Preparation method for polylactic acid porous microspheres and use thereof in stem cell seeding

Abstract

The present invention relates to the technical field of functional composite materials, and disclosed are a preparation method for polylactic acid porous microspheres, and a use thereof in stem cell seeding. The method comprises the following steps: (1) dissolving chitosan in an acetic acid aqueous solution, adding bioactive glass nanoparticles, and performing ultrasonic treatment, to obtain an aqueous phase solution; (2) dissolving PLGA or PLA in dichloromethane and adding an emulsifier, to obtain an oil phase solution; (3) adding the aqueous phase solution dropwise into the oil phase solution, to obtain a water-in-oil initial emulsion; (4) adding the water-in-oil primary emulsion dropwise into a PVA aqueous solution, to obtain a double emulsion; (5) stirring the double emulsion, then performing centrifugation and drying, followed by crosslinking treatment in a glutaraldehyde solution, to obtain porous microspheres. The present invention prepares porous microspheres by means of a double emulsion solvent evaporation method, resulting in more uniform pore size distribution and higher bioactivity, which can thus act as an excellent carrier for stem cell seeding, and support high-efficiency cell adhesion and proliferation.

Description

A method for preparing polylactic acid porous microspheres and their application in stem cell seeding. Technical Field

[0001] This invention relates to the technical field of functional composite materials, and in particular to a method for preparing polylactic acid porous microspheres and their application in stem cell seeding. Background Technology

[0002] In tissue engineering and regenerative medicine, scaffold materials play a crucial role in promoting cell attachment, proliferation, and differentiation. Polylactic-co-glycolic acid copolymer (PLGA), polylactic acid (PLA), and polycaprolactone (PCL) are all biodegradable polymers that have been widely used in the biomedical field. Combining hydroxyapatite (HA) or bioactive glass (BAG) with PLGA, PLA, or PCL can improve their biocompatibility and osteoconductivity. However, further promoting cell attachment and proliferation remains a challenge.

[0003] Patent CN 111905151 A discloses a mesoporous bioactive glass / polylactic acid-glycolic acid copolymer composite microsphere and its preparation method and application. Specifically, it discloses: first, mesoporous bioactive glass is prepared using microemulsion technology combined with the sol-gel template method; then, PLGA, Span 80, and the mesoporous bioactive glass are sequentially added to dichloromethane, stirred, and ultrasonically dispersed to form an S / O emulsion; this emulsion is then sequentially added to polyethylene solutions W1 and W2 of different concentrations, stirred, and the solvent is evaporated. After washing, centrifugation, and freeze-drying, the microspheres are obtained. However, the bioactivity of the composite microspheres prepared by this method is still relatively low, and it cannot meet the requirements for high-proportion cell attachment and proliferation. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method for preparing polylactic acid porous microspheres and their application in stem cell seeding. The method involves preparing polylactic acid / chitosan composite porous microspheres containing bioactive glass via a double emulsion solvent evaporation method. These microspheres exhibit a uniform pore size distribution and higher bioactivity, enhancing the adsorption capacity of proteins or peptides on the microsphere surface, thereby enabling the attachment and proliferation of a high proportion of cells.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] In a first aspect, the present invention provides a method for preparing polylactic acid porous microspheres, comprising the following steps:

[0007] (1) Chitosan was dissolved in an aqueous acetic acid solution, and bioactive glass nanoparticles were added and then sonicated to obtain an aqueous solution;

[0008] (2) Dissolve PLGA or PLA in dichloromethane and add an emulsifier to obtain an oil phase solution;

[0009] (3) Add the aqueous phase solution dropwise to the oil phase solution to obtain a water-in-oil primary emulsion;

[0010] (4) Add the water-in-oil primary emulsion dropwise into the PVA aqueous solution to obtain a double emulsion;

[0011] (5) Stir the double emulsion, then centrifuge and dry it, and then immerse it in glutaraldehyde solution for cross-linking treatment to obtain porous microspheres.

[0012] This invention prepares porous microspheres via a dual-emulsion solvent evaporation method. In the primary emulsion (W / O), the aqueous phases of chitosan and bioactive glass (BAG) are dispersed in an organic solvent containing PLGA or PLA, forming a droplet structure where water droplets are dispersed in the oil phase. The water-in-oil primary emulsion is then dropwise added to an aqueous PVA solution to obtain a dual emulsion (W / O / W). During emulsification and organic solvent evaporation, the inner aqueous phase (chitosan / BAG) gradually migrates from the system to the outer aqueous phase, forming internal pores.

