure that the microspheres are easy to transplant or inject, and can ensure that the cells are evenly distributed and effectively act in the body. Finally, through photopolymerization, under mild and cell-friendly conditions, the gel microspheres are rapidly solidified to form hydrogel microspheres with a stable cross-linked network structure. Finally, the gel microspheres are prepared into non-spherical microspheres by extrusion from a 100 μm screen. After washing, drying, and quality inspection, they are sterile packaged and stored for future use. The gel microspheres prepared by this method have excellent biocompatibility and osteogenic differentiation promotion ability, providing an efficient treatment solution for bone defect repair.

[0046] The specific steps include: 1. Material preparation (1) Ethylene glycol diacrylate (PEGDA): PEGDA with a molecular weight greater than 3000 Dalton is selected as the main excipient, which has good water solubility to ensure that it forms a stable cross-linked network structure in the hydrogel while maintaining good biocompatibility and mechanical properties. The concentration range is 2%-10% (w / v).

[0047] (2) Amniotic membrane: The amniotic membrane powder is freeze-dried and strictly processed and sterilized. It is used as the component of the gel. It is rich in various growth factors and extracellular matrix components, which help cell growth and differentiation and provide a suitable environment for the adhesion, proliferation and differentiation of stem cells. The concentration range is 1 wt%-3 wt%.

[0048] (3) Sarpagin: a naturally occurring plant extract that promotes cell differentiation into osteoblasts through its specific biological activity, thereby contributing to the repair and regeneration of bone tissue. The concentration range is 0.05 wt%-0.1 wt%.

[0049] (4) Mannitol: It plays an auxiliary role in promoting osteogenic differentiation. It indirectly promotes osteogenic differentiation by affecting the cell environment or metabolic pathways. The concentration range is 0.1wt%-0.5wt%.

[0050] (5) L-Arginine: An essential amino acid that participates in protein synthesis and has a positive effect on cartilage repair. It can improve arthritis and bone erosion by 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%.

[0051] (6) Meglumine: An alkaline substance that can inhibit the production of pro-inflammatory factors such as interleukin-1β, reduce joint inflammation, and help provide a good microenvironment for cell growth, thereby promoting tissue repair and regeneration. The concentration range is 0.05wt%-0.1wt%.

[0052] (7) Phenanthroline: As a metal ion chelator, it can reduce the expression of MMP3 and MMP13 mRNA. These two matrix metalloproteinases are related to inflammatory response and tissue damage, and play a role in inhibiting inflammatory response. The concentration range is 0.01wt%-0.05wt%.

[0053] (8) Acesulfame Potassium: As a chemically synthesized substance, it has multiple 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. The concentration range is 0.01wt%-0.06wt%.

[0054] (9) Amniotic membrane mesenchymal stem cells (hA-MSC): hA-MSC are isolated and cultured from the amniotic membrane of healthy donors and undergo rigorous testing and screening to ensure that they have good multidirectional differentiation potential and biological activity.

[0055] (10) 1-Hydroxycyclohexylphenyl ketone: As an initiator for photopolymerization, it should be accurately weighed and properly stored to avoid its ineffectiveness or degradation.

[0056] 2. Preparation process Step 1: Solution preparation 1) Dissolve PEGDA in an appropriate amount of deionized water to form a PEGDA solution.

[0057] 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.

[0058] 3) Sodium sapolisar, mannitol and L-arginine are dissolved in a solvent respectively to form an osteogenic differentiation factor solution.

[0059] 4) Dissolve meglumine, o-phenanthroline and acesulfame potassium in a solvent to form an anti-inflammatory regulator solution.

[0060] Step 2: Mixing and homogenization 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-MSCs.

[0061] 2) Use a magnetic stirrer or ultrasonic processor to disperse the mixture evenly to ensure that all components are fully mixed.

[0062] Step 3: Add photopolymerization initiator 1) Dissolve 1-hydroxycyclohexyl phenyl ketone in a small amount of solvent, then add it to the above mixture and stir evenly.

[0063] Step 4: Preparation of gel droplets using microfluidics technology 1) Using a microfluidic device, inject the mixture in step 3 into a microchannel at a certain flow rate to form stable droplets.

[0064] 2) Gel droplets with uniform size and morphology are prepared by precisely controlling the geometry and flow rate of the microchannel.

[0065] Step 5: Photopolymerization 1) Placing the microfluidic device containing the gel droplets under a UV light source to initiate a photopolymerization reaction.

[0066] 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.

[0067] Step 6: Preparation of gel microspheres 1) Collecting the polymerized hydrogel microspheres from the microfluidic device.

