Porous microsphere for filling alveolar fossa and preparation method thereof

Abstract

The invention belongs to the field of biomedical materials and tissue engineering, and particularly relates to a porous microsphere for filling alveolar fossa and a preparation method of the porous microsphere. The preparation method comprises the following steps: (1) modifying sodium alginate polypeptide; (2) preparing a sodium alginate-rich phase solution and a casein-rich sodium phase solution; (3) preparing sodium alginate / sodium caseinate composite microspheres by electrostatic spraying; (4) removing a sodium caseinate phase; and (5) in-situ mineralization. The microsphere prepared by the invention has injectability, a micron-sized pore structure penetrating capability, a cell collection capability and an osseointegration capability, and can effectively promote repair and regeneration of alveolar bone defects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and tissue engineering, and relates to a porous microsphere for alveolar socket filling and its preparation method. Background Technology

[0002] Alveolar bone resorption after tooth extraction is a common and challenging problem in the field of dentistry. After tooth extraction, due to the loss of periodontal ligament and physiological occlusal stimulation, the remaining alveolar ridge undergoes irreversible progressive atrophy. Studies have shown that approximately 70% to 80% of bone resorption occurs within the first three months after extraction, resulting in a significant loss of alveolar ridge width and height, which seriously affects the long-term stability and final outcome of subsequent prosthetic restorations and implant treatments.

[0003] Currently, bone graft materials (such as bone powder) are commonly used in clinical practice to fill alveolar sockets in order to slow down bone resorption and promote bone tissue regeneration. However, traditional bone powder materials still have some shortcomings in application: they lack active cell recruitment capabilities, have limited osteogenic induction activity, and their degradation rate often does not match that of new bone formation. These problems limit their restorative effects in clinical practice.

[0004] With the interdisciplinary integration of tissue engineering and biomaterials, the development of novel alveolar socket filling materials that combine good biocompatibility, injectability, and biological activity has become a research hotspot. However, many of the materials reported so far still have certain functional limitations. For example, collagen sponges have excellent biocompatibility and hemostatic properties, but they are difficult to conform to complex alveolar socket morphologies and lack osteogenic activity; ceramic materials such as hydroxyapatite and β-tricalcium phosphate, although possessing osteoconductive properties, generally suffer from slow degradation, high brittleness, and insufficient guidance for cell adhesion; injectable materials such as calcium sulfate can initially adapt to alveolar socket morphology, but after curing, they form a dense structure that cannot provide the necessary three-dimensional interconnected pores for cell migration and tissue ingrowth. Overall, most of the above materials have limited functionality, lacking specific biological signals to actively recruit endogenous stem cells, and are also difficult to achieve precise filling and osteogenic microenvironment construction in irregular extraction sockets. Therefore, developing a novel alveolar socket filling material that can simultaneously achieve active cell recruitment, precise morphological adaptation, and efficient bone activation is of great significance for improving the preservation and regeneration of alveolar bone. Summary of the Invention

[0005] To address the aforementioned challenges, this invention proposes a porous microsphere for alveolar socket filling and its preparation method, aiming to meet the needs of irregular alveolar socket morphology and promote stem cell homing and osteogenic differentiation, providing a new treatment strategy for clinically solving the problems of alveolar ridge resorption and bone regeneration.

[0006] Firstly, the preparation method of porous microspheres with both bone regeneration and osteodegeneration / osteogenesis functions includes the following steps: (1) Modification of sodium alginate with peptides: short peptides were grafted onto the surface of sodium alginate to prepare modified sodium alginate; (2) Preparation of sodium alginate-rich phase solution and sodium caseinate-rich phase solution: A two-phase system is constructed using the modified sodium alginate and sodium caseinate, and the two-phase system is separated to obtain sodium alginate-rich phase solution and sodium caseinate-rich phase solution. (3) Electrostatic spraying preparation of sodium alginate / sodium caseinate composite microspheres: a mixture of sodium alginate-rich solution and sodium caseinate-rich solution is electrostatically sprayed to form droplets, and the droplets are cross-linked and solidified to form sodium alginate / sodium caseinate composite microspheres. (4) Removal of sodium caseinate phase: Soak the sodium alginate / sodium caseinate composite microspheres in water to dissolve sodium caseinate and form porous microspheres; (5) In-situ mineralization: The porous microspheres are placed in a mineralization solution and in-situ mineralization is carried out to obtain porous microspheres with both bone regeneration and osteodegeneration-osteogenesis functions.

[0007] In an optional embodiment, the short peptide is an arginine-glycine-aspartic acid tripeptide; preferably, the grafting molar ratio of the arginine-glycine-aspartic acid tripeptide to the sodium alginate monosaccharide unit is 1:(50~1000).

[0008] In an optional embodiment, the mass-volume concentration of sodium alginate in the aqueous two-phase system is 15-30 mg / mL, and the mass-volume concentration of sodium caseinate is 20-50 mg / mL.

[0009] In an optional embodiment, the pore size of the porous microspheres is controlled by adjusting the volume ratio of the sodium alginate-rich phase solution to the sodium caseinate-rich phase solution.

[0010] In an optional embodiment, the volume ratio of the sodium alginate-rich phase solution to the sodium caseinate-rich phase solution is 10:90 to 90:10, preferably 50:50 to 90:10.

[0011] In an optional embodiment, short peptides are grafted onto sodium alginate molecular chains via a carbodiimide chemical method. Preferably, sodium alginate is dissolved in a 2-morpholine ethanesulfonic acid buffer solution with a pH of 5.5-6.5, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are added sequentially for carboxyl activation. After activation, an aqueous solution of the short peptide is added, and the reaction is allowed to proceed completely at room temperature. The reaction solution is then dialyzed and freeze-dried to obtain short peptide-modified sodium alginate. More preferably, the molar ratio of sodium alginate, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide is 1:(0.2-2):(0.1-1). Further preferably, the reaction temperature is room temperature, and the reaction time is 12-24 hours.