[0013] This process is similar to "aqueous phase extraction." Chitosan carries BAG nanoparticles and gradually migrates outward. Due to the subsequent evaporation of the solvent and the phase separation of the aqueous phase inside the microspheres, channels and cavities are left in the PLGA matrix, forming a porous structure of the microspheres. PLGA or PLA then solidifies as a structural support material. BAG nanoparticles can also embed themselves in the PLGA matrix. BAG is rich in calcium ions and can release calcium ions in an aqueous environment. Calcium ions can form ionic bonds with the negatively charged parts (such as carboxyl or phosphate groups) of proteins or peptides, thereby enhancing the adsorption capacity of proteins or peptides on the surface of the microspheres and increasing the bioactivity of the microspheres. Simultaneously, due to the aqueous phase extraction process, chitosan exists in both the outer and inner aqueous phases and is also embedded in the PLGA or PLA matrix. Chitosan molecules can interact with PLGA or PLA, and the three-dimensional network structure formed by the molecular chains can provide support during the solidification process of the microspheres, preventing the microsphere structure from collapsing due to voids. Furthermore, during the evaporation of organic solvents, chitosan present in the external aqueous phase adsorbs onto the surface of PLGA or PLA microspheres. Chitosan molecules are rich in amino groups, and the modification effect of chitosan molecules on the surface of microspheres can enhance the adsorption of proteins or peptides, which helps to better adsorb biomolecules in the aqueous environment and improve surface activity.

[0014] Chitosan is a hydrophilic material, while PLGA or PLA is a hydrophobic material. Single-emulsion methods (such as the W / O method) are not conducive to the uniform mixing of the two materials. However, the dual-emulsion method (W / O / W) of this invention helps to uniformly distribute chitosan within the PLGA or PLA matrix, forming a porous structure. Simultaneously, it allows BAG particles to be embedded within the microspheres. The distribution of chitosan and BAG within the microspheres enhances their bioactivity. Furthermore, in single-emulsion methods, the porosity and pore size of the microspheres are often difficult to control, easily leading to uneven pore size. The dual-emulsion method of this invention, however, is more advantageous in controlling pore size and porosity through parameter adjustments, resulting in microspheres with a more uniform pore size distribution.

[0015] Preferably, in step (1), the mass concentration of the acetic acid aqueous solution is 1~2%; the ratio of the amount of chitosan, bioactive glass nanoparticles and acetic acid aqueous solution added is 1~4g:1~2g:100mL.

[0016] Preferably, in step (1), the degree of deacetylation of the chitosan is 70-80%, the average molecular weight is 20-100 kDa, and the solution viscosity is 20-100 mPa·s (1% aqueous solution, w / w, 25℃); the average particle size of the bioactive glass nanoparticles is 200-1000 nm; and the ultrasonic conditions are 30-50 kHz for 20-30 min.

[0017] Chitosan has a degree of deacetylation of 70-80%, exhibiting good hydrophilicity and biocompatibility. It floats in a flocculent state in dilute acetic acid solution, which is beneficial for adsorbing bioactive glass nanoparticles and for forming a better water-in-oil primary emulsion, facilitating its escape. Conversely, excessive deacetylation results in a clear, oily solution in dilute acetic acid, causing it to aggregate at the oil-water interface and hindering chitosan escape during aqueous extraction. The molecular weight of chitosan also affects the aqueous extraction process; excessively high molecular weight leads to excessive viscosity. Appropriate viscosity helps ensure uniform dispersion in the aqueous phase, guaranteeing the stability of the emulsion process and facilitating escape. A suitable molecular weight provides good mechanical strength and solubility, promoting pore formation.

[0018] Preferably, in step (2), the ratio of PLGA or PLA to dichloromethane is 4~6g: 500mL; the average molecular weight of PLGA or PLA is 10~50 kDa.

[0019] Preferably, in step (2), the emulsifier is PVA or Tween 80; the oil phase solution contains 1~5 g / mL of emulsifier.

[0020] Preferably, in step (3), the volume ratio of the aqueous phase solution to the oil phase solution is 1~1.3:5.

[0021] Preferably, in step (3), the aqueous phase solution is added dropwise to the oil phase solution while stirring and sonicating simultaneously; the stirring conditions are: 1000~1500 rpm, 30~40 min; the sonication conditions are: 30~50 kHz, 20~30 min.

