Injectable porous microsphere mesenchymal stem cell compound as well as preparation method and application thereof

Abstract

The invention provides an injectable porous microsphere mesenchymal stem cell compound as well as a preparation method and application thereof, and relates to the field of biomedical materials and regenerative medicine. The injectable porous microsphere mesenchymal stem cell compound comprises a carrier and mesenchymal stem cells loaded on the carrier, the carrier is dMBG / PLGA porous microspheres, the dMBG / PLGA porous microspheres are prepared from dMBG and PLGA, the dMBG is dendritic mesoporous bioactive glass, and the PLGA is a polylactic acid-glycolic acid copolymer; the mesenchymal stem cells enter the three-dimensional through hole channels of the carrier and are planted in the three-dimensional through hole channels. According to the injectable porous microsphere mesenchymal stem cell compound, efficient planting and local retention of MSC can be achieved, high activity of cells after injection and cryopreservation resuscitation is guaranteed, and endogenous osteogenesis can be promoted without exogenous factors; meanwhile, the system has the functions of promoting angiogenesis, regulating immunity and repairing nerves, can fill irregular defects in a minimally invasive mode, can be applied in a large scale, is adaptive to various repairing scenes, and meets the clinical application requirements.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials and regenerative medicine, and in particular to an injectable porous microsphere mesenchymal stem cell complex, its preparation method, and its application. Background Technology

[0002] In the regenerative treatment of irregular defects such as bone and alveolar bone, restorative solutions must possess three key characteristics simultaneously: stable cell source, minimally invasive delivery capability, and the formation of a durable and adjustable local microenvironment after implantation, in order to meet the treatment needs of complex defects. However, current mainstream treatment approaches all have insurmountable limitations and cannot adequately meet this requirement.

[0003] On the one hand, while traditional bulk or sheet-like scaffolds can provide structural support, they cannot adapt to the morphological characteristics of irregular defects: they are difficult to fill complex cavities uniformly and conformally using minimally invasive methods, and cannot achieve precise repair by conforming to the defect contour; furthermore, their dense structure hinders cell permeability within the scaffold, impeding nutrient delivery and limiting the removal of metabolic waste, directly affecting cell survival and function, and thus reducing overall repair efficiency. On the other hand, although mesenchymal stem cells (MSCs) are considered ideal repair cells due to their potential for osteogenic differentiation, angiogenesis, and immunomodulation, naked cell form faces multiple challenges in clinical applications: during injection, liquid shear force directly damages the cell structure, leading to a significant decrease in activity; after entering the body, MSCs lack a carrier support, resulting in low retention rates and poor colonization effects in local tissues, and are prone to phenotypic drift due to the lack of spatial support, losing their original repair function; simultaneously, naked cells require a large amount of exogenous inducing factors to maintain repair activity, and the reproducibility of the preparation process is poor, making it difficult to guarantee stable clinical results for each application.

[0004] Based on the above pain points, the industry has turned to the research and development approach of "cell-material composites"—pre-combining MSCs with injectable microscale carriers to balance support and cell function. However, existing composites still have significant drawbacks: most adopt a surface adhesion design of "MSCs + inert polymer microspheres," where MSCs can only attach to the outer surface of the microspheres. This not only limits the cell load per unit particle but also makes them prone to detachment during injection or in vivo migration. The signal transduction at the composite interface is singular, lacking the ability to autonomously drive osteogenic and angiogenesis, and still requires exogenous factors to initiate the repair process. Furthermore, the ability to regulate the macrophage-mediated immune microenvironment after injection is insufficient, failing to alleviate the inflammatory response during the repair process and making it difficult to synergistically promote the regeneration and repair of bone defects.

[0005] In summary, neither traditional scaffolds, naked mesenchymal stem cells (MSCs), nor existing cell-material complexes can simultaneously meet the core requirements of "minimally invasive delivery, cell survival and colonization, and adaptation to the immune microenvironment." Clinically, there is an urgent need for a new treatment approach that can overcome these limitations. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and provide an injectable porous microsphere mesenchymal stem cell complex, its preparation method and application. It aims to overcome the core limitations of the prior art that cannot simultaneously meet the requirements of "minimally invasive delivery, cell survival and colonization, and adaptation to the immune microenvironment", and to improve the cell injection survival rate and site retention rate without sacrificing minimal invasiveness, reduce dependence on exogenous factors, and promote osteogenic, angiogenesis and immune microenvironment remodeling simultaneously through the synergistic effect of cells and materials.

[0007] This invention provides an injectable porous microsphere mesenchymal stem cell complex, comprising a carrier and mesenchymal stem cells loaded on the carrier; the carrier is a dMBG / PLGA porous microsphere, which is prepared from dMBG and PLGA, wherein dMBG is dendritic mesoporous bioactive glass and PLGA is polylactic acid-glycolic acid copolymer; the mesenchymal stem cells enter and colonize in the three-dimensional through-pores of the carrier, forming a cell-material composite microunit.

[0008] Furthermore, in the dMBG / PLGA porous microspheres, dMBG accounts for 5-20 wt% of PLGA by mass; the average particle size of the porous microspheres is 150-300 μm, the porosity is 80%-90%, and the pore size is 12-25 μm; the ratio of lactic acid to glycolic acid monomers in the PLGA is 1:1.

[0009] Furthermore, the mesenchymal stem cells are cells passaged to the 10th to 15th generation, with a cell confluence of approximately 80% and a trypan blue staining viability of ≥95%.

[0010] Furthermore, the loading ratio of the mesenchymal stem cells to dMBG / PLGA porous microspheres is 1×10⁻⁶. 6 Each cell corresponds to 5 mg of dMBG / PLGA porous microspheres.

[0011] The present invention also provides a method for preparing the above-mentioned injectable porous microsphere mesenchymal stem cell complex, comprising the following steps:

[0012] (1) Preparation of dMBG / PLGA porous microspheres: dMBG particles and PLGA were dissolved in an organic solvent and porous microspheres were prepared by emulsification-solvent evaporation method. After freeze drying, they were sterilized for later use.

[0013] (2) Cell loading: The mesenchymal stem cell suspension was mixed with the dMBG / PLGA porous microspheres from step (1) in the proportion specified in claim 4, and incubated in a 37°C, 5% CO2 incubator for 24 h to allow the mesenchymal stem cells to be fully adsorbed into the pores of the dMBG / PLGA porous microspheres.

[0014] (3) Composite culture: The mesenchymal stem cell-dMBG / PLGA porous microsphere system from step (2) was transferred into a T25 culture flask and the complete culture medium was added to 5 mL. During the culture period, the complex was gently blown every 12 h to avoid aggregation and to promote the mesenchymal stem cells to enter the internal pores of the dMBG / PLGA porous microspheres. Fresh culture medium was replaced every 2 days. After the culture was completed, the injectable porous microsphere mesenchymal stem cell complex was obtained.

[0015] Furthermore, the specific steps of step (1) above are as follows:

[0016] S1 organic phase preparation: 0.2 g PLGA and 20 mg dMBG were dissolved in 10 mL of dichloromethane or a mixture of dichloromethane and ethyl acetate to form an organic dispersion system of dMBG / PLGA.

[0017] Preparation of S2 colostrum: Add 2.5 mL of 8% Gel aqueous solution to the organic dispersion system obtained in S1, and homogenize at a high speed of 10000 rpm for 60 s to form WGel / OdMBG / PLGA colostrum;

[0018] Preparation of S3 double emulsion: The colostrum obtained from S2 was slowly added to 100 mL of 0.1% PVA external aqueous phase and stirred at 350 rpm to form a WGel / OdMBG / PLGA / WPVA double emulsion system;

[0019] S4 Evaporation and Pore Formation and Curing: The multi-emulsion system was placed in a 50 ℃ water bath and stirred at low speed for 8 min to promote the evaporation of organic solvents and achieve pore formation and curing of dMBG / PLGA microspheres.

[0020] S5 post-processing: The S4-cured dMBG / PLGA microsphere system was centrifuged at 400 g for 5 min, and the microsphere precipitate was collected; it was washed three times with deionized water to remove residual reagents, and finally freeze-dried to obtain dMBG / PLGA porous microspheres.

[0021] Furthermore, the sterilization described in step (1) above is performed by cobalt-60 irradiation sterilization.

[0022] The above-mentioned injectable porous microsphere mesenchymal stem cell complex is used in bone defect repair.

[0023] The present invention also provides an injectable formulation containing the above-described injectable porous microsphere mesenchymal stem cell complex, or the injectable porous microsphere mesenchymal stem cell complex obtained by the above-described preparation method.

[0024] The present invention also provides a kit comprising the above-described injectable formulation, or the above-described injectable porous microsphere mesenchymal stem cell complex, or the injectable porous microsphere mesenchymal stem cell complex obtained by the above-described preparation method.

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] 1. Three-dimensional microenvironment construction and efficient colonization: The through-channels of dMBG / PLGA porous microspheres provide a stable three-dimensional growth space for mesenchymal stem cells (MSCs), enabling MSCs to form a "micro-tissue" structure within the pores. This not only facilitates nutrient exchange and metabolic waste removal but also enhances the local retention and closure of MSCs at bone defect sites, solving the technical pain point of insufficient naked cell colonization.