[0012] In an optional embodiment, the crosslinking agent used for crosslinking comprises a polyvalent metal cation; preferably, the crosslinking agent is selected from one or more of calcium chloride, barium chloride, and zinc chloride, or a mixture thereof.

[0013] In an optional embodiment, the process parameters for electrostatic spraying are: voltage 4~10 kV, receiving distance 5~15 cm, solution propulsion rate 0.1~1.0 mL / h, and needle inner diameter 20~26 G.

[0014] In an optional embodiment, the mineralization solution is selected from at least one of simulated body fluid, calcium-phosphorus alternating mineralization solution, and supersaturated calcium phosphate solution; preferably, the pH of the mineralization solution is 7-7.5; more preferably, the reaction temperature of in-situ mineralization is 30-40°C, and the reaction time is 1-14 days.

[0015] Secondly, the present invention provides the application of porous microspheres with both bone regeneration and osteodegeneration / osteogenesis functions obtained by the preparation method described above in the preparation of dental restorative materials.

[0016] The present invention has the following beneficial effects: To address the shortcomings of existing alveolar socket filling materials, such as weak cell recruitment capacity, low osteogenic induction activity, and poor morphological adaptability, this invention provides a porous microsphere for alveolar socket filling and its preparation method. This method aims to accommodate irregular alveolar socket morphologies and promote stem cell homing and osteogenic differentiation, offering a new treatment strategy for clinically addressing alveolar ridge resorption and bone regeneration challenges. The microspheres prepared by this invention possess injectability, a permeable micron-sized pore structure, cell recruitment capabilities, and osteointegration capacity, effectively promoting the repair and regeneration of alveolar bone defects. Attached Figure Description

[0017] Figure 1 This is a flowchart illustrating the preparation process of the injectable self-mineralized porous microspheres (pAlg-R / P) of the present invention.

[0018] Figure 2 These are dark field microscopy and scanning electron microscopy (SEM) images of porous microspheres.

[0019] Figure 3 The pore size parameters are obtained by mercury intrusion porosimetry of the microspheres. Among them, (a) the cumulative pore area-pore size distribution curve of the porous microspheres; (b) the differential mercury intrusion curve of the porous microspheres, reflecting their pore size distribution characteristics; and (c) the key pore structure parameters calculated from the mercury intrusion data, including the total mercury intrusion volume, median pore size (volume basis) and porosity.

[0020] Figure 4The X-ray diffraction (XRD) pattern of the self-mineralized porous microspheres, compared with unmineralized microspheres and commercial bone powder, proves that it forms a bone-like apatite layer with low crystallinity.

[0021] Figure 5 The results show the cell culture results after 7 days on the materials of Example 1 (Alg, non-porous), Example 3 (pAlg, porous), Example 4 (pAlg-R, porous and RGD modified), Example 5 (pAlg-P, porous and mineralized), and Example 6 (pAlg-R / P, porous, RGD modified and mineralized). Among them, (a) results of cell liveness and death fluorescence staining; (b) cell proliferation activity detected by MTT assay (scale bar: 200 µm).

[0022] Figure 6 The alkaline phosphatase (ALP) staining results (scale bar: 50 µm) are shown after 7 days of cell culture on the materials of Example 1 (Alg, non-porous), Example 3 (pAlg, porous), Example 4 (pAlg-R, porous and RGD modified), Example 5 (pAlg-P, porous and mineralized), and Example 6 (pAlg-R / P, porous, RGD modified and mineralized).

[0023] Figure 7 The results of Alizarin Red S (ARS) staining (scale bar: 50 µm) are shown after 7 days of cell culture on materials from Examples 1 (Alg, non-porous), 3 (pAlg, porous), 4 (pAlg-R, porous and RGD modified), 5 (pAlg-P, porous and mineralized), and 6 (pAlg-R / P, porous, RGD modified and mineralized).

[0024] Figure 8 Animal experiment results: (a) Comparison of three-dimensional reconstructed images of alveolar bone of rats in the experimental group (implanted with the self-mineralized porous microspheres of this invention) and the control group (implanted with commercial bone powder) at 1 week and 3 weeks after surgery (scale bar: 1 cm); (b) Quantitative statistical analysis of new bone volume fraction (BV / TV). Detailed Implementation

[0025] The present invention is further illustrated by the following embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the present invention. The following exemplarily illustrates the method for preparing porous microspheres for alveolar socket filling.

[0026] Sodium alginate peptide modification. Modified sodium alginate is prepared by grafting short peptides onto the surface of sodium alginate. Sodium alginate is peptide-modified to enhance its cell adhesion and recruitment capabilities. In an optional embodiment, arginine-glycine-aspartic acid (RGD) tripeptides are grafted onto sodium alginate via a carbodiimide chemical method to prepare modified sodium alginate with cell adhesion function.

[0027] Among many short peptides, arginine-glycine-aspartic acid (RGD) tripeptide is preferred for grafting sodium alginate. As a tripeptide composed of only three amino acids, RGD has a small molecular weight and simple structure. This has a huge advantage in chemical modification: (1) less steric hindrance. It is easier to graft onto the molecular chain of sodium alginate via the carbodiimide method. (2) less impact on biological activity. The conformation and activity of short peptides are less likely to be lost due to steric or chemical factors during and after grafting. In contrast, long-chain polypeptides or whole proteins (such as fibronectin) are more likely to denature and become inactive during modification. (3) high grafting density. Due to its small molecular size, a higher grafting density can be achieved on a unit area of ​​sodium alginate material, thereby providing more binding sites for cells. In addition, it has extremely high broad-spectrum and effectiveness. The RGD sequence can be recognized by integrins of various cells, including fibroblasts, osteoblasts, endothelial cells, chondrocytes, etc. However, other cell adhesion peptides (such as YIGSR or IKVAV sequences derived from laminin) generally exhibit greater specificity. For example, IKVAV tends to promote the adhesion and differentiation of neuronal cells. RGD's broad-spectrum nature, on the other hand, allows it to recruit and adhere to a variety of desired cell types, which is crucial for biomaterials used to repair different tissues.