[0022] Preferably, in step (4), the concentration of the PVA aqueous solution is 10~12g / L; the volume ratio of the water-in-oil primary emulsion to the PVA aqueous solution is 6~8:50.

[0023] Preferably, in step (4), the water-in-oil primary emulsion is added dropwise to the PVA aqueous solution while stirring; the stirring conditions are: 600~800 rpm, 60~90 min.

[0024] The porosity and pore size of microspheres can be adjusted by regulating the water-to-oil ratio, stirring speed, and solvent evaporation time. The ratio of the aqueous to the oil phase directly affects porosity and pore size; an excessively high aqueous ratio leads to more pore formation, affecting the mechanical properties and structural stability of the microspheres, while an excessively low aqueous ratio may result in fewer pores, affecting the effectiveness of stem cell seeding. The stirring speed affects the emulsification effect and the size of the microspheres, thus impacting their structural stability. The solvent evaporation rate affects the solidification speed of PLGA / PLA and the effectiveness of aqueous phase extraction; a suitable evaporation rate promotes the formation of a uniform pore structure, provides sufficient migration time for the internal aqueous phase, and allows chitosan to better modify the surface of PLGA or PLA microspheres.

[0025] Preferably, in step (5), the dual emulsion is stirred at room temperature to promote the volatilization of dichloromethane. During this process, PLGA or PLA gradually solidifies to form porous PLGA or PLA microspheres. The microspheres are separated by centrifugation and then washed with deionized water to remove residual PVA and solvent. The washed microspheres are freeze-dried, and the dried microspheres are immersed in glutaraldehyde solution for crosslinking treatment. Then they are washed and dried again to obtain porous microspheres.

[0026] Freeze-drying can be used to further remove moisture from the microspheres and prevent the porous structure from collapsing during subsequent drying.

[0027] During freeze-drying, the sublimation of water leaves behind more pores and helps maintain the porous network structure inside the microspheres. Compared to conventional drying methods, freeze-drying better preserves the porosity and pore size distribution of the microspheres.

[0028] Glutaraldehyde can crosslink with chitosan molecules on the surface of porous microspheres, improving the mechanical strength of the microspheres and making them more stable and resistant to swelling in humid environments, thereby helping to maintain the structural integrity of the microspheres.

[0029] Preferably, in step (5), the stirring conditions at room temperature are: 400~500 rpm for 12~14 h; the centrifugation conditions are: 3000~5000 rpm for 10~20 min; and the freeze drying is: drying at -20~-30℃ for 24~48 h.

[0030] Preferably, in step (5), the mass concentration of the glutaraldehyde solution is 0.5-1%; and the crosslinking treatment time is 40-60 min.

[0031] Secondly, the present invention also provides an application of the porous microspheres prepared by the above preparation method in stem cell seeding, wherein the porous microspheres serve as stem cell seeding carriers.

[0032] The porous microspheres prepared by this invention have a more uniform pore size distribution and higher biological activity, enhancing the adsorption capacity of proteins or peptides on the surface of the microspheres, thus enabling them to serve as excellent stem cell seeding carriers to meet the needs of high-proportion cell attachment and proliferation.

[0033] Preferably, the porous microspheres are surface-functionalized before being used as stem cell seeding carriers. The surface functionalization steps include: immersing the dried porous microspheres in an aqueous solution containing hydroxyl or amino surfactants for activation treatment, then transferring the activated microspheres to an aqueous solution containing extracellular matrix proteins or peptides for incubation, followed by centrifugation.

[0034] Preferably, the concentration of the hydroxyl or amino activator in the aqueous solution is 1-5 mg / mL; the activation conditions are: a temperature of 4°C or room temperature, and a time of 1-2 h; the concentration of the extracellular matrix protein in the aqueous solution is 0.1-1 mg / mL, and the concentration of the polypeptide is 0.01-0.1 mg / mL; the incubation conditions are: a temperature of 4°C or room temperature, and a time of 12-24 h.

[0035] Preferably, the surface-functionalized microspheres are dispersed in a suspension of mesenchymal stem cells (MSCs) or osteoblasts at a cell concentration of 1×10⁻⁶. 5 ~1×10 6 Cells / mL, gently stirred or incubated on a shaker for 2-4 hours at 37°C and 5% CO2.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] (1) Porous microspheres were prepared by double emulsion solvent evaporation method. While improving the dispersion uniformity of bioactive glass and chitosan in polylactic acid matrix, the porosity and pore size of the microspheres could be adjusted by adjusting the water-oil ratio, stirring speed and solvent evaporation time.