[0027] 2. Injection shear protection and high cell load: The porous structure of dMBG / PLGA porous microspheres forms a mechanical buffer during the injection-reflux process, significantly reducing the damage of shear force to MSC activity and ensuring cell survival during delivery. Compared with solid microspheres or surface-adhesive complexes, the three-dimensional channels of dMBG / PLGA porous microspheres provide a larger cell load space, allowing each particle to carry more MSCs, and the cells are evenly distributed inside and outside the channels, maintaining high metabolic activity and proliferation capacity.

[0028] 3. Cryopreservation-resuscitation friendliness and large-scale application: The complex can be directly cryopreserved at −80℃ or in liquid nitrogen using commercial cryopreservation reagents without DMSO, without the need for complicated cooling procedures; it still maintains good rheological properties after revival, can be smoothly injected through an 18G needle, and cell viability is not affected. This enables centralized preparation, long-term storage and cross-institutional distribution of the complex, reducing the threshold for clinical application.

[0029] 4. Endogenous osteogenic and functional synergy: dMBG components continuously release Ca / Si / P ions, which promote osteogenic differentiation and mineralization nodule formation of MSCs by upregulating the expression of osteogenic marker genes or proteins such as Runx2, OCN, and COL1A1, without relying on exogenous osteogenic inducing factors, thus achieving endogenous osteogenic formation; at the same time, the PLGA matrix provides controllable degradation support, and the two work together to construct a microenvironment conducive to bone regeneration.

[0030] 5. Multidimensional microenvironment regulation and repair synergy: The porous microsphere-mesenchymal stem cell complex has multidimensional functions of promoting angiogenesis, immune regulation and nerve repair: On the one hand, it can enhance the migration and tube formation of endothelial cells through paracrine effects, and promote angiogenesis in the defect area; on the other hand, it can inhibit excessive M1 polarization of macrophages, promote their transition to a reparative phenotype, reduce the inflammatory peak, and reshape a mild immune microenvironment; in addition, it can promote the growth of DRG nerve axons, which is conducive to the regeneration of nerve fibers and the reconstruction of the nerve-bone interface in the defect area, and realize the repair of bone defects.

[0031] 6. Minimally invasive plasticity and clinical adaptability: The composite has good injectability and shaping ability, and can be delivered through a needle via minimally invasive means. It can fill irregular defects such as bone and alveolar bone in a conformal manner, solving the problem that traditional block scaffolds are difficult to adapt to complex defects. At the same time, the preparation process can be scaled up, with good batch-to-batch consistency. Furthermore, the stability of the indwelling can be improved by methods such as in-situ curing with GelMA after injection, which meets the needs of clinical minimally invasive treatment and the trend of industrial application.

[0032] 7. Scalability and application flexibility: Pore-forming agents, stabilizers, sterilization methods, injection media and MSC sources can all be equivalently replaced without changing the core technology framework, thus adapting to different bone defect repair scenarios and having a wide range of application flexibility and technical expansion space. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of the complex construction and application process;

[0034] Figure 2 The process for preparing dMBG / PLGA porous microspheres;

[0035] Figure 3 For the identification of porous microspheres; among which Figure 3 Images A and B in the image are electron microscope images: Figure 3 C represents the particle size. Figure 3 D represents the aperture. Figure 3 In this context, E represents porosity. Figure 3 F in the middle represents FTIR; Figure 3 G in the middle is EDS mapping;

[0036] Figure 4 This is a schematic diagram of the morphology and three-dimensional cell distribution of the complex;

[0037] Figure 5 This illustrates the cellular loading capacity of the complex. Figure 5 A in the middle is CCK-8; Figure 5 B in the diagram is a structural schematic. Figure 5 C in the middle section is DAPI stained after freezing; Figure 5 In the middle, D represents the staining count of the section;

[0038] Figure 6 For evaluation under injection shear conditions; Figure 6 In the diagram, A represents a flowchart; Figure 6 In the middle B, cell viability is measured by CCK-8, and the arrow indicates cell viability loss;

[0039] Figure 7 This is a diagram illustrating cryopreservation and thawing (using commercial cryopreservation reagents for direct cryopreservation at −80 °C and the thawing process). Figure 7 A in the diagram represents cryopreservation; Figure 7 In section B, cell viability before and after cryopreservation is used as an analogy with cell suspension.

[0040] Figure 8 The diagram shows the osteogenic related testing process and representative results. Figure 8 In the middle section, A represents Col1a1 / OSX immunofluorescence staining; Figure 7 B in the middle represents ARS staining;

[0041] Figure 9 Image showing the cell identity assessment results of the complex system (single-cell sequencing). Figure 9 In the middle, A represents the UMAP dimensionality reduction cluster; Figure 8 B represents quasi-time series analysis; Figure 8 In the middle C, the cell identity distribution of MSCs cultured on PLGA for seven days is shown. Figure 8 In the middle D, the cell identity distribution of MSCs cultured on dMBG / PLGA for seven days is shown.

[0042] Figure 10 A diagram showing the pro-angiogenic assessment process and representative results; Figure 10 A in the middle represents a scratch; Figure 10 B represents proliferation, stained with Ki67 immunofluorescence. Figure 10 C represents the tube forming experiment;

[0043] Figure 11 A flowchart illustrating the immune regulation assessment process and representative results; Figure 11 In the middle section, A represents CD86 (M1 polarization marker) / EMR and CD206 (M2 polarization marker) / EMR (human macrophage marker) immunofluorescence staining. Figure 11 B is a CD86 stream cytometer; Figure 11 C in the middle is CD206 stream cytometer;

[0044] Figure 12 The diagram shows the neurological assessment process and representative results (axonal growth analysis of DRG).

[0045] Figure 13 A schematic diagram of the experimental procedure for an in vivo bone defect model (μCT and histological / immunomarker illustration). Figure 13 In the middle, A stands for micro-ct. Figure 13B in the middle is for micro-ct quantification, BV / TV; Figure 13 The middle C represents the quantitative measurement using micro-CT, and the trabecular spacing between bone volume (BV) and trabecular bone (TV). Figure 13 D represents micro-CT quantitative analysis of trabecular bone thickness; Figure 13 E in the middle is staining with masson. Detailed Implementation

[0046] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0047] Example 1: Preparation of dMBG / PLGA injectable porous microsphere carriers

[0048] This embodiment aims to prepare dMBG / PLGA injectable porous microsphere carriers through a specific process, and to clarify its formulation and operating parameters.

[0049] 1. Raw materials and proportioning parameters

[0050] PLGA: The ratio of lactic acid to glycolic acid is 1:1, preferably with a relative molecular mass ≈ 1.1 × 10⁻⁶. 5 ;

[0051] dMBG powder: a functional component of microspheres;

[0052] Auxiliary reagents: gelatin (porogen), PVA (stabilizer), organic phase solvent (dichloromethane or dichloromethane / ethyl acetate mixture, volume ratio 3:1~1:1).

[0053] (2) Optimal proportions

[0054] dMBG dosage: 5–20 wt% of PLGA mass;

[0055] Pore-forming agent: Gel aqueous solution concentration of 5–10 wt%;

[0056] External aqueous phase: PVA aqueous solution concentration of 0.05–1.0 wt%.

[0057] 2. Experimental Grouping

[0058] In this embodiment, three types of comparative microspheres were prepared based on the same W1 / O / W2 emulsification-poration-curing process:

[0059] ①PLGA porous microspheres: without adding dMBG, only using the dual emulsification pore-forming process of PLGA+Gel+PVA;

[0060] ②dMBG / PLGA porous microspheres: dMBG particles (10 wt% of PLGA) were added to the PLGA organic phase, and other pore formation and emulsification conditions were the same as ①.

[0061] ③dMBG / PLGA solid microspheres: without adding gelatin, a single emulsification process (W / O→W) is used to obtain solid microspheres without through-holes.

[0062] The core process route is: W1 / O / W2 re-emulsification—pore formation—solvent evaporation—curing—washing—drying, with gelatin as a dedicated pore-forming agent.

[0063] 3. Optimized operating procedures

[0064] Organic phase preparation: 0.2 g PLGA and 20 mg dMBG (corresponding to 10 wt% of PLGA mass) were dissolved / dispersed in 10 mL of the above organic phase to form a uniform dMBG / PLGA organic dispersion system.

[0065] Preparation of colostrum: Add 2.5 mL of 8% Gel aqueous solution (preferred concentration) to the above organic dispersion system, and homogenize at a high speed of 10,000 rpm for 60 s to form WGel / OdMBG / PLGA colostrum;

[0066] Preparation of double emulsion: The colostrum was slowly added to 100 mL of 0.1% PVA in the external aqueous phase and stirred at 350 rpm to form a WGel / OdMBG / PLGA / WPVA double emulsion system;

[0067] Volatilization and Curing: The multi-emulsion system was placed in a 50 ℃ water bath and stirred at low speed for 8 min to promote the volatilization of organic solvents and achieve pore formation and microsphere curing.

[0068] Post-processing: The solidified microsphere system was centrifuged at 400 g for 5 min, and the microsphere precipitate was collected; it was washed three times with deionized water to remove residual reagents, and finally freeze-dried to obtain dMBG / PLGA porous microspheres.