[0028] Preferably, the grafting molar ratio of RGD peptide to sodium alginate monosaccharide units is 1:(50~1000). Controlling the grafting molar ratio within this range means that on average, one RGD peptide molecule is grafted onto every 50 to 1000 sodium alginate monosaccharide units. Different cell types and tissue engineering applications require adhesion signals of varying strengths. The lower limit (1:1000) is considered the “minimum effective dose” for initiating effective cell adhesion. Below this ratio, RGD sites may be too sparse to effectively overcome the anti-adhesion properties of sodium alginate, leading to cell adhesion failure. The upper limit (1:50) is close to the level of saturated grafting. Further increasing the RGD ratio will significantly alter the charge, hydrophobicity, and interchain interactions of sodium alginate molecules, thereby affecting their gelling properties, mechanical strength, and degradation rate. Excessively high grafting ratios after achieving the desired biological effect will result in unnecessary cost waste. Therefore, this range is suitable for osteoblasts requiring rapid and strong adhesion, or specific cells requiring moderate adhesion to facilitate migration. RGD peptide modification of sodium alginate introduces cell surface integrin recognition sites, significantly enhancing the cell adhesion function of the sodium alginate matrix. The modified microspheres efficiently recruit endogenous stem cells and promote initial cell adhesion, full spread, and subsequent proliferation, laying a crucial cellular foundation for bone tissue regeneration.

[0029] The inventors recognized that a high density of cell recognition signals is required on the material surface to achieve effective adhesion and recruitment of BMSCs. Given that unmodified sodium alginate itself lacks cell-specific recognition sites, this background must be covered by a high grafting ratio to impart strong biological activity. Therefore, in embodiments of the invention aimed at providing the highest level of cell activity, a high molar ratio close to saturation grafting, such as 1:50, is preferred. Those skilled in the art will understand that, based on the preparation method disclosed in this invention, it is feasible to adjust the ratio within the range of 1:50 to 1:1000 to obtain different levels of cell activity. However, to demonstrate the upper limit of the effect achievable by the invention in enhancing cell behavior, the examples highlight the superior performance achieved using a high ratio of 1:50.

[0030] As an example, the method for grafting RGD peptides onto modified sodium alginate includes: dissolving sodium alginate (Alg) in a 2-morpholine ethanesulfonic acid (MES) buffer solution at pH 5.5-6.5; sequentially adding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) for carboxyl activation; after activation, adding an aqueous solution of RGD peptides; reacting at room temperature; dialyzing the reaction solution; and freeze-drying to obtain RGD-modified sodium alginate. The molar ratio of Alg, EDC, and NHS can be 1:(0.2-2):(0.1-1). The reaction temperature can be room temperature, and the reaction time can be 12-24 hours. The dialyzing time of the reaction solution can be 3-7 days.

[0031] Prepare sodium alginate-rich phase solutions and sodium caseinate-rich phase solutions. In an optional embodiment, construct a modified sodium alginate and sodium caseinate aqueous two-phase system, and separate the sodium alginate-rich phase solution and sodium caseinate-rich phase solution by centrifugation. Use non-toxic, harmless, environmentally friendly, and aqueously separated polysaccharide (sodium alginate) and protein (sodium caseinate). Sodium alginate is a natural polysaccharide, widely available, low-cost, and has good biocompatibility, without causing immune responses or cytotoxicity; sodium caseinate is derived from milk, a natural protein that is readily available and inexpensive.

[0032] In the aqueous two-phase system, the mass-volume concentration of sodium alginate can be 15~30 mg / mL, and the mass-volume concentration of sodium caseinate can be 20~50 mg / mL.

[0033] The separation of the aqueous two-phase system was carried out at room temperature. The sodium alginate-rich phase and the sodium caseinate-rich phase could be separated by centrifugation. For example, a centrifugal force of 10,000 × g and a centrifugation time of 3 hours could be used.

[0034] Electrostatic spraying was used to prepare sodium alginate / sodium caseinate composite microspheres. For example, a mixture of sodium alginate-rich solution and sodium caseinate-rich solution was electrostatically sprayed to form droplets, and the droplets were then cross-linked and solidified to form sodium alginate / sodium caseinate composite microspheres.

[0035] The pore size of porous microspheres can be controlled by adjusting the volume ratio of the sodium alginate-rich phase solution to the sodium caseinate-rich phase solution. In an optional embodiment, the pore size of the obtained porous microspheres can be controlled by adjusting the volume ratio of the sodium alginate-rich phase to the sodium caseinate-rich phase to 50:50~90:10 (e.g., 90:10, 70:30, 50:50).

[0036] As an example, but not limited to, the process parameters of the electrostatic spraying include: voltage 4~10 kV, receiving distance 5~15 cm, solution propulsion rate 0.1~1.0 mL / h, and needle inner diameter 20~26 G.

[0037] The mixture of sodium alginate-rich solution and sodium caseinate-rich solution can form hydrogel microspheres through ionic cross-linking with polyvalent metal cations. Suitable cross-linking agents include, but are not limited to, calcium chloride (CaCl2), barium chloride (BaCl2), and zinc chloride (ZnCl2). The concentration of the cross-linking agent aqueous solution can be 100~500 mM.

[0038] Remove the sodium caseinate phase. The sodium alginate / sodium caseinate composite microspheres are soaked in water to dissolve the sodium caseinate, forming porous microspheres. For example, microspheres obtained by electrostatic spraying are placed in deionized water for 5-9 days to dissolve the sodium caseinate phase, thereby obtaining porous microspheres.

