A method for preparing a chitosan hydrogel loaded with gingival mesenchymal stem cell-derived exosomes and the hydrogel

Abstract

The application relates to a preparation method of a chitosan hydrogel loaded with gingival mesenchymal stem cell-derived exosomes and the hydrogel, which comprises the following steps: collecting and culturing, extracting exosomes, configuring an exosome-chitosan precursor solution, then mixing a beta-GP aqueous solution with the exosome-chitosan mixed solution to prepare a primary gel, then treating the primary gel through multiple freeze-thaw cycle treatments to promote the formation of a dense physical crosslinking network of chitosan molecular chains, and simultaneously realizing the synergistic effect of ion crosslinking of beta-GP, finally placing the hydrogel in a body temperature environment for preservation, so that the structure of the hydrogel is further stabilized, and finally the chitosan hydrogel loaded with gingival mesenchymal stem cell-derived exosomes is obtained. The application not only avoids the defects of a complex chemical crosslinking process, high cost and toxicity residues, but also improves the loading efficiency of the hydrogel on the exosomes, realizes controllable slow release of active ingredients, avoids the problem of burst release, and further improves the binding stability and release controllability.

Description

Technical Field

[0001] This invention relates to a method for preparing a chitosan hydrogel loaded with exosomes derived from gingival mesenchymal stem cells, and to the hydrogel itself, which is applicable to the field of biomedical technology. Background Technology

[0002] Periodontitis is a common chronic infectious oral disease, mainly caused by the destruction of periodontal tissues by bacteria in dental plaque. This destruction affects the periodontal supporting tissues (including the gingiva, periodontal ligament, alveolar bone, and cementum), and in severe cases, it can lead to tooth loss. Current clinical treatment mainly involves mechanical debridement (scaling and root planing) combined with local antibiotic delivery. However, this approach can only control the progression of inflammation and cannot achieve functional regeneration of periodontal tissues. In novel biological therapy strategies, stem cell exosomes have become a research hotspot in periodontal regeneration therapy due to their anti-inflammatory, immunomodulatory, and osteogenic induction activities.

[0003] Currently, there are two main types of treatments for periodontitis using gingival mesenchymal stem cell-exosomes (GMSC-EXO). One method involves directly isolating and culturing gingival mesenchymal stem cells and extracting exosomes, which are then applied locally to the periodontitis lesions via injection or application. While this method delivers a relatively high drug concentration and is relatively convenient, exosomes, being nanovesicles, are easily degraded by enzymes and phagocytes in the body or lost through diffusion in body fluids, making it difficult for them to exert their effects at the lesion site for an extended period. Furthermore, free exosomes can easily diffuse into non-target tissues, leading to insufficient drug concentration in the target tissue and affecting anti-inflammatory and osteogenic effects.

[0004] Secondly, by using alginate, gelatin, or ordinary chitosan hydrogel as carriers, stem cell exosomes are loaded through physical mixing or simple cross-linking. This method can overcome the problems of poor targeting ability, easy loss, and short residence time that exist in direct injection or application. However, most existing hydrogels are designed for bone marrow mesenchymal stem cell exosomes (BMSC-EXO) or adipose-derived mesenchymal stem cell exosomes (ADSC-EXO), and have not been optimized for the physicochemical properties of GMSC-EXO, such as particle size and surface charge. As a result, their loading efficiency is low (usually below 50%). If a high concentration of carrier is used to pursue high loading, it will lead to a decrease in gel porosity, which will affect the exosome release efficiency. Furthermore, if the cross-linking between the existing carrier and the exosome is carried out by simple physical cross-linking methods such as embedding and electrostatic adsorption, it is difficult to ensure the binding stability and burst release problem is likely to occur. On the other hand, if chemical cross-linking is used to ensure binding stability and controllable release, there are problems such as complex processes, high labor and material costs in the preparation process, chemical toxicity that can destroy the exosome activity, and easy residue. Summary of the Invention

[0005] To address the shortcomings of the existing technology, this invention proposes a method for preparing a chitosan hydrogel loaded with exosomes derived from gingival mesenchymal stem cells, and the hydrogel itself. The technical solution adopted by this invention is as follows: On one hand, this invention provides a method for preparing a chitosan hydrogel loaded with exosomes derived from gingival mesenchymal stem cells, comprising:

[0006] S1. Collect and culture gingival mesenchymal stem cells, and extract exosomes from them to obtain exosome samples;

[0007] S2. Add chitosan to the acetic acid solution and stir to dissolve, to obtain the chitosan precursor solution;

[0008] S3. Mix the exosome sample with phosphate-buffered saline (PBS) to form an exosome solution, then add the exosome solution to the chitosan precursor solution and stir to mix to obtain an exosome-chitosan mixture.