[0069] 4. Statistics and Data Processing

[0070] Unless otherwise stated, data are expressed as mean ± standard deviation (mean ± SD), with n ≥ 3 independent replicates; Shapiro–Wilk normality and Levene homogeneity of variance tests were performed first; two-tailed t-tests were used for comparisons between two groups, and one-way ANOVA with Tukey post-hoc tests were used for comparisons of three or more groups; Mann–Whitney U tests were used for non-normal data. The significance threshold α = 0.05. Figures and tables may be generated by GraphPad Prism or equivalent software. All images / data are for illustrative purposes only and do not constitute a limitation on the claims, hereinafter the same.

[0071] 5. Experimental Results

[0072] Multidimensional characterization of dMBG / PLGA microspheres with different ratios revealed that the microspheres exhibited the best overall performance when the amount of dMBG was 10 wt% of the mass of PLGA, the concentration of gel aqueous solution was 8 wt%, and the concentration of PVA in the external aqueous phase was 0.1 wt%.

[0073] Example 2 Characterization of dMBG / PLGA microsphere carriers

[0074] 1. Characterization purpose and sample range

[0075] This embodiment uses multi-dimensional characterization techniques to systematically verify the structural integrity, component compositeness, and key performance parameters of the microsphere carrier, providing a quality basis for subsequent applications.

[0076] Characterization samples: The three types of microspheres prepared in Example 1 include ① PLGA porous microspheres, ② dMBG / PLGA porous microspheres, and ③ dMBG / PLGA solid microspheres.

[0077] 2. Sample pretreatment

[0078] The three types of microspheres were dried to constant weight and then prepared for use, and then processed according to different characterization requirements:

[0079] Surface morphology observation: Dry microspheres were directly attached to the surface of the conductive adhesive;

[0080] Cross-sectional structure observation: The dried microspheres were rapidly fractured in liquid nitrogen, and the fresh cross-section was immediately attached to the conductive adhesive.

[0081] SEM morphology observation: Au / Pd sputtering coating with a thickness of 5–8 nm was used; EDS elemental analysis was performed using a carbon film coating to avoid interference from gold element peaks on the detection results.

[0082] 3. Core Representation Items and Operating Procedures

[0083] (1) SEM morphology characterization (three-dimensional through-hole verification)

[0084] Test conditions: accelerating voltage 5–10 kV, working distance 8–12 mm, vacuum degree better than 10. -3 Pa.

[0085] Testing requirements: Collect surface and cross-sectional images of each type of microsphere at magnifications of 100×, 500×, and 1000×, respectively. Randomly select ≥5 fields of view for each type of sample to ensure data representativeness.

[0086] (2) Statistical analysis of particle size and pore size

[0087] Particle size detection (D10 / D50 / D90): Laser particle size analyzer (dry method) is preferred for measurement. If microsphere agglomeration occurs, the test effect can be optimized by short-term airflow dispersion for 30 s. The auxiliary method is microscopic image analysis. ≥1000 microspheres are randomly selected, calibrated according to the image scale, and then Otsu thresholding, morphological denoising and connected component identification are performed using ImageJ software. The microsphere particle size is expressed as equivalent circle diameter (ECD).

[0088] Pore ​​size detection: Based on the analysis of SEM cross-sectional images, ≥300 pores are randomly selected, and the equivalent circle diameter (ECD) is extracted using the same ImageJ processing method as particle size statistics. The final output is the mean ± standard deviation (SD) of the pore size, the frequency distribution curve, and the coefficient of variation (CV).

[0089] (3) Porosity determination (ethanol water displacement method / liquid displacement method)

[0090] Test principle: Anhydrous ethanol with no expansion and low surface tension is selected as the replacement liquid. The open pore volume and the overall volume of the microspheres are calculated by the volume difference, and then the porosity is obtained.

[0091] Operating steps: Add a quantitative amount of anhydrous ethanol to the graduated cylinder and record the initial volume V1; accurately weigh a dry microsphere with a mass of m, add it to the graduated cylinder, gently shake it, and place it in a vacuum environment for 5–10 min to ensure that the ethanol fully wets and enters the microsphere channels, and record the liquid level volume V2 at this time; carefully remove the ethanol-saturated microsphere with tweezers to avoid bringing out excess liquid, and record the remaining ethanol volume V3 in the graduated cylinder.

[0092] Calculation formula: Porosity (%) = (V1 – V3) / (V2 – V3) × 100

[0093] Formula explanation: (V2–V3) represents the total volume of the microspheres, and (V1–V3) represents the volume of the open pores of the microspheres. At least three parallel experiments were conducted for each batch of samples, and results are expressed as mean ± SD. If further conversion of volume fraction or apparent density is required, the alcohol density and apparent density of the sample at the test temperature can be measured simultaneously.

[0094] (4) FTIR infrared spectroscopy analysis

[0095] Test conditions: ATR-FTIR mode (diamond crystal), resolution 4 cm. -1 The scan was performed 32 times, and the scan range covered the key feature peak intervals.

[0096] Characteristic peak identification: PLGA characteristic peak: C=O stretching vibration peak located at 1750–1760 cm⁻¹ -1 The –OH stretching vibration peak is located at 3500 cm⁻¹. -1 Range; dMBG characteristic peak: Si–O–Si stretching vibration peak located at 1080 cm⁻¹ -1 Range; Composite microsphere determination: The infrared spectrum of dMBG / PLGA microspheres must simultaneously show the characteristic peaks of PLGA and dMBG to prove that the components are successfully composited.

[0097] (5) EDS elemental mapping analysis (composite homogeneity verification)

[0098] Test objects: Select the cross-sectional area of ​​microspheres for surface scanning analysis of Si (dMBG characteristic element) and C (PLGA characteristic element). ≥3 different microspheres are tested for each type of sample to ensure the reliability of the uniformity evaluation.

[0099] Judgment criteria: Distribution uniformity: Si and C elements are continuously and uniformly distributed in the cross section of the microspheres, without obvious enrichment or depletion bands; Element content: Statistical analysis of the mean atomic percentage (at.%) of Si and the coefficient of variation (CV) across particles. Preferably, the at.% of Si is in the representative range of 6–12%.

[0100] 4. Qualification Standards

[0101] A batch of microspheres that meets all of the following indicators can be deemed qualified and proceed to the subsequent cell recombining stage:

[0102] Structural features: a three-dimensional through-hole with continuous hole walls and interconnected holes;

[0103] Particle size parameters: D50 is in the range of 150–300 μm;

[0104] Pore ​​size parameters: The average pore size is in the range of 12–25 μm;

[0105] Porosity: 80–90% as determined by the ethanol replacement method;

[0106] Component homogeneity: EDS elemental mapping shows that Si and C elements are uniformly distributed in the cross section.

[0107] 5. Experimental Results

[0108] Among the three types of microspheres, dMBG / PLGA porous microspheres performed best, with the following characteristics: they have a continuous and complete pore wall structure and three-dimensional interconnected channels; Si and C elements are uniformly distributed as confirmed by EDS mapping, and the composition is well-composite; key structural parameters meet the preset standards.

[0109] Specifically, such as Figure 3 As shown, FTIR: Fourier Transform Infrared, where the PLGA microspheres are at 3455 cm⁻¹. -1 The stretching vibration peak of -OH is generated at 2985 cm⁻¹. -1 The peak at 1640 cm⁻¹ represents the stretching vibration of saturated -CH. -1 The absorption peak is generated by the C=O stretching vibration, 1330~1050 cm⁻¹ -1 Multiple stretching vibration absorption peaks were observed, with a strong asymmetric absorption peak, confirming the characteristic structure of PLGA. Simultaneously, testing of dMBG / PLGA porous microspheres revealed new, weak characteristic peaks of dMBG. EDS: Quantitative characterization using energy dispersive spectroscopy (EDS) mapping of the three microsphere systems revealed significant differences in elemental distribution. The PLGA porous microspheres exhibited a carbon-dominated distribution, highly consistent with the carbon chain-based molecular structure of PLGA. Notably, significant characteristic signals of silicon were detected in both types of dMBG / PLGA microspheres (porous and solid). Further analysis indicated that this characteristic silicon distribution is closely related to the dMBG component in the experimental design. Since the main chemical component of dMBG is a silica matrix, its successful introduction into the three microsphere systems inevitably leads to an enhancement of the characteristic silicon signal. The excellent agreement between experimental data and theoretical expectations fully confirms the successful loading of the dMBG component in the three microsphere systems, providing crucial compositional information for subsequent functional studies of the materials. Figure 3As can be seen, the three types of microspheres are generally approximately spherical, with a particle size D50 of around 200 μm. The average particle sizes of the porous PLGA microspheres, dMBG / PLGA porous microspheres, and solid dMBG / PLGA microspheres are approximately 195 μm, 209 μm, and 215 μm, respectively, with no significant differences, ensuring comparability in subsequent injection and mechanical behavior. Both porous PLGA and porous dMBG / PLGA microspheres exhibit interconnected three-dimensional channels on their surface and cross-section, with an average pore size of approximately 13–16 μm and a porosity of approximately 80–90%, which is beneficial for cell entry and nutrient exchange. In contrast, the solid dMBG / PLGA microspheres have a smooth surface and almost no internal interconnected pore structures. EDS elemental mapping shows that the Si element signal inside the porous and solid dMBG / PLGA microspheres is significantly stronger than that in the pure PLGA group, and the distribution is uniform, confirming the successful introduction and good dispersion of dMBG in the composite microspheres. In summary, the dMBG / PLGA porous microspheres obtained in this embodiment meet the preset indicators in terms of particle size, pore size, porosity and component uniformity, making them a preferred carrier for subsequent cell loading and in vivo applications.