[0039] This invention utilizes a sodium alginate / sodium caseinate aqueous two-phase system combined with electrostatic spraying to construct porous microspheres. The preparation of porous structures based on an aqueous two-phase system is a simple and mild process requiring no complex post-treatment. The preparation process is carried out in aqueous solution, avoiding organic solvents or harsh chemical conditions, thus preventing damage to cells and environmental pollution from solvent residues, conforming to green chemistry principles. The method is simple to operate, achieving pore formation through a single crosslinking and elution step, requiring no complex equipment, and is easily reproducible and scalable for large-scale production.

[0040] Aqueous two-phase technology provides controllable pore structures for materials. By adjusting key parameters such as the volume ratio of the two phases and polymer concentration, the pore size, porosity, and pore connectivity of the hydrogel can be precisely controlled, thereby customizing the most suitable growth microenvironment for different types of cells. The resulting three-dimensional interconnected macroporous structure not only effectively promotes nutrient transport, cell migration and spatial distribution, but also facilitates the ingrowth of host blood vessels and nerves, significantly enhancing the biological function of the scaffold. Furthermore, it possesses high molding flexibility, enabling the fabrication of both uniformly shaped porous microspheres and the construction of porous hydrogel scaffolds with various complex shapes, demonstrating broad application prospects in cutting-edge fields such as cell therapy, tissue engineering, and in vitro disease models.

[0041] In-situ mineralization. The porous microspheres are placed in a mineralization solution and subjected to in-situ mineralization to obtain porous microspheres with both bone regeneration and osteodegeneration / osteogenesis functions.

[0042] The in-situ mineralization method includes placing the porous microspheres in a mineralization solution. The mineralization solution is selected from at least one of simulated body fluid, an alternating calcium-phosphorus mineralization solution, and a supersaturated calcium phosphate solution. The pH value of the mineralization solution can be 7.4. In-situ mineralization forms a bone-like apatite layer on the surface and within the pores of the microspheres. The mineralization reaction temperature can be 37°C, and the mineralization reaction time can be 1–14 days. In-situ mineralization constructs a bone-like apatite layer on the surface of the microspheres, enhancing osteogenic capacity.

[0043] During the development of the mineralization process, the inventors systematically adjusted and optimized the mineralization conditions to determine the optimal process parameters, ensuring the integrity of the microsphere structure and the mineralization effect. Alternating calcium and phosphorus mineralization is a key step. In an optional embodiment, calcium (Ca) in the mineralization solution... 2+ ) and phosphorus (PO4) 3- The molar ratio of calcium (Ca) in the mineralizing solution needs to be maintained at approximately 1.6–1.7. This clarifies the concentration of calcium (Ca) in the mineralizing solution. 2+ ) and phosphorus (PO4) 3- The molar ratio of disodium hydrogen phosphate (Na₂HPO₄) needs to be maintained at approximately 1.67 to simulate the stoichiometry of hydroxyapatite and promote the effective deposition of bone-like apatite. However, initial experiments revealed that if the concentration of disodium hydrogen phosphate (Na₂HPO₄) is too high (e.g., significantly exceeding 10 mM), its phosphate ions will competitively bind with calcium ions in the calcium alginate microspheres, leading to partial "deprivation" of calcium from the microsphere network. This, in turn, causes the microsphere structure to soften, break, or even disintegrate, making it impossible to maintain a complete spherical morphology. Therefore, the concentration of disodium hydrogen phosphate (Na₂HPO₄) is preferably below 10 mM.

[0044] Based on the above observations, the optimal mineralization solution concentration was determined through repeated experiments: a 16.7 mM calcium chloride (CaCl2) solution and a 10 mM disodium hydrogen phosphate (Na2HPO4) solution. This ratio maintains a suitable calcium-to-phosphorus ratio while avoiding excessive erosion of the microsphere matrix. Simultaneously, the single mineralization soaking time was also investigated. It was found that if the soaking time exceeded 30 minutes, the microspheres would swell significantly, resulting in a decrease in morphological integrity. Therefore, the soaking time for each session was limited to within 30 minutes (e.g., 30 minutes). In an optional implementation, the number of cycles is 2 to 4 (preferably 3).

[0045] As an example, the solution is immersed in 16-17 mM calcium chloride solution (5-30 minutes) and 5-15 mM disodium hydrogen phosphate solution (5-30 minutes) in a cycle 2-5 times.

[0046] In addition, other mineralization systems were explored as alternatives or supplementary solutions. For example, slow mineralization can be achieved using simulated body fluid (SBF), by soaking the microspheres at physiological temperature (37°C) and pH conditions for 14–21 days, allowing calcium and phosphorus minerals to gradually deposit on the surface and within the pores of the microspheres. This method has a slower mineralization rate, but the resulting mineral layer composition and structure are closer to natural bone, making it suitable for applications requiring higher bioactivity of the mineralized material. In summary, the current calcium-phosphorus alternating mineralization process parameters (concentration, time, number of cycles) were determined after multiple rounds of experimental optimization, enabling effective mineralization while maximizing the morphological integrity and structural stability of the porous microspheres.

[0047] In-situ mineralization constructed a bone-like apatite layer inside and on the surface of the microspheres. This mineralized layer has low crystallinity, and its degradation rate is more closely matched to the new bone formation process; while commercial bone powders (such as Bio-Oss) ® High crystallinity and slow degradation can hinder complete bone replacement if left for extended periods. Furthermore, the microspheres of this invention continuously release osteogenic calcium and phosphorus ions during degradation, actively promoting osteogenic differentiation and achieving a functional leap from a "passive spatial scaffold" to "active bio-induction." In contrast, commercial bone powder primarily provides physical support with weak osteoinductive capabilities. These characteristics give the material of this invention significant advantages in bioactivity and degradability compared to traditional commercial bone powder, potentially enabling faster bone defect repair. In an optional embodiment, the porous microspheres have a pore size of 10–200 µm and a volumetric porosity of 50%–80%.