[0068] 2) Screening or other methods are used to obtain gel microspheres with non-spherical microsphere morphology and ensure that their average size is in the range of 80 to 200 microns.

[0069] Step 7: Wash and dry 1) Wash the gel microspheres with sterile saline or distilled water to remove unreacted residues.

[0070] 2) Drying the washed gel microspheres to remove excess water.

[0071] Step 8: Quality Inspection and Packaging 1) Conduct quality inspection on the prepared gel microspheres, including inspection of appearance, size, biocompatibility and other indicators.

[0072] 2) The qualified gel microspheres are aseptically packaged and stored under appropriate conditions for future use.

[0073] Through the above preparation, the present invention successfully prepared a gel microsphere carrying amniotic mesenchymal stem cells, which has excellent biocompatibility, osteogenic differentiation promotion and anti-inflammatory regulation ability, and can be used as a filler for soft tissue reconstruction, effectively promoting the rapid repair of cartilage defects. At the same time, the preparation is simple, controllable, and suitable for large-scale production, providing an innovative and efficient treatment solution for bone defect repair. Example 2

[0074] This embodiment discloses a preparation and application experiment of gel microspheres carrying amniotic mesenchymal stem cells.

[0075] 1. Material preparation PEGDA: PEGDA with a molecular weight of 3000 Dalton and a concentration of 5% (w / v) was selected.

[0076] Amniotic membrane powder: concentration is 2 wt%.

[0077] Sodium sapolimate: concentration is 0.075 wt%.

[0078] Mannitol: concentration is 0.3 wt%.

[0079] L-arginine: concentration is 0.1 wt%.

[0080] Meglumine: concentration is 0.075 wt%.

[0081] O-phenanthroline: concentration is 0.03 wt%.

[0082] Acesulfame potassium: concentration is 0.03 wt%.

[0083] Amniotic membrane mesenchymal stem cells (hA-MSC): 1×10 per ml of solution 6 The ratio of cells added.

[0084] Photopolymerization initiator: 1-hydroxycyclohexyl phenyl ketone, added at a ratio of 0.5%.

[0085] 2. Preparation 1) Dissolve PEGDA in deionized water and heat to 40°C to accelerate dissolution to form a uniform solution.

[0086] 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.

[0087] 3) Dissolve saprolidine sodium, mannitol, L-arginine, meglumine, o-phenanthroline and acesulfame potassium in deionized water to form respective solutions.

[0088] 4) Mix all the above solutions and add hA-MSCs, stirring gently to avoid damaging the cells.

[0089] 5) Add photopolymerization initiator and perform ultrasonic treatment for 5 minutes to achieve uniform dispersion of all components.

[0090] 6) The above liquid was used as the inner phase of the microfluidic device, and the flow rate was controlled at 8 μL / min. The intensity and irradiation time of the ultraviolet light were adjusted at the outlet through the microfluidic device.

[0091] 7) Expose the microfluid under ultraviolet light for 5 minutes to initiate a photopolymerization reaction to form a hydrogel.

[0092] 8) The non-spherical microspheres were screened using a sieve, washed three times with deionized water, and then freeze-dried.

[0093] 9) After quality inspection, sterile packaging is performed and stored at -20°C until use.

[0094] 3. Experimental part 1) Cell viability detection: The prepared gel microspheres are placed in a cell culture medium. After a period of culture, the cells are stained with trypan blue dye to distinguish live cells from dead cells and count them. This is a cell activity detection method.

[0095] 2) Evaluation of osteogenic differentiation ability: The gel microspheres were implanted into the rat animal model (bone defect), and samples were collected at predetermined time points (such as 4 weeks, 8 weeks, and 12 weeks) for histological, biochemical, and molecular biological analyses to evaluate the osteogenic differentiation ability and the effect of bone repair.

[0096] 4. Experimental results: 1) Please refer to Figure 2 ,The cell survival rate test results showed that the prepared gel microspheres had good cell compatibility in the culture medium, and the cell survival rate was higher than 90%.

[0097] 2) Please refer to Figure 3 In vitro cell detection experiments showed that gel microspheres can significantly promote the repair of bone defects, have strong osteogenic differentiation ability, and their RUNX2 indicators are better than those of the untreated control group. Example 3

[0098] This embodiment discloses a preparation and application experiment of gel microspheres carrying amniotic mesenchymal stem cells.

[0099] 1. Material preparation PEGDA: PEGDA with a molecular weight of 5000 Dalton and a concentration of 8% (w / v) was selected.