[0110] Example 3 Construction and culture of cell-microsphere complex

[0111] 1. Experimental Objective

[0112] Using the dMBG / PLGA porous microspheres prepared in Example 1 as a carrier, mesenchymal stem cells (MSCs) were efficiently loaded and stably cultured in vitro, and a bioactive cell-microsphere complex was constructed to provide a functional unit for subsequent bone repair applications. The quality control standards of the complex were also defined.

[0113] 2. Core Processes and Operating Procedures

[0114] (1) Cell source and preparation

[0115] Cell selection: MSCs from passages 10–15 (P10–P15) were selected. Subsequent operations were performed when the confluence of cells reached about 80% under a microscope.

[0116] Digestion and collection: Digest with CTS™ TrypLE™ Select digestion solution for 3–5 min, and collect the cell pellet by centrifugation at 300g for 5 min after digestion is terminated.

[0117] Quality assessment: Trypan blue staining and counting; only cells with a viability of ≥95% can be used for loading.

[0118] (2) Microsphere pretreatment

[0119] The porous microspheres of Example 1 were subjected to a stepwise treatment:

[0120] Sterilization: Cobalt-60 irradiation (25 kGy) ensures sterility and absence of pyrogens;

[0121] Disinfection: Soak in 75% ethanol overnight;

[0122] Washing: Wash twice with PBS (30 min each time) to remove residue;

[0123] Pre-wetting: Pre-wet the target culture medium for 30 min to remove air bubbles in the pores and improve wettability.

[0124] (3) Cell loading and culture

[0125] Initial loading (low adhesion 24-well plate): Add 5 mg of pretreated microspheres to each well, seeding with 1 × 102 6 Add 1 cell / well and 2 mL of culture medium (optional: serum-free MSC medium or α-MEM + 10% FBS, containing 1% penicillin and streptomycin), and incubate at 37 ℃ in a 5% CO2 incubator for 24 h.

[0126] Composite culture (T25 flask): Transfer the complex into a T25 flask and add culture medium to 5 mL; gently pipette every 12 h to avoid aggregation and promote cell entry into the pores; replace with fresh culture medium every 2 days.

[0127] (4) Complex characterization (confocal imaging and 3D reconstruction)

[0128] On day 7, samples were taken and stained with Calcein-AM / PI for both live and dead cells. Z-stack images (step size 1–3 μm) were acquired using a laser confocal microscope. The three-dimensional distribution and depth of cells in the microsphere channels were reconstructed using software to visually present the cell loading effect.

[0129] 3. Qualification Standards

[0130] Cell viability: Cell survival rate (Calcein-positive cells / total nuclei) ≥80% on day 7;

[0131] Injectability: The complex can be successfully injected through an 18 G needle without needle blockage.

[0132] 4. Experimental Results

[0133] Depend on Figure 4As can be seen, mesenchymal stem cells (MSCs) can adhere and extend well on both the surface and inside the pores of the dMBG / PLGA porous microspheres. The cells exhibit a spindle-shaped or multi-protrusion morphology, forming a continuous cell layer along the pore walls. Confocal 3D reconstruction shows that a large number of live cell signals are distributed throughout the pores inside the microspheres, indicating that the porous structure of this embodiment not only provides a large external surface area but also forms a true "three-dimensional micro-tissue" unit. In contrast, solid microspheres can only form a single cell layer on the outer surface and are unlikely to form a volumetric cell network. These results demonstrate that the porous microspheres of this invention can provide a stable three-dimensional growth space for MSCs, which is beneficial for improving local colonization and long-term function.

[0134] Example 4: Assessment of the cell loading capacity of the complex

[0135] 1. Experimental Objective

[0136] By performing section staining and quantitative analysis on the cell-microsphere complex of Example 3 at different culture time points, the cell loading efficiency of porous microspheres and the distribution characteristics of cells inside and outside the microspheres were clarified, providing data support for its application potential as a cell carrier.

[0137] 2. Experimental Procedures and Operating Specifications

[0138] (1) Sample preparation and staining

[0139] Sampling time points: Samples were taken at 1 day, 3 days, 5 days, and 7 days after cell loading;

[0140] Fixation and embedding: After fixation with 4% paraformaldehyde for 15 min, sucrose dehydration was followed by OCT embedding to prepare 15 μm thick frozen sections.

[0141] Staining and Imaging: DAPI staining of cell nuclei was performed, and images of ≥10 fields of view were randomly acquired for each sample under uniform fluorescence microscopy parameters.

[0142] (2) Cell entry determination and efficiency calculation

[0143] Entry criteria: The shortest distance d from each cell nucleus to the outer surface of the microsphere is measured using ImageJ software. When d is greater than 1 / 10 of the microsphere radius, it is determined as "entering the interior of the microsphere".

[0144] Load efficiency calculation:

[0145] Loading efficiency (%) = Number of cells loaded on a single porous microsphere / Number of cells loaded on a single solid microsphere × 100.

[0146] Calculation of the internal / external distribution ratio:

[0147] Internal / external distribution ratio = number of pore nuclei / number of surface nuclei

[0148] 3. Qualification Standards

[0149] The load efficiency is ≥195%, and the internal / external distribution ratio is close to 1.

[0150] 4. Experimental Results

[0151] like Figure 5 As shown, under the same inoculation conditions, the cell loading per unit particle of porous microspheres is significantly higher than that of solid microspheres. At 7 days, the total loading of porous dMBG / PLGA microspheres is approximately twice that of solid microspheres. Further statistical analysis by dividing the microspheres into an "inner region" and an "outer shell" reveals that the ratio of inner to outer cell nuclei in porous microspheres is close to 1, indicating a relatively balanced distribution of cells within the pores and on the surface. In contrast, cells in solid microspheres are mainly concentrated on the outer surface, with almost no cells entering the interior. These results demonstrate that the porous microspheres of this invention, without altering the overall particle size, significantly expand the usable cell attachment area and effective loading space, enabling the formation of cell-material composite "micro-tissue" units within each microsphere—a structural advantage difficult to achieve with ordinary solid microspheres.

[0152] Porous microspheres (PLGA porous microspheres or dMBG / PLGA porous microspheres) exhibit significant advantages over solid microspheres: higher cell loading per unit particle and more even distribution of cells inside and outside the microsphere; the cells of solid microspheres are mainly confined to the outer surface, resulting in poorer loading efficiency and distribution uniformity.

[0153] Example 5: Injection Shear Protection Assessment

[0154] 1. Experimental Objective

[0155] By simulating clinical injection scenarios, the cell-microsphere complex's ability to retain cell viability under injection shear force was evaluated, clarifying its practicality as an injectable carrier.

[0156] 2. Experimental Procedures and Operating Specifications

[0157] (1) Preparation of injection device and sample

[0158] Injection device: 5 mL Luer-lock syringe with 18 G needle (20–22 G needles can be used for gradient testing).

[0159] Experimental sample: Cell-microsphere complex cultured for 7 days as described in Example 3;

[0160] Control group: Complex of MSCs and solid microspheres;

[0161] Suspension medium: Cell culture medium preheated to 37 °C, 2 mL in volume.

[0162] (2) Injection procedure and rhythm

[0163] Three reciprocating injections were performed at 37 ℃, with each injection lasting 3–5 seconds and an interval of 6 hours between injections. Samples were taken at four time points: 0, 1, 2, and 3 injections. The samples were gently collected after each injection to avoid damage from cumulative shear forces.

[0164] (3) Cell viability evaluation

[0165] Cellular metabolic activity was determined using the CCK-8 assay. With the absorbance A0 of the 0-injection group as the baseline, residual activity at each time point was calculated: R n (%)=A n / A0×100

[0166] 3. Experimental Results

[0167] Depend on Figure 6 As shown, under simulated clinical injection conditions (three reciprocating injections with an 18G needle), the porous dMBG / PLGA microsphere-cell complex of this invention can significantly reduce the damage to cell viability caused by shear stress: compared with before injection, the residual cell viability remained at a high level after three injections, and the loss of viability caused by injection was reduced by about 40% compared with the solid microsphere group; compared with naked MSC suspension, the porous microsphere group showed higher cell metabolic activity and survival rate after each injection. These results indicate that the three-dimensional pore walls of the porous microspheres act as a mechanical buffer during the injection process, effectively protecting mesenchymal stem cells from needle shearing damage, which is a key structural basis for realizing "injectable viable grafts".

[0168] Example 6: Cryopreservation and thawing of cell-microsphere complexes

[0169] 1. Experimental Objective

[0170] To establish a convenient cryopreservation and efficient resuscitation method for cell-microsphere complexes, and to clarify the quality indicators of the complexes after resuscitation, such as cell viability, injectability, and sterility, so as to provide technical support for the clinical transport and immediate application of the complexes.

[0171] 2. Experimental Materials

[0172] Cryopreservation reagents: Use commercially available tissue / cell cryopreservation reagents (such as Cyclone tissue cryopreservation solution; the trade name is for illustrative purposes only and does not constitute a product limitation). Prioritize cryopreservation solutions without DMSO to reduce cytotoxicity.