[0048] In summary, this invention relates to a porous microsphere with green, safe, and highly biocompatible raw materials and its preparation method. The method involves grafting an arginine-glycine-aspartic acid (RGD) tripeptide onto sodium alginate via a carbodiimide chemical method to prepare modified sodium alginate with cell adhesion function. Then, a two-phase aqueous system is constructed using the modified sodium alginate and sodium caseinate, and sodium alginate microspheres are prepared by electrostatic spraying. The microspheres are washed to dissolve the sodium caseinate phase, yielding porous microspheres. Finally, the porous microspheres are subjected to in-situ mineralization. The porous microspheres obtained by this method can be used for alveolar socket filling.

[0049] The following examples further illustrate the present invention in detail. It should also be understood that the following examples are only for further explanation of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are within the scope of protection of the present invention. The specific process parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make appropriate selections within the appropriate range based on the description herein, and are not intended to be limited to the specific values ​​in the examples below.

[0050] Example 1: Preparation of 1.5% Alg microspheres (non-porous) (1) Weigh 300 mg of sodium alginate and dissolve it in 20 mL of deionized water. Stir the solution at 500 rpm for 12 hours at room temperature using a magnetic stirrer to obtain an Alg solution.

[0051] (2) An electrostatic spraying device was used, with a voltage of 5 kV, a propulsion rate of 0.1 mL / h, a needle of 22 G, and a receiving distance of 8 cm. A 500 mM calcium chloride solution was used as the receiving bath to crosslink and solidify Alg microspheres.

[0052] Example 2: Preparation of porous microspheres with an Alg to Cas volume ratio of 9:1 (1) Weigh 300 mg of sodium alginate and 1.0 g of sodium caseinate (Cas), and dissolve them together in 20 mL of deionized water. Stir with a magnetic stirrer at 500 rpm for 12 hours at room temperature to obtain a homogeneous Alg / Cas aqueous two-phase solution.

[0053] (2) Transfer the above solution to a centrifuge tube and centrifuge at 10,000×g for 3 hours at room temperature. After centrifugation, the system clearly separates into layers. Collect the upper sodium alginate-rich phase (Alg-rich) and the lower sodium caseinate-rich phase (Cas-rich), and store at 4℃ or use immediately.

[0054] (3) Measure 9 mL of Alg-rich phase and 1 mL of Cas-rich phase at a volume ratio of 9:1, and mix them evenly to prepare an aqueous two-phase solution. Use an electrostatic spray device with a voltage of 5 kV, a propulsion rate of 0.1 mL / h, a needle of 22 G, and a receiving distance of 8 cm. Use 500 mM calcium chloride solution as the receiving bath to crosslink and solidify to form Alg / Cas composite microspheres.

[0055] (4) The composite microspheres were soaked in deionized water for 9 days to dissolve sodium caseinate and form a porous structure. Subsequently, calcium-phosphorus alternating mineralization was performed, i.e., the microspheres were successively soaked in 16.7 mM calcium chloride solution (30 min) and 10 mM disodium hydrogen phosphate solution (30 min) three times. After mineralization, the microspheres were washed with deionized water and freeze-dried to obtain the final product (pAlg-1).

[0056] Example 3: Preparation of porous microspheres with an Alg to Cas volume ratio of 7:3 (1) Weigh 300 mg of sodium alginate and 1.0 g of sodium caseinate and dissolve them together in 20 mL of deionized water. Stir with a magnetic stirrer at 500 rpm for 12 hours at room temperature to obtain a homogeneous Alg / Cas aqueous two-phase solution.

[0057] (2) Transfer the above solution to a centrifuge tube and centrifuge at 10,000×g for 3 hours at room temperature. After centrifugation, the system clearly separates into layers. Collect the upper sodium alginate-rich phase (Alg-rich) and the lower sodium caseinate-rich phase (Cas-rich), and store at 4℃ or use immediately.

[0058] (3) 7 mL of Alg-rich phase and 3 mL of Cas-rich phase were measured at a volume ratio of 7:3 and mixed evenly to prepare an aqueous two-phase solution. An electrostatic spraying device was used, with a voltage of 5 kV, a propulsion rate of 0.1 mL / h, a needle of 22 G, and a receiving distance of 8 cm. A 500 mM calcium chloride solution was used as the receiving bath to crosslink and solidify the Alg / Cas composite microspheres.

[0059] (4) The composite microspheres were soaked in deionized water for 9 days to dissolve sodium caseinate and form a porous structure. Subsequently, calcium-phosphorus alternating mineralization was performed, that is, the microspheres were soaked in 16.7 mM calcium chloride solution (30 min) and 10 mM disodium hydrogen phosphate solution (30 min) three times in succession. After mineralization, the microspheres were washed with deionized water and freeze-dried to obtain the final product (pAlg-2).

[0060] In optimizing the volume ratio of sodium alginate-rich phase to sodium caseinate-rich phase, the inventors discovered that the concentration of sodium alginate is the decisive factor in maintaining a stable gel structure formed through calcium ion crosslinking. Specifically, when the volume ratio of sodium alginate-rich phase to sodium caseinate-rich phase is too low, the sodium alginate content in the mixed system is insufficient to form a complete three-dimensional network structure, resulting in failure to form during calcium ion crosslinking and thus failing to obtain a stable gel material with practical value. Based on this discovery, the present invention excludes volume ratios less than 50:50 (e.g., 30:70, 10:90). The volume ratio of the sodium alginate-rich phase solution to the sodium caseinate-rich phase solution must be higher than 50:50, preferably 70:30, 90:10, or any ratio between them, to ensure the acquisition of biomaterials with the desired structure and properties.

[0061] Figure 2 The results showed that all samples exhibited a regular spherical morphology. Their microstructure was observed by scanning electron microscopy. Alg microspheres were a continuous, uniform, and dense gel structure with no obvious visible pores. pAlg-1 and pAlg-2 had a porous structure with a wider pore size range and a denser pore distribution. Moreover, the pores were interconnected, forming a three-dimensional interconnected micron-scale pore system.