[0038] (2) Porous microspheres have a more uniform pore size distribution and higher biological activity, which enhances the adsorption capacity of proteins or peptides on the surface of microspheres, thus enabling them to serve as excellent stem cell seeding carriers to meet the needs of high proportion of cell attachment and proliferation. Detailed Implementation

[0039] The technical solution of the present invention will be illustrated below with specific embodiments, but the scope of protection of the present invention is not limited thereto. Example 1

[0040] 1. Preparation of porous polylactic acid microspheres containing bioactive glass:

[0041] (1) Dissolve 2 g of chitosan (degree of deacetylation 80%, viscosity 80 mPa·s) in 100 mL of 1% acetic acid aqueous solution and stir until completely dissolved. Add 1 g of bioactive glass nanoparticles (average particle size 500 nm) and treat with an ultrasonic processor at 40 kHz for 20 min to uniformly disperse them in the chitosan solution to obtain an aqueous solution.

[0042] (2) Dissolve 5 g PLGA (average molecular weight 30 kDa) in 500 mL dichloromethane, stir until completely dissolved, add emulsifier (Tween 80) and stir evenly to obtain an oil phase solution containing 2 g / mL emulsifier.

[0043] (3) The aqueous phase solution is slowly added dropwise to the oil phase solution while stirring at 1000 rpm for 30 min and ultrasonically treated at 40 kHz to form a uniform water-in-oil (W / O) primary emulsion.

[0044] (4) Dissolve 50 g of polyvinyl alcohol in 5 L of deionized water to obtain a polyvinyl alcohol aqueous solution. Slowly add the water-in-oil (W / O) primary emulsion to the polyvinyl alcohol aqueous solution while stirring at 800 rpm for 60 min to form a water-in-oil (W / O / W) double emulsion.

[0045] (5) The double emulsion was stirred at 500 rpm for 12 h at room temperature to form porous PLGA microspheres. The microspheres were separated by centrifugation at 3000 rpm for 15 min, and then washed three times with 5 L of deionized water each time. The washed microspheres were freeze-dried at -30℃ for 24 h.

[0046] (6) The dried microspheres were immersed in a 0.5% glutaraldehyde solution for 1 hour for cross-linking treatment, and then washed and dried again to obtain porous microspheres.

[0047] 2. Surface functionalization of porous microspheres (adsorption of proteins / peptides):

[0048] (1) The dried porous microspheres were immersed in a solution containing 3 mg / mL of hydroxyl surfactant (3-aminopropyltriethoxysilane APTES, Sigma-Aldrich 440140) to activate the surface activity. The immersion time was 2 h and the temperature was 4°C.

[0049] (2) The activated microspheres were transferred to a solution containing 0.5 mg / mL extracellular matrix protein (Type I Collagen, Bovine Type I Collagen C9785 from Sigma-Aldrich), and gently stirred to ensure uniform adsorption. The incubation time was 18 h and the temperature was 4°C.

[0050] (3) After incubation, the microspheres are recovered by centrifugation or filtration and washed multiple times with PBS (phosphate buffer) to remove unadsorbed proteins.

[0051] 3. Stem cell inoculation:

[0052] (1) The surface-functionalized microspheres were dispersed in a mesenchymal stem cell suspension at a cell concentration of 4.8×10^5 cells / mL.

[0053] (2) Incubate the microsphere and cell mixture in a shaker for 3 hours at 37°C and 5% CO2. Example 2

[0054] 1. Preparation of porous polylactic acid microspheres containing bioactive glass:

[0055] (1) Dissolve 3 g of chitosan (degree of deacetylation 80%, viscosity 80 mPa·s) in 100 mL of 1% acetic acid aqueous solution and stir until completely dissolved. Add 1.6 g of bioactive glass nanoparticles (average particle size 500 nm) and treat with an ultrasonic processor at 40 kHz for 30 min to uniformly disperse them in the chitosan solution to obtain an aqueous solution.

[0056] (2) Dissolve 5 g PLGA (average molecular weight 30 kDa) in 500 mL dichloromethane, stir until completely dissolved, add emulsifier (Tween 80) and stir evenly to obtain an oil phase solution containing 2 g / mL emulsifier.

[0057] (3) The aqueous phase solution is slowly added dropwise to the oil phase solution while stirring at 1200 rpm for 40 min and ultrasonically treated at 40 kHz to form a uniform water-in-oil (W / O) primary emulsion.