[0173] Samples to be frozen: Take the cell-microsphere complex cultured on day 7 of Example 3, collect it by low-speed centrifugation (to avoid microsphere breakage), discard the supernatant, resuspend it with a small amount of fresh culture medium, and adjust the complex concentration to ≥5 mg / mL;

[0174] Equipment preparation: sterile cryovials, 15 mL conical tubes, 37 ℃ constant temperature water bath. All equipment should be sterilized in advance.

[0175] 3. Experimental Procedures and Operating Specifications

[0176] (1) Cryopreservation process

[0177] Sample aliquoting and equilibration: Perform aseptic operations in a biosafety cabinet, slowly add the complex suspension to the cryopreservation solution at a volume ratio of 1:10, and gently mix while adding to avoid generating air bubbles; place the mixed sample on ice for 5 minutes to equilibrate and further eliminate air bubbles in the system.

[0178] Direct freezing at -80 ℃: Aliquot the equilibrated sample into sterile cryovials, labeling each tube with the batch number, freezing time, complex mass (mg), and cell count (×10⁻⁶). 6 Immediately place the cryovials in a standard -80°C freezer and store them upright. If long-term storage is required, the cryovials can be transferred to the liquid nitrogen vapor phase zone after 24 hours.

[0179] (2) Recovery process and refrigerant removal

[0180] Rapid rewarming: Remove the cryovial from the -80 ℃ freezer and immediately place it in a 37 ℃ constant temperature water bath for rapid rewarming. Gently shake the cryovial during this process to accelerate thawing. Control the rewarming time to 30–60 s. Remove the cryovial after the sample inside has completely thawed.

[0181] Serial dilution: Transfer the resuscitated sample into a 15 mL conical tube, add fresh culture medium pre-warmed to 37 ℃ and perform serial dilution (first dilute 1-fold, let stand for 3 min; then dilute to 3-fold, let stand for 3 min; finally dilute to 10-fold) to avoid sudden changes in osmotic pressure that could damage the cells;

[0182] Cryolysis removal: Centrifuge the diluted sample at 200 g for 5 min, discard the supernatant containing cryolysis, resuspend the complex in 5 mL of fresh culture medium to complete the resuscitation operation.

[0183] 4. Post-recovery quality evaluation

[0184] (1) Evaluation indicators and testing methods

[0185] Cell viability: Cell viability was observed using the Calcein-AM / PI live-dead cell double staining method, and cell metabolic activity was measured using the CCK-8 assay.

[0186] Injectability: The resuscitated complex suspension was drawn into a syringe equipped with an 18 G needle and manually injected to observe the smoothness. It was considered qualified if there was no obvious needle blockage and the injection resistance was uniform.

[0187] Pollution monitoring: Place the revived complex in an incubator for 7 days, and observe under a microscope every day for signs of contamination such as bacteria and fungi.

[0188] (2) Control setting and judgment criteria

[0189] The revived complex needs to meet all the following criteria to be qualified:

[0190] The cell viability immediately after revival ≥ 80–85%;

[0191] The cell metabolic activity 24 h after revival ≥ 70–90% of the level before cryopreservation;

[0192] The passing rate of injection with a 18G needle = 100%;

[0193] There is no significant difference in cell viability between the complex group and the simple MSC control group (p > 0.05).

[0194] 5. Experimental results

[0195] As Figure 7 shown, after direct cryopreservation at -80 °C using a DMSO-free commercial cryopreservation solution, the porous microsphere-cell complex still maintained good particle morphology and three-dimensional pore structure after revival, and no obvious aggregation or fragmentation was observed. Live / dead staining and CCK-8 detection showed that the cell viability and metabolic activity of the porous microsphere group were comparable to those of traditional cell suspension cryopreservation / revival immediately after revival and after short-term culture, and were significantly better than those of the solid microsphere group, in which the surface cells were prone to large-area shedding during the freezing and thawing process. This result proves that the porous microsphere-cell complex of the present invention is suitable for simple direct cryopreservation at -80 °C, and can maintain high cell activity and good injectability after revival, laying a foundation for the realization of centralized preparation, long-term storage and "ready-to-use" scale application.

[0196] Example 7 Osteogenesis-related evaluation of cell-microsphere complex

[0197] 1. Experimental purpose

[0198] Systematically evaluate the osteogenic differentiation potential of the dMBG / PLGA microsphere-cell complex from four dimensions of enzyme activity, mineralization ability, gene expression and protein level, clarify the osteogenic induction effect of the dMBG component, and provide a functional basis for its bone repair application.

[0199] 2. Experimental system and culture conditions

[0200] (1) Grouping setting

[0201] Experimental group: dMBG / PLGA microsphere-cell complex (qualified sample taken on the 7th day of culture in Example 3);

[0202] Control group: MSC+PLGA porous microsphere complex (without dMBG component, other preparation conditions are the same as the experimental group).

[0203] (2) Training Program

[0204] Two culture systems were set up for both groups of samples, and experiments were conducted in parallel:

[0205] Basal culture group: using basal culture medium (without exogenous osteogenic inducers) to assess the spontaneous osteogenic tendency of the complex;

[0206] Induction culture group: Differentiation capacity under osteogenic induction conditions was evaluated using osteogenic induction medium;

[0207] Sampling time points: Samples were taken on the 7th and 14th days of culture for testing various indicators.

[0208] 3. Core Evaluation Indicators and Testing Methods

[0209] (1) Alkaline phosphatase (ALP) activity detection

[0210] Commercial ALP detection kits (such as Beyotime ALP kits) were used for detection: complexes from each group were collected, lysed, and total protein was extracted. The enzyme activity was measured according to the kit instructions. The results were normalized to the protein activity per mg to eliminate the influence of protein quantity differences on the results.

[0211] (2) Detection of mineralized nodule (ARS) formation

[0212] The samples were stained with Alizarin Red S staining solution (pH=4.2), and washed with distilled water to remove excess staining solution; the formation of mineralized nodules was observed by optical microscopy to qualitatively determine osteogenic mineralization capacity.

[0213] (3) Osteogenesis-related gene expression detection (RT-qPCR)

[0214] Genes detected: Key genes for osteogenic differentiation were selected, including alkaline phosphatase gene (ALPL), osteoblast-specific transcription factor gene (RUNX2), and osteogenic-associated transcription factor gene (OSX). Internal control: GAPDH; 2–ΔΔCt; Immunofluorescence: COL1A1, OSX.

[0215] 4. Qualification Standards

[0216] Using "all osteogenic indicators in the experimental group were significantly higher than those in the control group, and the differences between the groups were statistically significant (p<0.05)" as the passing criterion, it was confirmed that dMBG components can effectively promote the differentiation of MSCs into osteoblasts.

[0217] 5. Experimental Results

[0218] Figure 8 The results showed that, under basal culture conditions without the addition of exogenous osteogenic inducers, MSCs loaded on dMBG / PLGA porous microspheres exhibited higher ALP activity and stronger mineralized nodule formation ability on days 7 and 14. The expression levels of osteogenic-related genes such as ALP, RUNX2, and OSX were all higher than those in the PLGA porous microsphere control group, and the immunofluorescence signals of osteogenic proteins such as COL1A1 were also significantly enhanced. Under osteogenic induction culture conditions, these differences were further amplified. This indicates that the sustained-release Ca / Si ions from the dMBG component can continuously provide osteogenic induction signals in the three-dimensional microsphere environment, enabling the complex of this invention to have a significant endogenous osteogenic tendency even in the absence of exogenous growth factors.

[0219] Example 8 Cell Identity Assessment of Cell-Microsphere Complex

[0220] 1. Experimental Objective

[0221] Using single-cell RNA sequencing (scRNA-seq) technology, we analyzed the heterogeneity, differentiation status, and functional gene expression characteristics of MSCs in the dMBG / PLGA microsphere-cell complex, clarified the regulatory role of the complex on the identity and functional lineage of MSCs, and provided a molecular mechanism basis for their potential in bone repair and tissue regeneration.

[0222] 2. Experimental Procedure

[0223] (1) Preparation of single-cell suspension

[0224] A two-step mild enzymatic digestion method was used to dissociate the cell-microsphere complex, preserving cell viability and integrity to the greatest extent. The specific steps are as follows:

[0225] Primary digestion: Treat the complex with Tryple digestive solution for 15 minutes and collect the digestive solution;

[0226] Secondary digestion: For the remaining incompletely dissociated complex, add a mixed enzyme solution of collagenase I, collagenase II and neutral protease, and continue digestion for 30 minutes;

[0227] Filtration and quality control: The digestion products from both digestion processes were combined and filtered through a 40 μm cell filter to remove microsphere fragments, and a single-cell suspension was prepared. Cell viability was detected by methylene blue staining, and only samples with a viability of ≥85% were retained for subsequent experiments.

[0228] (2) Library construction and sequencing

[0229] Sequencing platform: A droplet-based high-throughput single-cell sequencing platform was used to construct single-cell cDNA libraries;

[0230] Sequencing depth: Set the sequencing depth to at least 30,000 reads / cell, or follow the standard parameters recommended by the selected sequencing platform to ensure sufficient transcript coverage to meet the needs of subsequent gene expression analysis.