[0062] Figure 3 The results show that the pore size and porosity of porous microspheres can be effectively controlled by adjusting the proportion of Cas in the aqueous two-phase system. With increasing Cas proportion, both the median pore size and total porosity of the microspheres show a significant upward trend. (Cumulative mercury ingress curve) Figure 3 a) This shows that mercury first enters the largest accessible pores in the material. For pAlg-2, the curve rises sharply in the 10–100 µm pore size range and peaks at 0.2 cm. 2 g -1 The plateau at this point indicates that its pores are primarily composed of interconnected macropores. In contrast, the cumulative mercury ingress of Alg and pAlg-1 is lower than that of pAlg-2, with a final total mercury ingress of 0.038 cm⁻¹. 2 g -1 and 0.035 cm 2 g -1 This indicates that both types of cells are predominantly composed of smaller pores, resulting in a low overall porosity. Based on the pore structure parameters calculated from the cumulative mercury ingress data, pAlg-2 exhibits a porosity (φ) of 68.43% and a median pore size (d). 50 The value is 199 µm; pAlg-1 is φ = 58.50%, d 50 = 176 µm; while Alg has the lowest corresponding values, φ = 42.11% and d = 176 µm. 50 = 129.8 µm (porosity φ is calculated as the ratio of total mercury ingress volume to apparent sample volume, median pore diameter d) 50 (Taken from the pore size value corresponding to a cumulative mercury ingress of 50%).

[0063] The corresponding differential pore size distribution diagram ( Figure 3 (b) Further analysis revealed the differences in pore size distribution among the three. pAlg-2 exhibited a significant and sharp macropore peak at 225.49 µm, confirming the successful construction of a macroporous structure; pAlg-1 showed a macropore peak at 180.62 µm, with a smaller pore size than pAlg-2; while Alg showed a micropore peak near 144.26 µm. Combined with the cumulative mercury ingress curves, it can be seen that pAlg-2 has higher porosity and larger pore size than pAlg-1 and Alg, indicating that the pore structure of porous microspheres can be effectively controlled by adjusting the proportion of Cas in the aqueous two-phase system.

[0064] Example 4: Preparation of RGD-modified calcium alginate porous microspheres (1) Dissolve 1g of sodium alginate in MES buffer at pH 6.0, add EDC and NHS (molar ratio of sodium alginate carboxyl group: EDC: NHS = 1:1:0.5) and activate for 30 minutes. Add 40 mg of RGD peptide (grafting molar ratio 1:50) and react for 12 hours. Dialyze and lyophilize to obtain modified sodium alginate (Alg-RGD).

[0065] (2) Weigh 300 mg of modified sodium alginate and 1.0 g of sodium caseinate, and dissolve them together in 20 mL of deionized water. Stir with a magnetic stirrer at 500 rpm for 12 hours at room temperature to obtain a homogeneous Alg / Cas aqueous two-phase solution.

[0066] (3) Transfer the above solution to a centrifuge tube and centrifuge at 10,000×g for 3 hours at room temperature. After centrifugation, the system will separate into layers. Collect the upper sodium alginate-rich phase (Alg-rich) and the lower sodium caseinate-rich phase (Cas-rich), and store at 4℃ or use immediately.

[0067] (4) Measure 7 mL of Alg-rich phase and 3 mL of Cas-rich phase at a volume ratio of 7:3, and mix them evenly to prepare an aqueous two-phase solution. Use an electrostatic spray device with a voltage of 5 kV, a propulsion rate of 0.1 mL / h, a needle of 22 G, and a receiving distance of 8 cm. Use 500 mM calcium chloride solution as the receiving bath to crosslink and solidify to form Alg / Cas composite microspheres.

[0068] (5) The composite microspheres were soaked in deionized water for 9 days to dissolve sodium caseinate and form a porous structure (pAlg-R).

[0069] Example 5: Preparation of self-mineralized porous microspheres (1) Weigh 300 mg of sodium alginate and 1.0 g of sodium caseinate and dissolve them together in 20 mL of deionized water. Stir with a magnetic stirrer at 500 rpm for 12 hours at room temperature to obtain a homogeneous Alg / Cas aqueous two-phase solution.

[0070] (2) Transfer the above solution to a centrifuge tube and centrifuge at 10,000×g for 3 hours at room temperature. After centrifugation, the system clearly separates into layers. Collect the upper sodium alginate-rich phase (Alg-rich) and the lower sodium caseinate-rich phase (Cas-rich), and store at 4℃ or use immediately.

[0071] (3) 7 mL of Alg-rich phase and 3 mL of Cas-rich phase were measured at a volume ratio of 7:3 and mixed evenly to prepare an aqueous two-phase solution. An electrostatic spraying device was used, with a voltage of 5 kV, a propulsion rate of 0.1 mL / h, a needle of 22 G, and a receiving distance of 8 cm. A 500 mM calcium chloride solution was used as the receiving bath to crosslink and solidify the Alg / Cas composite microspheres.

[0072] (4) The composite microspheres were soaked in deionized water for 9 days to dissolve sodium caseinate and form a porous structure (pAlg).

[0073] (5) Subsequently, calcium-phosphorus alternating mineralization was performed, that is, the product was successively immersed in 16.7 mM calcium chloride solution (30 minutes) and 10 mM disodium hydrogen phosphate solution (30 minutes) three times. After mineralization, the product was washed with deionized water and freeze-dried to obtain the final product (pAlg-P).

[0074] Example 6: Preparation of RGD-modified self-mineralized calcium alginate porous microspheres (1) Dissolve 1g of sodium alginate in MES buffer at pH 6.0, add EDC and NHS (sodium alginate carboxyl: EDC: NHS molar ratio = 1:1:0.5) and activate for 30 minutes. Add 40 mg of RGD peptide (grafting molar ratio 1:50) and react for 12 hours. Dialyze and lyophilize to obtain modified sodium alginate (Alg-RGD).