[0009] S4. Slowly add the aqueous solution of sodium β-glycerophosphate (β-GP) dropwise to the exosome-chitosan mixture and stir magnetically to obtain the initial gel.

[0010] S5. Place the initial gel in a sterile mold and perform 2 to 4 physical cross-linking treatments. The physical cross-linking treatments include: first freezing at -25℃ to -15℃ for 10 to 14 hours, and then thawing at room temperature for 5 to 7 hours.

[0011] S6. The product obtained in step S5 is stored at a constant temperature of 37°C to obtain a chitosan hydrogel loaded with gingival mesenchymal stem cell exosomes.

[0012] In the above steps, the chitosan molecular chain contains amino groups, which can be protonated in acetic acid solution to form a positively charged acidic precursor solution. At the same time, the exosome sample (usually lyophilized powder) is added to PBS for reconstitution to restore the biological activity of the exosomes and to disperse them in the exosome solution. Then, the exosome solution is mixed with the chitosan precursor solution. The membrane surface of GMSC-EXO is rich in acidic lipids such as phosphatidylserine, which are negatively charged. After mixing, a stable "exosome-chitosan" complex can be formed through electrostatic adsorption, which improves the loading efficiency and also makes the exosomes uniformly dispersed in the precursor solution, avoiding uneven distribution or leakage. Then, by mixing β-GP with the "exosome-chitosan" complex, the thermosensitive gelation of chitosan is triggered to produce the characteristics of "low-temperature flow and body temperature gelation", which not only facilitates injection administration but also facilitates adhesion to lesions and avoids the diffusion and loss of exosomes. When added slowly under magnetic stirring, β-GP can be evenly dispersed in the mixture. During the gelation process, chitosan molecules slowly cross-link to form a three-dimensional network, embedding GMSC-EXO in the pores. However, this simple cross-linking has weak stability. When applied to periodontal lesions, it is prone to leakage of some exosomes during gel swelling. Therefore, in step S5, the initial gel produced in the previous step undergoes a freeze-thaw cycle physical cross-linking treatment. First, the initial gel is frozen in a low-temperature environment of -25℃ to -15℃ for 10 to 14 hours to allow water in the gel to crystallize, pushing the chitosan molecular chains closer together to form intermolecular hydrogen bonds and hydrophobic interactions. Then, the initial gel is allowed to thaw naturally at room temperature for 5 to 7 hours. After the ice crystals in the initial gel sublimate, the chitosan molecular chains retain the three-dimensional network structure formed by cross-linking. This freeze-thaw process is repeated 2 to 4 times to achieve gel solidification. The growth size of ice crystals can be controlled by adjusting the freezing temperature and freezing time to adjust the pore size of the porous structure within the hydrogel. Finally, the β-GP thermosensitive action is triggered by body temperature at 37℃ to complete gelation, thus stably embedding GMSC-EXO in the hydrogel network. The above preparation process not only avoids the problems of poor targeting ability, easy loss and short residence time of direct injection or application of exosomes, but also optimizes the physicochemical properties of GMSC-EXO by combining ionic crosslinking and physical crosslinking. This not only improves the loading efficiency of exosomes, but also avoids the disadvantages of chemical crosslinking such as complex process, high cost and toxic residues. At the same time, it also improves binding stability and release controllability.

[0013] Furthermore, in step S1, the method for collecting and culturing gingival mesenchymal stem cells includes:

[0014] a. Take gingival tissue from healthy individuals, rinse repeatedly with phosphate buffer, and mince to 1 mm. 3 Next, a mixed solution of 0.25% trypsin and 0.1% collagenase I was added, and then digested at 37°C for 40-80 min to obtain the first product; wherein, the volume ratio of trypsin to collagenase I in the mixed solution was 1:1.