[0231] (3) Comparison settings

[0232] The MSC+PLGA porous microsphere complex served as a control group, and was prepared, digested, and subsequently processed in the same batch as the experimental group (dMBG / PLGA cell-microsphere complex) to ensure consistency of experimental conditions.

[0233] 3. Data Analysis Process and Standards

[0234] The sequencing data were processed using a standardized bioinformatics analysis workflow, with the following steps and standards:

[0235] (1) Data quality control: Low-quality cells were removed to improve data reliability. The removal criteria were ① cells with <200 expressed genes; ② cells with >10–15% mitochondrial gene expression.

[0236] (1) Data normalization: The gene expression data were normalized using the LogNormalize method or the SCTransform algorithm to eliminate the interference of sequencing depth differences and technical noise on the results;

[0237] (2) Dimensionality reduction and cell clustering: First, linear dimensionality reduction is performed by principal component analysis (PCA) to screen core principal components; then, nonlinear dimensionality reduction is performed using UMAP or t-SNE algorithms to visualize the cell population; cell clustering analysis is performed based on the dimensionality reduction results, with the clustering resolution parameter set to 0.6–1.2 to accurately identify different cell subpopulations.

[0238] (3) Differential gene expression analysis: The FindMarkers function combined with the Wilcoxon rank-sum test was used to identify differentially expressed genes among different cell subpopulations or between experimental and control groups; the Benjamini-Hochberg (BH) method was used to correct the detection results and control the false detection rate (FDR).

[0239] (4) Pseudo-time series analysis: Based on the expression characteristics of marker genes, cell subpopulations that may have upstream and downstream differentiation relationships are screened out. Pseudo-time series analysis algorithms such as Monocle3 or Slingshot are used to construct cell differentiation trajectories and predict the differentiation direction of MSCs.

[0240] (5) Cell annotation: Combine the differentiation trajectory of cell subpopulations, differential gene expression profiles and known cell type marker genes to accurately name and define the identity of each cell subpopulation.

[0241] 4. Analysis Content

[0242] Focusing on the differences between the experimental group (dMBG / PLGA complex) and the control group (MSC+PLGA complex), the study particularly examines the expression changes and pathway enrichment of three types of functional genes:

[0243] Osteogenic differentiation marker genes: RUNX2, OSX, ALPL, etc.;

[0244] Paracrine function genes: VEGF, ANGPT1 and other angiogenesis-related factor genes;

[0245] Immune regulation genes: anti-inflammatory and immune regulation genes such as IL-10 and TGF-β.

[0246] 5. Experimental Results

[0247] Depend on Figure 9 Single-cell transcriptome analysis revealed that MSCs loaded on porous PLGA and porous dMBG / PLGA microspheres could be divided into four main subpopulations. The PLGA group was dominated by a population of dry / activated MSCs maintaining high SFRP2 expression, while the dMBG / PLGA group significantly enriched an osteogenic subpopulation expressing osteogenic marker genes such as ALPL and COL1A1, accompanied by upregulation of cell cycle-related genes, suggesting the presence of a proliferating osteogenic precursor population. Pseudo-temporal analysis showed that the cell lineage trajectory gradually transitioned from the dry MSC subpopulation to the dMBG / PLGA-related osteogenic subpopulation, accompanied by continuous activation of osteogenic and matrix secretion-related pathways. These results validate, at the single-cell level, the osteogenic lineage bias of the dMBG / PLGA porous microspheres of this invention on MSCs.

[0248] Example 9: Evaluation of the pro-angiogenic effect of cell-microsphere complex

[0249] 1. Experimental Objective

[0250] Using human umbilical vein endothelial cells (HUVECs) as the research object, this study systematically evaluated the ability of the dMBG / PLGA microsphere-cell complex to regulate vascular endothelial cell function through paracrine effects from three core dimensions: proliferation, migration, and tube formation. This study aimed to clarify its pro-angiogenic potential and provide experimental evidence for blood supply reconstruction during bone repair.

[0251] 2. Experimental System and Core Detection Methods

[0252] (1) Evaluation of HUVEC proliferation capacity (Transwell co-culture system)

[0253] System construction: Transwell chambers with a pore size of 0.4 μm were selected, and HUVECs were seeded in the lower chamber (seedling density 2 × 10⁻⁶). 4–5×10 4 (cells / well), placed in an incubator to adhere to the walls overnight; cell-microsphere complexes are placed in the upper chamber to ensure that the complexes do not have direct contact with HUVECs and only exert their effects through paracrine factors;

[0254] Culture and staining: After co-culturing for 24 h, the Transwell chamber was removed, and the HUVECs in the lower chamber were fixed, permeabilized, and blocked with PFA. They were then incubated with anti-Ki67 primary antibody, labeled with fluorescent secondary antibody, and finally counterstained with DAPI to the cell nuclei.

[0255] Quantitative analysis: ≥5 fields of view / samples were randomly collected using confocal microscopy or fluorescence microscopy, and the Ki67 index was calculated: Ki67 index (%) = (Ki67 + number of cell nuclei) / total number of DAPI nuclei × 100. The higher the Ki67 index, the stronger the cell proliferation activity.

[0256] (2) Assessment of HUVEC migration ability (confocal cell + scratch method)

[0257] Scratch model construction: HUVECs were seeded in glass-bottomed confocal microplates and cultured to 95–100% confluence. Then, a 1000 μL pipette tip was used to make a scratch perpendicular to the bottom of the plate to form a uniform cell-free band. The cells were then gently washed twice with PBS to remove airborne cells and low-serum culture medium was added.

[0258] Paracrine exposure: Place a confocal chamber / compartment in the same dish, place the cell-microsphere complex in the chamber, and ensure that the complex does not come into direct contact with the HUVEC monolayer;

[0259] Imaging and Analysis: Optical microscopy imaging was performed at 0 h, 12 h, and 24 h to record changes in the scratched area; scratch width or cell-free area was measured, and migration-related indicators were calculated.

[0260] Scratch closure rate (%) = [1 − width (t) / width (0)] × 100;

[0261] Optional metrics: Extract cell leading edge migration velocity (μm / h) and migration directionality parameters to comprehensively evaluate migration ability.

[0262] (3) Assessment of HUVEC tube formation ability (conditioned medium + Matrigel tube formation model)

[0263] Preparation of conditioned medium (CM): The cell-microsphere complex was cultured in serum-free or low-serum medium for 24 h. The culture supernatant was collected and filtered through a 0.22 μm filter membrane to remove impurities, thus obtaining the conditioned medium from which the complex was derived.

[0264] Tube formation model construction: Matrigel matrix was spread in a glass-bottomed confocal dish and incubated at 37 °C for 30 min to allow it to solidify; HUVECs (2 × 10⁻⁶) were seeded. 4 (each well contains 10 cells / well), and the prepared conditioned medium is added (with blank medium set as a control).

[0265] Imaging and quantification: Confocal images were acquired at 6 h, 12 h, and 24 h, respectively. Quantitative analysis was performed using ImageJ software or the Angiogenesis Analyzer plugin. The core parameters included total tube length, number of branches / nodes, and number of grids. Higher parameter values ​​indicated stronger tube formation ability.

[0266] 3. Judgment Criteria

[0267] Proliferation indicator: Ki67 index;

[0268] Migration metric: Scratch closure rate;

[0269] Pipeline performance indicators: Total pipe length, number of branches / nodes, and number of grids have significantly improved.

[0270] 4. Experimental Results

[0271] Figure 10 The results showed that, in the HUVEC co-culture system, the MSC-porous microsphere complex of this invention significantly enhanced the proliferation, migration, and tube formation ability of endothelial cells. Compared with the blank control, iMSC-PLGA porous microspheres and iMSC-dMBG / PLGA porous microspheres increased the proportion of Ki67-positive endothelial cells by approximately 2.55-fold and 2.26-fold, respectively, and improved the scratch closure rate by approximately 46.6% and 37.6%, respectively. In the Matrigel tube formation experiment, both groups significantly increased the number of branch points and the total tube length, with the PLGA porous group showing a slightly larger increase, but the dMBG / PLGA porous group was still significantly better than the cell-free or material-only control groups. This indicates that under osteogenic induction conditions, the complex of this invention can still maintain strong pro-angiogenic paracrine function, providing support for vascular reconstruction in the defect area.

[0272] Example 10: Evaluation of the immune regulation of cell-microsphere complexes

[0273] 1. Experimental Objective

[0274] Using human mononuclear cell line THP-1 derived macrophages as a model, we verified their tissue repair potential from the perspective of immune regulation, while distinguishing the contributions of paracrine secretion and material ion release.

[0275] 2. Construction of the core experimental system

[0276] (1) Macrophage phenotype induction

[0277] Macrophage preparation: Logarithmic growth phase THP-1 cells were treated with 50–100 nM PMA for 24 h to induce adherent differentiation; the supernatant was discarded, the cells were washed with PBS, and then replaced with fresh culture medium and allowed to stand for 24 h to recover, thus obtaining unpolarized M0 phenotype macrophages.

[0278] Macrophage induction: M0 cells were co-stimulated with 100 ng / mL LPS + 20 ng / mL IFNγ for 24 h to induce the M1 phenotype;

[0279] Macrophage control (optional): M0 cells were stimulated with 20 ng / mL IL-4 + 20 ng / mL IL-13 for 24–48 h to induce the M2 phenotype, serving as a control.