[0058] (4) Dissolve 50 g of polyvinyl alcohol in 5 L of deionized water to obtain a polyvinyl alcohol aqueous solution. Slowly add the water-in-oil (W / O) primary emulsion to the polyvinyl alcohol aqueous solution while stirring at 800 rpm for 90 min to form a water-in-oil (W / O / W) double emulsion.

[0059] (5) The double emulsion was stirred at 400 rpm for 14 h at room temperature to form porous PLGA microspheres. The microspheres were separated by centrifugation at 3000 rpm for 15 min, and then washed three times with 5 L of deionized water each time. The washed microspheres were freeze-dried at -30℃ for 24 h.

[0060] (6) The dried microspheres were immersed in a 0.8% glutaraldehyde solution for 50 min for cross-linking treatment, and then washed and dried again to obtain porous microspheres.

[0061] 2. Surface functionalization of porous microspheres (adsorption of proteins / peptides):

[0062] (1) The dried porous microspheres were immersed in a solution containing 3 mg / mL of hydroxyl surfactant (3-aminopropyltriethoxysilane APTES, Sigma-Aldrich 440140) to activate the surface activity. The immersion time was 2 h and the temperature was 4°C.

[0063] (2) The activated microspheres were transferred to a solution containing 0.5 mg / mL extracellular matrix protein (Type I Collagen, Bovine Type I Collagen C9785 from Sigma-Aldrich), and gently stirred to ensure uniform adsorption. The incubation time was 18 h and the temperature was 4°C.

[0064] (3) After incubation, the microspheres are recovered by centrifugation or filtration and washed multiple times with PBS (phosphate buffer) to remove unadsorbed proteins.

[0065] 3. Stem cell inoculation:

[0066] (1) The surface-functionalized microspheres were dispersed in a mesenchymal stem cell suspension at a cell concentration of 4.8×10^5 cells / mL.

[0067] (2) Incubate the microsphere and cell mixture in a shaker for 3 hours at 37°C and 5% CO2. Example 3

[0068] 1. Preparation of porous polylactic acid microspheres containing bioactive glass:

[0069] (1) Dissolve 4 g of chitosan (degree of deacetylation 80%, viscosity 80 mPa·s) in 100 mL of 2% acetic acid aqueous solution and stir until completely dissolved. Add 2 g of bioactive glass nanoparticles (average particle size 500 nm) and treat with an ultrasonic processor at 40 kHz for 30 min to uniformly disperse them in the chitosan solution to obtain an aqueous solution.

[0070] (2) Dissolve 6 g PLGA (average molecular weight 30 kDa) in 500 mL dichloromethane, stir until completely dissolved, add emulsifier (Tween 80) and stir evenly to obtain an oil phase solution containing 3 g / mL emulsifier.

[0071] (3) The aqueous phase solution is slowly added dropwise to the oil phase solution while stirring at 1000 rpm for 30 min and ultrasonically treated at 40 kHz to form a uniform water-in-oil (W / O) primary emulsion.

[0072] (4) Dissolve 60 g of polyvinyl alcohol in 5 L of deionized water to obtain a polyvinyl alcohol aqueous solution. Slowly add the water-in-oil (W / O) primary emulsion to the polyvinyl alcohol aqueous solution while stirring at 800 rpm for 60 min to form a water-in-oil (W / O / W) double emulsion.

[0073] (5) The double emulsion was stirred at 400 rpm for 14 h at room temperature to form porous PLGA microspheres. The microspheres were separated by centrifugation at 3000 rpm for 15 min, and then washed three times with 5 L of deionized water each time. The washed microspheres were freeze-dried at -30℃ for 24 h.

[0074] (6) The dried microspheres were immersed in a 0.5% glutaraldehyde solution for 1 hour for cross-linking treatment, and then washed and dried again to obtain porous microspheres.

[0075] 2. Surface functionalization of porous microspheres (adsorption of proteins / peptides):

[0076] (1) The dried porous microspheres were immersed in a solution containing 3 mg / mL of hydroxyl surfactant (3-aminopropyltriethoxysilane APTES, Sigma-Aldrich 440140) to activate the surface activity. The immersion time was 2 h and the temperature was 4°C.

[0077] (2) The activated microspheres were transferred to a solution containing 0.5 mg / mL extracellular matrix protein (Type I Collagen, Bovine Type I Collagen C9785 from Sigma-Aldrich), and gently stirred to ensure uniform adsorption. The incubation time was 18 h and the temperature was 4°C.