[0075] (2) Weigh 300 mg of modified sodium alginate and 1.0 g of sodium caseinate, and dissolve them together in 20 mL of deionized water. Stir with a magnetic stirrer at 500 rpm for 12 hours at room temperature to obtain a homogeneous Alg / Cas aqueous two-phase solution.

[0076] (3) Transfer the above solution to a centrifuge tube and centrifuge at 10,000×g for 3 hours at room temperature. After centrifugation, the system clearly separates into layers. Collect the upper sodium alginate-rich phase (Alg-rich) and the lower sodium caseinate-rich phase (Cas-rich), and store at 4℃ or use immediately.

[0077] (4) Measure 7 mL of Alg-rich phase and 3 mL of Cas-rich phase at a volume ratio of 7:3, and mix them evenly to prepare an aqueous two-phase solution. Use an electrostatic spray device with a voltage of 5 kV, a propulsion rate of 0.1 mL / h, a needle of 22 G, and a receiving distance of 8 cm. Use 500 mM calcium chloride solution as the receiving bath to crosslink and solidify to form Alg / Cas composite microspheres.

[0078] (5) The composite microspheres were soaked in deionized water for 9 days to dissolve sodium caseinate and form a porous structure (pAlg-R).

[0079] (6) Subsequently, calcium-phosphorus alternating mineralization was performed, that is, the product was successively immersed in 16.7 mM calcium chloride solution (30 min) and 10 mM disodium hydrogen phosphate solution (30 min) three times. After mineralization, the product was washed with deionized water and freeze-dried to obtain the final product (pAlg-R / P).

[0080] The X-ray diffraction (XRD) analysis results in Figure 4 show that the self-mineralized porous microspheres prepared in this invention successfully formed a low-crystallinity bone-like apatite layer. The XRD pattern exhibits a broadened and low-intensity diffuse peak at 32.1° (2θ), and also a weak diffraction signal at 25.6°. This significantly broadened peak and low intensity indicate that the formed calcium phosphate mineral has low crystallinity, small crystal size, and contains more amorphous phases, consistent with the structural characteristics of low-crystallinity bone-like apatite formed during biomimetic mineralization. This is beneficial for its gradual degradation in vivo and participation in bone metabolism. In contrast, unmineralized microspheres did not show obvious apatite diffraction peaks, while commercial bone powder (Bio-Oss) showed... ® The microspheres exhibit sharp hydroxyapatite characteristic peaks, indicating high crystallinity. This structural feature allows the microspheres of this invention to exhibit more ideal degradation performance in vivo, with their degradation rate better matching the new bone formation process.

[0081] Example 7: Evaluation of the recruitment of bone marrow mesenchymal stem cells (BMSCs) by RGD-modified self-mineralized calcium alginate porous microspheres Porous microspheres from Examples 1 (Alg, non-porous), 3 (pAlg, porous), 4 (pAlg-R, porous and RGD modified), 5 (pAlg-P, porous and mineralized), and 6 (pAlg-R / P, porous, RGD modified and mineralized) were placed in 96-well plates, with 20 microspheres per well and seeded with 2 × 10⁶ microspheres. 4 BMSCs were cultured at 37°C and 5% CO2 for 7 days. Upon reaching the predetermined time point, the culture medium was discarded, and the cells were washed three times with PBS. Staining working solution was prepared according to the kit instructions, and 100 μL was added to each well. The cells were incubated at 37°C in the dark for 30 minutes. After staining, the cells were washed with PBS and observed immediately using a fluorescence microscope.

[0082] Figure 5 Fluorescent staining results showed that only a few cells attached to the Alg group, while the introduction of the porous structure (pAlg) resulted in a small number of cells infiltrating into the interior. This indicates that the interconnected pores play a positive role in promoting cell migration and spatial distribution, but the overall cell recruitment capacity remains weak. Modification of sodium alginate with RGD peptides (pAlg-R) significantly enhanced cell recruitment capacity, and dense green fluorescent signals were observed on the surface of the microspheres. This indicates that RGD effectively promotes cell adhesion. Furthermore, the introduction of calcium phosphate coatings (pAlg-P and pAlg-R / P groups) not only enhanced cell viability but also promoted the formation of cell aggregates between adjacent microspheres, demonstrating a synergistic enhancement of biological effects. These observations are corroborated by the cell proliferation activity data measured by the MTT assay. Figure 5(b) Cellular metabolic activity increased with prolonged culture time in all groups, with the pAlg-R / P group showing a higher metabolic level.

[0083] Figure 6 The results of cellular alkaline phosphatase (ALP) activity assays showed that all material groups exhibited ALP-positive staining after culturing in osteogenic induction medium. The porous microsphere groups (pAlg, pAlg-R, pAlg-P, and pAlg-R / P) showed stronger ALP expression than the Alg group, highlighting the crucial role of the porous structure in supporting osteogenic differentiation. Compared to pAlg, the ALP activity of the pAlg-R and pAlg-P groups was significantly enhanced, indicating that both the biochemical signaling (RGD) and the mineralized ion-mediated microenvironment (calcium phosphate coating) effectively promote early osteogenic differentiation. Importantly, the pAlg-R / P group exhibited the strongest ALP staining intensity. The synergistic enhancement observed in this group stemmed from the continuous release of calcium ions from its dense amorphous calcium phosphate coating, providing essential mineral precursors. Simultaneously, the RGD peptides ensured strong cell adhesion and activated integrin-mediated signaling pathways, thereby constructing an inducible microenvironment for initiating osteogenic differentiation.