[0280] (2) Co-training model

[0281] The Transwell paracrine co-culture system was used (to avoid direct contact between cells and materials): THP-1 derived macrophages (M0 / M1 / M2 phenotype) were seeded in the lower chamber, and cell-microsphere complexes were placed in the upper chamber. Transwell chambers with a pore size of 0.4 μm were used; the co-culture time was 24 h.

[0282] Control groups: ① Blank control group (macrophages only, no complex); ② Vector blank group (no cells dMBG / PLGA microspheres, no MSCs).

[0283] (3) Evaluation indicators and testing methods

[0284] Flow cytometry: Collect lower chamber macrophages, exclude dead cells with DAPI or 7-AAD, and detect the expression of M1 marker CD86 and M2 marker CD206; provide detailed gating strategy, and report the percentage of positive cells and mean fluorescence intensity (MFI).

[0285] Immunofluorescence staining: Macrophages were stained with CD86+F4 / 80 or CD206+F4 / 80 double label (F4 / 80 is a macrophage-specific marker). Intracellular factor staining with iNOS (M1) / ARG1 (M2) was also performed. Phenotypic distribution was observed by confocal microscopy.

[0286] Cytokine detection (optional): The concentrations of M1-related cytokines (TNF-α, IL-6) and M2-related cytokines (IL-10) in the co-culture supernatant were quantitatively analyzed using ELISA or a multifactor detection kit;

[0287] NO detection (RAW264.7 specific): If the mouse macrophage cell line RAW264.7 is used for parallel validation, the nitrite content in the supernatant is detected by the Griess method, which indirectly reflects the NO secretion level of M1 macrophages.

[0288] (4) Parallel validation in mouse system (supplementary experiment)

[0289] A mouse model was established using RAW264.7 cells: the M1 phenotype was induced with 100 ng / mL LPS, following the Transwell co-culture procedure described above; the detection indicators included CD86 / CD206 flow cytometry analysis, IL-10 / IL-1β / STAT6 gene expression (qRT-PCR), and Griess method NO detection to verify the consistency of immune regulation under species differences.

[0290] 3. Qualification Standards

[0291] Compared with the M1 control group, the cell-microsphere complex group needed to meet the following criteria, and the difference was statistically significant (p<0.05):

[0292] Phenotype: CD86 + Decreased cell percentage and MFI, CD206 + Increased cell percentage and MFI;

[0293] Secretion profile: TNF-α and IL-6 concentrations decreased, while IL-10 concentration increased.

[0294] 4. Experimental Results

[0295] like Figure 11 As shown, in the human THP-1 macrophage model, the MSC-porous microsphere complex significantly downregulated the expression of LPS-induced M1 marker CD86 and upregulated the expression of M2 marker CD206. Compared with the material group alone, the MSC-loaded complex was more effective in inhibiting M1 polarization and promoting the M2 phenotype, suggesting that the "material ion release + MSC paracrine" in this invention has a synergistic immunomodulatory effect, which helps to alleviate inflammation in the defect area in a timely manner and transform it into a regenerative immune microenvironment.

[0296] By comparing with the blank control group, it was clarified that the immunomodulatory effect of the complex originated from the synergistic effect of MSC paracrine secretion and material ion release.

[0297] Example 11: Neurological Assessment of Cell-Microsphere Complexes

[0298] 1. Experimental Objective

[0299] Using the dorsal root ganglion (DRG) of fetal rats as a neural model, this study evaluated the ability of cell-microsphere complexes to promote neurite growth through paracrine effects, providing a basis for their application at the bone-nerve repair interface.

[0300] 2. Experimental Procedure and Methods

[0301] (1) DRG source and culture preparation

[0302] DRG acquisition: Fetal rats at gestation days 14–15 (E14–E15) were selected, and the dorsal root ganglia were isolated under aseptic conditions.

[0303] Culture base treatment: Glass-bottomed culture dishes are pre-coated with poly-D-lysine / laminin to enhance the initial adhesion ability of DRGs.

[0304] (2) Construction of a co-training system

[0305] The isolated DRGs were seeded into a pretreated glass dish. After the DRGs adhered, a confocal chamber was placed in the same culture dish, and the cell-microsphere complex was placed in the chamber. This achieved a paracrine exposure mode with shared culture medium and no direct contact through physical isolation.

[0306] (3) Staining, imaging and quantitative analysis

[0307] Staining treatment: On the 3rd day of co-culture, the neural cytoskeleton (Actin) was labeled with phalloidin, and the cell nuclei were counterstained with DAPI;

[0308] Imaging: Images are acquired using a confocal microscope, and neural processes and growth cones are identified based on the cytoskeleton's orientation;

[0309] Quantitative analysis: ImageJ software was used to analyze the average neural protrusion length; at least 3 independent DRGs were selected for each sample, and ≥3 visual fields were collected for each DRG to ensure data representativeness.

[0310] 3. Comparison settings

[0311] ① Blank control group (DRG only, no complex); ② Vector blank group (cell-free dMBG / PLGA microspheres).

[0312] 4. Experimental Results

[0313] Figure 12The results showed that in a rat fetal dorsal root ganglion (DRG) explant model, DRG axons exposed to paracrine factors from the MSC-porous microsphere complex were significantly longer and denser than those in the control group. Quantitative analysis showed that iMSC-PLGA porous microspheres and iMSC-dMBG / PLGA porous microspheres increased the average axon length by approximately 2.35-fold and 2.28-fold, respectively, with no significant difference between the two, indicating that the introduction of dMBG did not weaken the nerve growth-promoting capacity of MSCs. The complex of this invention, in addition to its osteogenic, angiogenic, and immunomodulatory functions, also possesses certain neural support functions, which is beneficial for the overall repair of the bone-nerve interface.

[0314] Example 12: In vivo assessment of bone defect repair using cell-microsphere complexes

[0315] 1. Experimental Objective

[0316] In a BALB / c nude mouse parietal bone critical bone defect model, the in vivo bone repair effect of cell-microsphere complex was verified, clarifying the role of "injectable microspheres + in situ fixation" in improving material retention rate and repair quality.

[0317] 2. Animal Models and Experimental Groups

[0318] Animal selection: Adult BALB / c nude mice, weighing 18–22 g;

[0319] Group design: Each group consisted of ≥6 animals to ensure statistical power. The specific groups were: ① Blank control group (bone defects only, no treatment); ② Naked MSC group (MSC suspension only); ③ Material control group (dMBG / PLGA porous microspheres only); ④ Experimental group (MSC + dMBG / PLGA complex).

[0320] 3. Surgical Procedure and Complex Delivery

[0321] (1) Complex fixative and preparation

[0322] To improve the in-situ stability of the microspheres after injection, the composite sample was suspended in a porous GelMA prepolymer solution (e.g., 5–10 wt%) as a fixative; after injection into the defect cavity, in-situ curing was performed to fix the microspheres.

[0323] (2) Surgical procedure

[0324] a. Bone defect preparation: After anesthetizing nude mice, the head was prepared and disinfected, and a critical bone defect with a diameter of 4 mm was prepared in the parietal bone using a trephine (critical defect refers to a defect size that cannot be repaired by itself).

[0325] b. Complex injection and curing: Inject 50–100 μL of GelMA prepolymer containing the complex into the defect cavity to ensure that the microspheres are uniformly embedded in the prepolymer; then add a small amount of photoinitiator (such as LAP 0.05–0.1 wt%) and cure the surface by irradiation with 405 nm light for 10–30 s, or cure by a mild method such as body temperature / ionic / enzymatic crosslinking;

[0326] c. Postoperative care: Routine suturing of the surgical incision, administration of analgesia, individual cage housing, free access to food and water, and observation of postoperative recovery.

[0327] 4. Evaluation (12 weeks post-surgery):

[0328] (1) Micro-CT imaging assessment: Obtain skull specimens, perform micro-CT scans, and quantitatively analyze bone repair-related parameters: ① Bone volume fraction (BV / TV); ② Trabecular thickness (Tb.Th); ③ Trabecular number (Tb.N); ④ Trabecular separation (Tb.Sp).

[0329] (2) Histological and immunomarker assessment:

[0330] Routine staining: HE staining to observe tissue morphology and new bone formation, and Masson trichrome staining to distinguish collagen fibers from mineralized tissue;

[0331] Osteogenic marker staining: Alkaline phosphatase (ALP) staining and osteocalcin (OCN) immunohistochemistry were used to verify osteoblast activity;

[0332] Functional markers: CD31 immunohistochemistry was used to label vascular endothelial cells to assess vascular density in the defect area; COL1A1 immunohistochemistry was used to label type I collagen to assess bone matrix synthesis.