[0078] (3) After incubation, the microspheres are recovered by centrifugation or filtration and washed multiple times with PBS (phosphate buffer) to remove unadsorbed proteins.

[0079] 3. Stem cell inoculation:

[0080] (1) The surface-functionalized microspheres were dispersed in a mesenchymal stem cell suspension at a cell concentration of 4.8×10^5 cells / mL.

[0081] (2) Incubate the microsphere and cell mixture in a shaker for 3 hours at 37°C and 5% CO2.

[0082] Comparative Example 1

[0083] The difference from Example 1 is that chitosan was not added during the preparation of polylactic acid porous microspheres.

[0084] (1) 1 g of bioactive glass nanoparticles (average particle size 500 nm) were added to 100 mL of aqueous solution and treated with an ultrasonic processor at 40 kHz for 20 min to obtain an aqueous solution.

[0085] (2) Dissolve 5 g PLGA (average molecular weight 30 kDa) in 500 mL dichloromethane, stir until completely dissolved, add emulsifier (Tween 80) and stir evenly to obtain an oil phase solution containing 2 g / mL emulsifier.

[0086] (3) The aqueous phase solution is slowly added dropwise to the oil phase solution while stirring at 1000 rpm for 30 min and ultrasonically treated at 40 kHz to form a uniform water-in-oil (W / O) primary emulsion.

[0087] (4) Dissolve 50 g of polyvinyl alcohol in 5 L of deionized water to obtain a polyvinyl alcohol aqueous solution. Slowly add the water-in-oil (W / O) primary emulsion to the polyvinyl alcohol aqueous solution while stirring at 800 rpm for 60 min to form a water-in-oil (W / O / W) double emulsion.

[0088] (5) The double emulsion was stirred at 500 rpm for 12 h at room temperature to form porous PLGA microspheres. The microspheres were separated by centrifugation at 3000 rpm for 15 min, and then washed three times with 5 L of deionized water each time. The washed microspheres were freeze-dried at -30℃ for 24 h.

[0089] (6) The dried microspheres were immersed in a 0.5% glutaraldehyde solution for 1 hour for cross-linking treatment, and then washed and dried again to obtain porous microspheres.

[0090] Comparative Example 2

[0091] The difference from Example 1 is that the concentration of chitosan in the acetic acid aqueous solution was too high during the preparation of polylactic acid porous microspheres.

[0092] (1) Dissolve 5 g of chitosan (degree of deacetylation 80%, viscosity 80 mPa·s) in 100 mL of 1% acetic acid aqueous solution and stir until completely dissolved. Add 1 g of bioactive glass nanoparticles (average particle size 500 nm) and treat with an ultrasonic processor at 40 kHz for 20 min to uniformly disperse them in the chitosan solution to obtain an aqueous solution.

[0093] (2) Dissolve 5 g PLGA (average molecular weight 30 kDa) in 500 mL dichloromethane, stir until completely dissolved, add emulsifier (Tween 80) and stir evenly to obtain an oil phase solution containing 2 g / mL emulsifier.

[0094] (3) The aqueous phase solution is slowly added dropwise to the oil phase solution while stirring at 1000 rpm for 30 min and ultrasonically treated at 40 kHz to form a uniform water-in-oil (W / O) primary emulsion.

[0095] (4) Dissolve 50 g of polyvinyl alcohol in 5 L of deionized water to obtain a polyvinyl alcohol aqueous solution. Slowly add the water-in-oil (W / O) primary emulsion to the polyvinyl alcohol aqueous solution while stirring at 800 rpm for 60 min to form a water-in-oil (W / O / W) double emulsion.

[0096] (5) The double emulsion was stirred at 500 rpm for 12 h at room temperature to form porous PLGA microspheres. The microspheres were separated by centrifugation at 3000 rpm for 15 min, and then washed three times with 5 L of deionized water each time. The washed microspheres were freeze-dried at -30℃ for 24 h.

[0097] (6) The dried microspheres were immersed in a 0.5% glutaraldehyde solution for 1 hour for cross-linking treatment, and then washed and dried again to obtain porous microspheres.

[0098] Comparative Example 3

[0099] The difference from Example 1 is that the amount of water phase added is too much compared to the oil phase during the preparation of polylactic acid porous microspheres.