[0015] b. Centrifuge the first product and inoculate the precipitate into α-MEM medium containing fetal bovine serum (FBS). Incubate the medium in an incubator, changing the medium every 3 days until cell confluence reaches 80-90%, yielding the second product. The FBS concentration in this second product is 10-20% (volume fraction). The incubator environment is 37°C and 5% CO2. After sampling, rinse the gingival tissue with PBS to remove blood, microorganisms, and other impurities. After mincing, hydrolyze the collagen and fibronectin between gingival tissue cells using a mixture of trypsin and collagenase I. Then, in α-MEM medium containing FBS, meet the proliferation requirements of mesenchymal stem cells and provide the necessary factors and substances for cell growth. Timely medium changes ensure timely nutrient replenishment, removal of metabolic waste, and stable cell growth, rapidly achieving the desired confluence.

[0016] In addition, after enzymatically isolating GMSCs from gingival tissue, their CD105 and CD90 positive expression and CD34 negative expression can be detected by flow cytometry to verify stemness. Their multi-lineage differentiation potential can also be identified through osteodifferentiation induction to ensure the high activity and purity of GMSCs. This results in obtaining gingival mesenchymal stem cells with a clear source and stable stemness, providing a highly functional cell source for the subsequent preparation of GMSC-EXO. It also eliminates contamination from hematopoietic stem cells and other contaminated cells, ensuring cell purity and avoiding interference from exosomes secreted by contaminated cells with subsequent treatment effects. It also ensures that GMSC-EXO has the osteogenic activity basis required for periodontal tissue repair.

[0017] Furthermore, in step S1, the method for extracting exosomes includes:

[0018] a. Passage the second product through culture. Cells at 80-90% confluence are cultured in serum-free medium for 24-48 hours, and the supernatant is collected. These cells are P3-P5, which are in a stable proliferative phase with high cell viability and uniform biological characteristics, ensuring both exosome yield and quality. Starving the cells in serum-free medium for 24-48 hours stimulates exosome secretion, further increasing yield.

[0019] b. Perform gradient centrifugation on the supernatant, collect the precipitate, and then resuspend the precipitate with phosphate buffer to obtain the third product. The gradient centrifugation conditions are as follows: first centrifuge at 600-900 rpm for 8-10 min, then centrifuge at 6000-10000 rpm for 30-60 min, and then centrifuge at 100000-120000 rpm for 1.5-2.5 h. The first centrifugation can remove large particulate impurities such as cell debris and apoptotic bodies, avoiding co-precipitation of impurities with exosomes during subsequent ultracentrifugation. The second centrifugation can remove microvesicles and protein aggregates, further purifying the exosome supernatant matrix. The third ultracentrifugation can accurately capture exosome vesicles and ensure sufficient precipitation of exosomes.

[0020] c. The third product was purified by ultrafiltration using an ultrafiltration membrane, and the resulting product was stored at -80℃ to obtain an exosome sample; specifically, a 100kDa ultrafiltration membrane was used. The molecular sieving effect of the 100kDa ultrafiltration membrane further retained GMSC-EXO and removed impurities such as small-molecule proteins and free nucleic acids, thereby further improving purity.

[0021] In addition, after GMSC-EXO extraction, exosomes can be identified to ensure their structural integrity and biological activity. Specifically, transmission electron microscopy is used to observe the cup-shaped or biconcave disc-shaped structure of exosomes and to observe whether there is membrane rupture or aggregation. The diffusion rate of particles is detected by NTA, and the particle size distribution and concentration are calculated. The protein concentration is quantitatively calculated by the absorbance value through the colorimetric reaction that occurs in BCA detection. Western blot is used to verify whether the CD9, CD81, and TSG101 markers are positively expressed, thus verifying that the product is an exosome.

[0022] Furthermore, in step S2, the acetic acid solution has a mass-volume concentration of 1% to ensure a suitable pH value in the weakly acidic environment, which promotes the dissolution of chitosan; the chitosan precursor solution has a mass-volume concentration of 2% to ensure the fluidity and operability of the precursor solution, which is also conducive to the moderate mechanical strength of the subsequently generated gel.

[0023] Furthermore, in step S2, after stirring and dissolving, the solution is sterilized using a 0.22μm sterile filter membrane to obtain chitosan precursor solution. The sterilization of the precursor solution is achieved by using a 0.22μm sterile filter membrane, while ensuring the homogeneity and integrity of the solution.