[0100] Amniotic membrane powder: concentration is 1.5 wt%.

[0101] Sodium sapolimate: concentration is 0.1 wt%.

[0102] Mannitol: concentration is 0.4 wt%.

[0103] L-arginine: concentration is 0.15 wt%.

[0104] Meglumine: concentration is 0.05 wt%.

[0105] O-phenanthroline: concentration is 0.02 wt%.

[0106] Acesulfame potassium: concentration is 0.05 wt%.

[0107] Amniotic membrane mesenchymal stem cells (hA-MSC): 2×10 per ml of solution 6 The ratio of cells added.

[0108] Photopolymerization initiator: 1-hydroxycyclohexyl phenyl ketone, added at a ratio of 1%.

[0109] 2. Preparation 1) Dissolve PEGDA and add amniotic membrane powder, and stir magnetically for 4 hours.

[0110] 2) Dissolve the other components separately, mix them and add hA-MSC and photopolymerization initiator.

[0111] 3) Ultrasonic treatment for 10 minutes to ensure uniform dispersion.

[0112] 4) Liquid was used as the internal phase of the microfluidic system, and the flow rate was controlled at 8 μL / min.

[0113] 5) Irradiate with ultraviolet light for 10 minutes to form a hydrogel.

[0114] 6) Screening, washing, drying, quality inspection, sterile packaging and storage.

[0115] 3. Experimental part 1) Cell viability detection: The prepared gel microspheres are placed in a cell culture medium. After a period of culture, the cells are stained with trypan blue dye to distinguish live cells from dead cells and count them. This is a cell activity detection method.

[0116] 2) Animal model experiment: The gel microspheres were implanted into the bone defect of the animal model to evaluate their repair effect.

[0117] IV. Experimental Results 1) Please refer to Figure 4 The prepared gel microspheres have good cell compatibility in the culture medium, and the cell survival rate is higher than 90%, even reaching more than 95%.

[0118] 2) Please refer to Figure 5 In vitro cell experiments found that the adjusted gel microspheres showed better performance in bone defect repair, and both histological scores and biochemical indicators were improved at the animal level. Example 4

[0119] This embodiment discloses a preparation and application experiment of gel microspheres carrying amniotic mesenchymal stem cells.

[0120] 1. Material preparation PEGDA: PEGDA with a molecular weight of 8000 Dalton and a concentration of 2% (w / v) was selected.

[0121] Amniotic membrane powder: concentration is 3 wt%.

[0122] Sodium sapolinate: concentration is 0.05 wt%.

[0123] Mannitol: concentration is 0.1 wt%.

[0124] L-arginine: concentration is 0.05 wt%.

[0125] Meglumine: concentration is 0.1 wt%.

[0126] O-phenanthroline: concentration is 0.01 wt%.

[0127] Acesulfame potassium: concentration is 0.06 wt%.

[0128] Amniotic membrane mesenchymal stem cells (hA-MSC): 0.5×10 per ml of solution 6 The ratio of cells added.

[0129] Photopolymerization initiator: 1-hydroxycyclohexyl phenyl ketone, added at a ratio of 0.25%.

[0130] 2. Preparation: 1) Dissolve PEGDA and add amniotic membrane powder, and stir magnetically for 3 hours.

[0131] 2) Dissolve the other components separately, mix them and add hA-MSC and photopolymerization initiator.

[0132] 3) Ultrasonic treatment for 8 minutes to ensure uniform dispersion.

[0133] 4) Liquid was used as the internal phase of the microfluidic system, and the flow rate was controlled at 8 μL / min.

[0134] 5) Irradiate with ultraviolet light for 8 minutes to form a hydrogel.

[0135] 6) Screening, washing, drying, quality inspection, sterile packaging and storage.

[0136] 3. Experimental part 1) Evaluation of cell differentiation direction: Using a specific differentiation medium, the gel microspheres are cultured together with hA-MSCs, and the ability of cells to differentiate into osteoblasts or chondrocytes is evaluated by detecting the expression of differentiation-related genes (such as RUNX2).

[0137] 2) Comparison of in vitro and in vivo experiments: The gel microspheres were used for in vitro cell culture experiments and implanted into animal models for in vivo experiments to compare the differences in cell differentiation, proliferation and repair effects.

[0138] IV. Experimental Results 1) Please refer to Figure 6 The results of cell differentiation direction evaluation showed that the prepared gel microspheres could significantly promote the differentiation of hA-MSCs into osteoblasts and chondrocytes, and the expression level of differentiation-related genes was higher than that of the untreated control group.