[0100] (1) Dissolve 3 g of chitosan (degree of deacetylation 80%, viscosity 80 mPa·s) in 150 mL of 1% acetic acid aqueous solution and stir until completely dissolved. Add 1.5 g of bioactive glass nanoparticles (average particle size 500 nm) and treat with an ultrasonic processor at 40 kHz for 30 min to uniformly disperse them in the chitosan solution to obtain an aqueous solution.

[0101] (2) Dissolve 5 g PLGA (average molecular weight 30 kDa) in 500 mL dichloromethane, stir until completely dissolved, add emulsifier (Tween 80) and stir evenly to obtain an oil phase solution containing 2 g / mL emulsifier.

[0102] (3) The aqueous phase solution is slowly added dropwise to the oil phase solution while stirring at 1000 rpm for 30 min and ultrasonically treated at 40 kHz to form a uniform water-in-oil (W / O) primary emulsion.

[0103] (4) Dissolve 50 g of polyvinyl alcohol in 5 L of deionized water to obtain a polyvinyl alcohol aqueous solution. Slowly add the water-in-oil (W / O) primary emulsion to the polyvinyl alcohol aqueous solution while stirring at 800 rpm for 60 min to form a water-in-oil (W / O / W) double emulsion.

[0104] (5) The double emulsion was stirred at 500 rpm for 12 h at room temperature to form porous PLGA microspheres. The microspheres were separated by centrifugation at 3000 rpm for 15 min, and then washed three times with 5 L of deionized water each time. The washed microspheres were freeze-dried at -30℃ for 24 h.

[0105] (6) The dried microspheres were immersed in a 0.5% glutaraldehyde solution for 1 hour for cross-linking treatment, and then washed and dried again to obtain porous microspheres.

[0106] Comparative Example 4

[0107] The difference from Example 1 is that the stirring speed for solvent evaporation was too fast and the evaporation time was too short during the preparation of polylactic acid porous microspheres.

[0108] (1) Dissolve 2 g of chitosan (degree of deacetylation 80%, viscosity 80 mPa·s) in 100 mL of 1% acetic acid aqueous solution and stir until completely dissolved. Add 1 g of bioactive glass nanoparticles (average particle size 500 nm) and treat with an ultrasonic processor at 40 kHz for 20 min to uniformly disperse them in the chitosan solution to obtain an aqueous solution.

[0109] (2) Dissolve 5 g PLGA (average molecular weight 30 kDa) in 500 mL dichloromethane, stir until completely dissolved, add emulsifier (Tween 80) and stir evenly to obtain an oil phase solution containing 2 g / mL emulsifier.

[0110] (3) The aqueous phase solution is slowly added dropwise to the oil phase solution while stirring at 1000 rpm for 30 min and ultrasonically treated at 40 kHz to form a uniform water-in-oil (W / O) primary emulsion.

[0111] (4) Dissolve 50 g of polyvinyl alcohol in 5 L of deionized water to obtain a polyvinyl alcohol aqueous solution. Slowly add the water-in-oil (W / O) primary emulsion to the polyvinyl alcohol aqueous solution while stirring at 800 rpm for 60 min to form a water-in-oil (W / O / W) double emulsion.

[0112] (5) The double emulsion was stirred at 1000 rpm for 6 h at room temperature to form porous PLGA microspheres. The microspheres were separated by centrifugation at 3000 rpm for 15 min, and then washed 3 times with 5 L of deionized water each time. The washed microspheres were freeze-dried at -30℃ for 24 h.

[0113] (6) The dried microspheres were immersed in a 0.5% glutaraldehyde solution for 1 hour for cross-linking treatment, and then washed and dried again to obtain porous microspheres.

[0114] The particle size, pore size, and porosity of the porous microspheres prepared in Examples 1-3 and Comparative Examples 1-4 were determined. After the surface-functionalized porous microspheres were seeded with stem cells, the proliferation rate of stem cells seeded on the microspheres was detected and calculated using the CCK-8 (Cell Counting Kit-8). The stem cell proliferation rate was calculated as: (Number of stem cells 72 hours after seeding / Number of stem cells at initial seeding) * 100%. The results are shown in Table 1.

[0115] Table 1

[0116] Average particle size / μm Average pore size / μm Porosity / % Stem cell proliferation rate / % Example 1 163 33 75 162 Example 2 188 2188 184 Example 3 139 278 4176 Comparative Example 1 154 256 6138 Comparative Example 2 167 409 2156 Comparative Example 3 169 379 3151 Comparative Example 4 165 316 3144

[0117] As shown in Table 1, porous microspheres prepared by the double emulsion solvent evaporation method have higher biological activity and enhanced adsorption capacity of proteins or peptides on the surface of the microspheres, thus serving as excellent stem cell seeding carriers. Furthermore, the microspheres have porosity and pore size more suitable for stem cell seeding, which can meet the requirements for high-proportion cell attachment and proliferation.