[0084] Figure 7 Alizarin Red S (ARS) staining results showed significant differences in mineralization levels among the groups. The pAlg-R / P group exhibited the densest and deepest calcium nodule deposition, indicating the highest level of late-stage mineralization and extracellular matrix maturity. In contrast, the Alg and pAlg groups showed only sporadic and weak staining. The introduction of RGD modification (pAlg-R) or calcium-phosphorus coating (pAlg-P) alone significantly promoted mineral deposition, suggesting that each possesses a certain osteogenic induction capacity. These results indicate that while porous structures facilitate initial cell migration and distribution, they are insufficient to independently drive the complete osteogenic differentiation process. The superior performance of the pAlg-R / P group stems from the synergistic effect between the biochemical adhesion signals provided by RGD and the continuous release of mineral ions from the calcium-phosphorus coating. This system, by integrating cell recognition sites and a biomimetic mineralization microenvironment, constructs an osteogenic induction platform that effectively guides the orderly progression of cells from early adhesion and proliferation to the late-stage mineralization, providing a crucial material basis for achieving functional bone regeneration.

[0085] Example 8: Alveolar Bone Repair Experiment in Rats Eighteen Sprague-Dürer (SD) rats were randomly divided into three groups (n=6). The experimental group was implanted with the pAlg-R / P porous microspheres of this invention; the control group was implanted with commercially available bone powder (Bio-Oss). ® The control group healed naturally. Rats were anesthetized, and the maxillary first molar was extracted. After hemostasis, the experimental and control groups were implanted with the corresponding materials, while the control group was left unfilled. The wounds were sutured, and prophylactic antibiotics were administered postoperatively.

[0086] Animals were sacrificed on postoperative days 7 and 21 to obtain maxillary bone samples, which were fixed with 4% paraformaldehyde. Micro-computed tomography (Micro-CT) was performed on the samples, and the new bone volume fraction (BV / TV) was calculated.

[0087] The Micro-CT analysis in Figure 8 shows that, 21 days post-operation, the porous microsphere group exhibited the best new bone volume and bone density in the alveolar bone defect area, with osteogenic efficacy comparable to that of commercial Bio-Oss. ® The bone meal was comparable and significantly superior to the blank control group. The porous microspheres degraded synchronously during osteoogenesis, leaving no residue; while Bio-Oss... ® A large number of undegraded particles remained in the sample. This demonstrates that the microspheres of the present invention, while achieving equivalent bone regeneration, possess the unique advantages of degradation-osteogenetic matching and avoiding long-term retention, making them a more promising bioactive bone repair material for clinical applications.

[0088] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing porous microspheres having both bone regenerative and bone-osteogenic functions, characterized by, The preparation method comprises the following steps: (1) sodium alginate polypeptide modification: short peptides are grafted to the surface of sodium alginate to prepare modified sodium alginate; (2) preparation of sodium alginate-rich phase solution and sodium caseinate-rich phase solution: a two-water phase system is constructed by using modified sodium alginate and sodium caseinate, and the two-water phase system is separated to obtain a sodium alginate-rich phase solution and a sodium caseinate-rich solution; (3) electrostatic spraying preparation of sodium alginate / sodium caseinate composite microspheres: a mixture of the sodium alginate-rich phase solution and the sodium caseinate-rich solution is used to form droplets by electrostatic spraying, and the droplets are crosslinked and solidified to form sodium alginate / sodium caseinate composite microspheres; (4) removal of the sodium caseinate phase: the sodium alginate / sodium caseinate composite microspheres are soaked in water to dissolve the sodium caseinate and form microspheres with a porous structure; (5) in-situ mineralization: the microspheres with a porous structure are placed in a mineralization solution to obtain microspheres with bone regeneration and bone-osteogenesis functions through in-situ mineralization.

2. The production method according to claim 1, characterized by, The short peptides are arginine-glycine-aspartic acid tripeptides; preferably, the grafting molar ratio of arginine-glycine-aspartic acid tripeptides to sodium alginate monosaccharide units is 1: (50-1000).

3. The production method according to claim 1 or 2, characterized by, The mass-volume concentration of sodium alginate in the two-water phase system is 15-30 mg / mL, and the mass-volume concentration of sodium caseinate is 20-50 mg / mL.

4. The production method according to any one of claims 1 to 3, characterized by, The pore size of the porous microspheres is adjusted by adjusting the volume ratio of the sodium alginate-rich phase solution to the sodium caseinate-rich phase solution.

5. The production method according to any one of claims 1 to 4, characterized by, The volume ratio of the sodium alginate-rich phase solution to the sodium caseinate-rich phase solution is 10:90-90:10, preferably 50:50-90:

10.

6. The production method according to any one of claims 1 to 5, characterized by, The short peptides are grafted to the sodium alginate molecular chain by a carbodiimide chemical method; preferably, the sodium alginate is dissolved in a 2-morpholinoethanesulfonic acid buffer with a pH of 5.5-6.5, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide are sequentially added for carboxyl activation, after the activation is completed, a short peptide aqueous solution is added, and after the reaction is completed at room temperature, the reaction solution is subjected to dialysis and freeze-drying to obtain short peptide-modified sodium alginate; more preferably, the molar ratio of sodium alginate, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide is 1: (0.2-2) : (0.1-1); further preferably, the reaction temperature is room temperature, and the reaction time is 12-24 hours.

7. The production method according to any one of claims 1 to 6, characterized by, The crosslinking agent for crosslinking comprises a multivalent metal cation; preferably, the crosslinking agent is selected from a mixture of one or more of calcium chloride, barium chloride and zinc chloride.

8. The production method according to any one of claims 1 to 6, characterized by, The process parameters of the electrostatic spraying are: voltage 4-10 kV, receiving distance 5-15 cm, solution propelling rate 0.1-1.0 mL / h, and needle inner diameter 20-26 G.

9. The production method according to any one of claims 1 to 8, characterized by, The mineralization solution is selected from at least one of a simulated body fluid, a calcium-phosphorus alternating mineralization solution and a supersaturated calcium phosphate solution; preferably, the pH of the mineralization solution is 7-7.5; more preferably, the reaction temperature of the in-situ mineralization is 30-40℃, and the reaction time is 1-14 days.

10. Use of the porous microspheres with both bone regeneration and bone-osteogenesis reduction functions obtained by the preparation method according to any one of claims 1 to 9 in preparation of a dental repair material.

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