[0333] 5. Experimental Results

[0334] like Figure 13As shown, in the BALB / c nude mouse model of a 4 mm critical skull defect, the blank control group still exhibited significant interosseous gaps and low parameters such as BV / TV in the defect area after 12 weeks; the group injected with only dMBG / PLGA porous microspheres showed a small amount of new bone ingrowth, but the repair was incomplete; the group injected with only MSCs showed some new bone formation, but the increase in bone volume was limited due to the lack of scaffold support and local retention. In contrast, the dMBG / PLGA porous microsphere-MSC complex of this invention showed on micro-CT that the defect was almost completely filled with new bone, with a significant increase in BV / TV and trabecular thickness, and a significant decrease in trabecular separation. Masson staining also confirmed the presence of continuous new bone bridging and mature collagen matrix deposition. These results indicate that the injectable porous microsphere-MSC complex of this invention can significantly promote bone defect repair in vivo, and its bone regeneration effect is superior to that of cells or materials alone.

[0335] Example 13 Application of Cell-Microsphere Complexes—Cell-Microsphere Complex Injectable Formulations and Kits

[0336] 1. Positioning and Core Components

[0337] This kit is a "ready-to-use" cell therapy formulation, developed based on the previously validated qualified cell-microsphere complex. It is suitable for basic research and preclinical evaluation of bone defect repair. The core components include five modules of AE, as follows:

[0338] (1) Carrier dry powder (optional)

[0339] Aseptic lyophilized dMBG / PLGA porous microspheres labeled fill weight (mg).

[0340] (2) Cryopreservation complex vials

[0341] MSC-loaded dMBG / PLGA microspheres were suspended in a commercially available cryopreservation solution without DMSO.

[0342] (3) Dilution / Resuspension Buffer

[0343] Isotonic calcium- and magnesium-free PBS or equivalent injection media may contain a small amount of albumin to enhance lubrication and anti-adsorption capacity.

[0344] (4) Injection assembly

[0345] Use a 5 mL syringe, 18 G needle, and connecting tubing (if necessary) to match the particle size of the complex and ensure smooth injection.

[0346] (5) Operation Manual

[0347] Cell seeding guidelines, thawing and removal of cryopreservation solution procedures, quality release standards and precautions.

[0348] 2. Storage, transportation and labeling requirements

[0349] Storage conditions: ① Carrier dry powder: Store at 2–8 ℃ away from light, shelf life 12–24 months; ② Frozen complex: Store at −80℃ for short period (≤6 months), or in the liquid nitrogen vapor phase region for long period (≥1 year), repeated freeze-thaw cycles are strictly prohibited;

[0350] Transportation requirements: Dry ice must be used to maintain a low-temperature environment during the transportation of frozen complexes;

[0351] Labeling guidelines: Each component must be labeled with batch number, production date, and expiration date; cryopreservation complexes must additionally label cell source / passage, microsphere content (mg), and cell count (×10⁻⁶). 6 The type of cryopreservation solution, sterility level, and endotoxin limit (if used for research purposes, it can be labeled "non-clinical").

[0352] 3. Core usage methods (two paths)

[0353] (1) Carrier dry powder path (for on-site compounding)

[0354] Suitable for scenarios requiring on-site cell loading: ① Perform cell loading and culture according to the method in Example 3; ② Resuspend the complex in a homogeneous suspension with buffer solution before injection; ③ Draw into a syringe and inject through an 18 G needle.

[0355] (2) Pathway for cryopreserved complexes (ready to use after thawing)

[0356] Suitable for rapid application scenarios, the core steps are "thawing - removing cryopreservation solution - quality control - injection", as detailed below:

[0357] Rapid rewarming: Remove the complex vial from −80 ℃ / liquid nitrogen and immediately place it in a 37 ℃ water bath and gently shake for 30–60 s until the complex is completely melted;

[0358] Stepwise dilution to remove cryopreservation solution: Transfer the thawed complex into a 15 mL conical tube and dilute stepwise with pre-warmed injection medium / culture medium at a ratio of 1→3→10, allowing each step to stand for 3 min to reduce osmotic pressure shock.

[0359] Centrifugation and washing: Centrifuge at 200 g for 5 min, discard the supernatant; repeat the dilution-centrifugation step ≥2 times until the residual frozen solution is <0.1% (v / v);

[0360] Quality control: Assess cell viability with trypan blue or Calcein-AM / PI staining (recommended ≥80–85%), observe whether the complex aggregates, and gently disperse them if necessary;

[0361] Injection preparation and procedure: Resuspend the complex to the target concentration with isotonic calcium- and magnesium-free PBS or an equivalent injection medium, draw 5 mL into a syringe, and inject through an 18 G needle; if in situ fixation is required, in situ fixation can be performed using non-cytotoxic instruments (such as medical absorbable mesh / plugs, scaffolds, or embolization materials), ensuring that it is cell-compatible and suitable for in vivo placement.

[0362] Important note: The cryopreservation solution contains cytotoxic components and must not be injected directly. It must be thoroughly washed away before use.

[0363] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. An injectable porous microsphere mesenchymal stem cell complex, characterized in that, The composition comprises a carrier and mesenchymal stem cells loaded on the carrier; the carrier is dMBG / PLGA porous microspheres prepared from dMBG and PLGA; the dMBG is dendritic mesoporous bioactive glass, and the PLGA is polylactic acid-glycolic acid copolymer; the mesenchymal stem cells enter and colonize in the three-dimensional through-pores of the carrier to form a cell-material composite micro-unit.

2. The injectable porous microsphere mesenchymal stem cell complex of claim 1, wherein, The mass percentage of dMBG in the dMBG / PLGA porous microspheres is 5-20 wt% of PLGA; the average particle size of the porous microspheres is 150-300 μm, the porosity is 80-90%, and the pore size is 12-25 μm; and the monomer ratio of lactic acid to glycolic acid of the PLGA is 1:

1.

3. The injectable porous microsphere mesenchymal stem cell complex of claim 1, wherein, The mesenchymal stem cells are cells passed to the 10th-15th generation, with a cell confluence of about 80% and a trypan blue staining viability of ≥95%.

4. The injectable porous microsphere mesenchymal stem cell complex of claim 1, wherein, The loading ratio of the mesenchymal stem cells to the dMBG / PLGA porous microspheres is 1×10 6 cells to 5 mg of dMBG / PLGA porous microspheres.

5. A method for preparing the injectable porous microsphere mesenchymal stem cell complex according to any one of claims 1 to 4, characterized in that, The composition comprises the following steps: (1) Preparation of dMBG / PLGA porous microspheres: dMBG particles and PLGA are dissolved in an organic solvent, and porous microspheres are prepared by emulsification-solvent evaporation method, and then freeze-dried and sterilized for standby use; (2) Cell loading: the mesenchymal stem cell suspension is mixed with the dMBG / PLGA porous microspheres of step (1) at the ratio in claim 4, and incubated in a 37℃, 5% CO2 incubator for 24 h to allow the mesenchymal stem cells to fully adsorb in the pores of the dMBG / PLGA porous microspheres; (3) Composite culture: the mesenchymal stem cell-dMBG / PLGA porous microsphere system of step (2) is transferred into a T25 culture flask, and the complete culture medium is supplemented to 5 mL; during the culture, the composite is gently blown every 12 h to avoid agglomeration and promote the mesenchymal stem cells to enter the internal pores of the dMBG / PLGA porous microspheres; the fresh culture medium is replaced every 2 days, and the injectable porous microsphere mesenchymal stem cell composite is obtained after the culture is completed.

6. The method for preparing the injectable porous microsphere mesenchymal stem cell complex according to claim 5, characterized in that, The specific steps of step (1) of claim 5 are as follows: S1 Organic phase preparation: 0.2 g of PLGA and 20 mg of dMBG are dissolved in 10 mL of dichloromethane or a mixture of dichloromethane and ethyl acetate organic phase to form a dMBG / PLGA organic dispersion system; S2 Colostrum preparation: To the organic dispersion system obtained in S1, 2.5 mL of 8% Gel aqueous solution was added, and homogenized at a speed of 10000 rpm for 60 s to form W Gel / Od MBG / PLGA Colostrum; S3 double emulsion preparation: The primary emulsion from S2 was slowly added to 100 mL of 0.1% PVA external water phase with stirring at 350 rpm to form W Gel / Od MBG / PLGA / W PVA double emulsion system; S4 Volatile pore-forming and solidification: the double emulsion system is placed in a 50℃ water bath, and low-speed stirring is performed for 8 min to promote the volatilization of the organic phase solvent and realize the pore-forming and solidification of the dMBG / PLGA microspheres; S5 Post-treatment: the dMBG / PLGA microsphere system after S4 solidification is centrifuged at 400 g for 5 min to collect the microsphere precipitate; The residual reagents are removed by washing with deionized water for 3 times, and finally freeze-dried to obtain the dMBG / PLGA porous microspheres.

7. The method for preparing the injectable porous microsphere mesenchymal stem cell complex according to claim 5, characterized in that, The sterilization in step (1) of claim 5 is performed by 60Co irradiation sterilization.

8. Use of the injectable porous microsphere mesenchymal stem cell composite according to any one of claims 1-4 in bone defect repair.

9. An injectable formulation characterized in that, The injectable porous microsphere mesenchymal stem cell complex according to any one of claims 1 to 4, or the injectable porous microsphere mesenchymal stem cell complex obtained by the preparation method according to any one of claims 5 to 6.

10. A kit characterized in that, The injectable preparation according to claim 9, or the injectable porous microsphere mesenchymal stem cell complex according to any one of claims 1 to 4, or the injectable porous microsphere mesenchymal stem cell complex obtained by the preparation method according to any one of claims 5 to 6.

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