[0118] Comparative Examples 1 and 2 show that the addition of chitosan provides structural support, forming a stable porous network as the main polymer component, and also promotes cell adhesion and proliferation. In Comparative Example 1, without chitosan, although a porous structure could be formed, the porosity, pore size uniformity, and structural stability decreased, as did the bioactivity of the microspheres, resulting in a significant decrease in stem cell proliferation rate. In Comparative Example 2, excessively high chitosan concentrations were also detrimental to the formation of a suitable porous structure, and excessively large and distributed pores could lead to pore collapse, hindering high-proportion stem cell seeding and reducing stem cell proliferation rate.

[0119] Similarly, in Comparative Example 3, an excessively high proportion of the internal aqueous phase leads to the formation of more pores, resulting in an unstable microsphere structure, which is detrimental to high-proportion stem cell seeding. In Comparative Example 4, the excessively fast stirring speed and short evaporation time during solvent evaporation prevent the aqueous phase from migrating and escaping from the microspheres, hindering the formation of a uniform pore distribution during aqueous phase extraction and resulting in low porosity, which is also unfavorable for high-proportion stem cell seeding.

[0120] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made using the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for preparing porous polylactic acid microspheres, characterized by, Includes the following steps: (1) Chitosan was dissolved in an aqueous acetic acid solution, and bioactive glass nanoparticles were added and then sonicated to obtain an aqueous solution; (2) Dissolve PLGA or PLA in dichloromethane and add an emulsifier to obtain an oil phase solution; (3) Add the aqueous phase solution dropwise to the oil phase solution to obtain a water-in-oil primary emulsion; (4) Add the water-in-oil primary emulsion dropwise into the PVA aqueous solution to obtain a double emulsion; (5) Stir the double emulsion, then centrifuge and dry it, and then immerse it in glutaraldehyde solution for cross-linking treatment to obtain porous microspheres.

2. The method for preparing polylactic acid porous microspheres according to claim 1, characterized in that, In step (1), the mass concentration of the acetic acid aqueous solution is 1~2%; the ratio of the amount of chitosan, bioactive glass nanoparticles and acetic acid aqueous solution added is 1~4g:1~2g:100mL.

3. The method for preparing polylactic acid porous microspheres according to claim 1, characterized in that, In step (1), the degree of deacetylation of the chitosan is 70-80%, and the viscosity is 20-100 mPa·s; the average particle size of the bioactive glass nanoparticles is 200-1000 nm.

4. The method for preparing polylactic acid porous microspheres according to claim 1, 2, or 3, characterized in that, In step (2), the ratio of PLGA or PLA to dichloromethane is 4~6g:500mL; the average molecular weight of PLGA or PLA is 10~50 kDa.

5. The method for preparing polylactic acid porous microspheres according to claim 1, characterized in that, In step (3), the volume ratio of the aqueous solution to the oil solution is 1~1.3:

5.

6. The method for preparing polylactic acid porous microspheres according to claim 1 or 5, characterized in that, In step (4), the concentration of the PVA aqueous solution is 10~12g / L; the volume ratio of the water-in-oil primary emulsion to the PVA aqueous solution is 6~8:

50.

7. The method for preparing polylactic acid porous microspheres according to claim 1, characterized in that, In step (5), the stirring conditions are: 400~500 rpm, 12~14h.

8. The method for preparing polylactic acid porous microspheres according to claim 1 or 7, characterized in that, In step (5), the mass concentration of the glutaraldehyde solution is 0.5-1%; the crosslinking treatment time is 40-60 min.

9. Use of porous microspheres prepared according to the process of any one of claims 1 to 8 for the seeding of stem cells, characterized in that, The porous microspheres serve as a stem cell inoculation carrier.

10. Use according to claim 9, characterized in that, Before using porous microspheres as stem cell seeding carriers, surface functionalization is performed. The surface functionalization steps include: immersing dried porous microspheres in an aqueous solution containing hydroxyl or amino surfactants for activation treatment, then transferring the activated microspheres to an aqueous solution containing extracellular matrix proteins or peptides for incubation, followed by centrifugation.