[0139] 2 Comparison of in vitro and in vivo experiments showed that gel microspheres can better promote the repair of bone defects in the in vivo environment, and the cell differentiation, proliferation and repair effects are better than the in vitro experimental results.

[0140] Of course, the above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by those skilled in the art within the essential scope of the present invention shall also fall within the protection scope of the present invention. The present invention does not elaborate on some of the known technologies of those skilled in the art.

Claims

1. A gel microsphere carrying amniotic mesenchymal stem cells, characterized in that: Includes the following ingredients: Excipients: 2wt%-10wt% of polyethylene glycol diacrylate and 1wt%-3wt% of powdered amniotic membrane; Osteodifferentiation promoting ingredients: 0.1wt%-0.5wt% of sapolisan sodium, 0.5wt%-2wt% of mannitol, and 0.1wt%-0.5wt% of L-arginine; Anti-inflammatory regulators: 0.1wt%-0.5wt% of meglumine, 0.05wt%-0.5wt% of o-phenanthroline, and 0.05wt%-0.5wt% of acesulfame potassium; Cell content: Cell concentration is 1×10 7 cells / mL of amniotic mesenchymal stem cells; Photopolymerization initiator: 0.1wt%-2wt% of 1-hydroxycyclohexyl phenyl ketone.

2. The gel microspheres carrying amniotic mesenchymal stem cells according to claim 1, characterized in that: The molecular weight of the polyethylene glycol diacrylate is greater than 3000 Daltons, the particle size of the powdered amniotic membrane is less than 50 microns, the osteogenic differentiation 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, and 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 the gel microspheres described in claim 1 or 2. By mixing the photopolymerization initiator 1-hydroxycyclohexyl phenyl ketone, microfluidic technology is used to accurately control the flow and interaction of the fluid in the microchannel, and ultraviolet light is used to induce a free radical polymerization reaction to form a hydrogel with a cross-linked network structure inside the droplets, and then the amniotic mesenchymal stem cells are wrapped in the formed new gel material, and the new 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: The specific steps include: S1. dissolving polyethylene glycol diacrylate in deionized water, adding powdered amniotic membrane to form a solution of a certain concentration; the mass volume ratio of the polyethylene glycol diacrylate is in the range of 2%-10%, and the mass volume ratio of the amniotic membrane is in the range of 1%-3%, to form a uniform excipient solution; S2. Adding the osteogenic differentiation component to the excipient solution in an amount of 5% to 20% of the total mass, wherein the osteogenic differentiation component includes sodium sapolisan, mannitol, and L-arginine, and stirring until completely dissolved; S3. adding an anti-inflammatory regulator to the solution in an amount of 1% to 5% of the total mass, and continuing to stir until uniformly mixed, wherein the anti-inflammatory regulator includes meglumine, o-phenanthroline, and acesulfame potassium; S4. Add amnion mesenchymal stem cell suspension and stir evenly; S5. 1-hydroxycyclohexyl phenyl ketone is used as a photopolymerization initiator to mix with the mixed solution obtained in S4, and then injected into the fluid channel of the microfluidic device, and the flow rate and flow rate are precisely controlled to ensure the stable flow of the fluid in the microchannel; an ultraviolet light source is set at the outlet of the microfluidic device, and when the mixed solution flows through the outlet, it is irradiated by ultraviolet light to initiate a free radical polymerization reaction to form a colloidal solution, and the colloidal solution reaches the screen to prepare a hydrogel; S6. Pushing the hydrogel out from the screen, thereby preparing novel gel microspheres.

5. The method for preparing gel microspheres carrying amniotic mesenchymal stem cells according to claim 4, characterized in that: The temperature of the excipient solution in step S1 does not exceed 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: After the amniotic membrane mesenchymal stem cell suspension is added in step S5, it is used 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-hydroxycyclohexyl phenyl ketone and the precursor, and the polymerization time does not exceed 8 minutes to ensure efficient polymerization while avoiding damage to the stem cells.

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 microns.

8. An application of gel microspheres carrying amniotic mesenchymal stem cells, characterized in that: The gel microspheres carrying amniotic mesenchymal stem cells are the gel microspheres described in claim 1 or 2 or the gel microspheres prepared by the preparation method described in any one of claims 3 to 7. The gel microspheres are implanted into the bone defect site by injection or surgery, have good compatibility, and will not cause severe immune response or rejection reaction.

9. The use of gel microspheres carrying amniotic mesenchymal stem cells according to claim 8, characterized in that: The gel microspheres are suitable for repairing bone defects including skull, spine and limb bones.

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