[0024] Furthermore, in step S3, the concentration of the exosome solution is 5 × 10⁻⁶. 10The particle / mL ratio matches the effective dosage for periodontitis treatment, ensuring therapeutic efficacy. Furthermore, the volume ratio of exosome solution to chitosan precursor solution is 1:1, which ensures uniform dispersion of exosomes, avoids local burst release or accumulation, maintains the stability of the mixed system, and guarantees gelation efficiency.

[0025] Furthermore, in step S3, the stirring and mixing conditions are 4°C and stirring for 20~40 minutes. Stirring and mixing at low temperature can prevent the chitosan solution from gelling prematurely, and at the same time ensure that the exosomes are evenly dispersed in the mixture, avoiding local aggregation.

[0026] Furthermore, in step S4, the mass-volume concentration of the β-glycerophosphate sodium aqueous solution is 50-60%, which can provide sufficient ionic strength to ensure stable and efficient gelation; and the volume ratio of the β-GP aqueous solution to the exosome-chitosan mixture is 7:3, which can precisely adjust the pH value of the initial gel to make the pH value close to the physiological pH, avoid irritating the periodontal tissue, and also ensure the effective concentration of β-GP to ensure sufficient cross-linking.

[0027] Furthermore, in step S4, the magnetic stirring conditions are 200~400 rpm for 20~40 min. Through gentle stirring at medium and low speed, the interfacial tension between the β-GP aqueous solution and the exosome-chitosan mixture can be broken to achieve uniform mixing at the molecular level, while avoiding excessive shear force that could damage the exosome structure and ensure the integrity of its active ingredients.

[0028] On the other hand, the present invention also provides a chitosan hydrogel loaded with exosomes derived from gingival mesenchymal stem cells, which is prepared by the above method.

[0029] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0030] The present invention provides a method for preparing a chitosan hydrogel loaded with exosomes derived from gingival mesenchymal stem cells (GMSCs) and the hydrogel thereof. This method avoids the problems of poor targeting ability, easy loss, and short residence time associated with direct injection or application of exosomes. Furthermore, by combining an ionic crosslinking method of mixing and stirring an exosome-chitosan mixture with β-GP with a physical crosslinking method involving multiple freeze-thaw cycles, the present invention not only avoids the drawbacks of chemical crosslinking, such as complex processes, high costs, and residual toxicity, but also optimizes the physicochemical properties of GMSCs by adjusting the pore size of the hydrogel, making the hydrogel structure highly compatible with GMSCs, thereby improving the loading efficiency of the hydrogel on exosomes and enabling controlled sustained release of active ingredients, avoiding burst release problems, and thus improving binding stability and controllable release. Detailed Implementation

[0031] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, 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.

[0032] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other. Example 1

[0033] On one hand, this embodiment provides a method for preparing a chitosan hydrogel loaded with exosomes derived from gingival mesenchymal stem cells, comprising:

[0034] S1. Collect and culture gingival mesenchymal stem cells, and extract exosomes from them to obtain exosome samples. The specific method is as follows:

[0035] a. Take approximately 0.5cm 3 Healthy human gingival tissue was rinsed three times with PBS and then minced to 1 mm. 3 Next, a mixed solution of 0.25% trypsin and 0.1% collagenase I was added, and then digested at 37°C for 60 minutes to obtain the first product. The volume ratio of 0.25% trypsin to 0.1% collagenase I in the mixed solution was 1:1. After sampling, the gingival tissue was rinsed with PBS to remove blood, microorganisms and other impurities. After being cut into small pieces, the collagen and fibronectin in the gingival tissue were hydrolyzed by the mixed solution of trypsin and collagenase I. Specifically, "0.25%" and "0.1%" here refer to the mass-volume concentration of the added components themselves, that is, the 0.25% mass-volume concentration of trypsin solution and the 0.1% mass-volume concentration of collagenase I solution were mixed to form this mixed solution.

[0036] b. Centrifuge the first product at 1000 rpm for 5 min, and seed the precipitate into α-MEM medium containing 15% fetal bovine serum. Incubate the medium at 37°C with 5% CO2, and change the medium every 3 days until the cell confluence reaches 80-90%, thus obtaining the second product. The α-MEM medium containing fetal bovine serum meets the proliferation requirements of mesenchymal stem cells and provides the factors and substances needed for cell growth. Changing the medium on time can replenish nutrients, remove metabolic waste, ensure stable cell growth, and quickly reach the required confluence.

[0037] The second product was detected by flow cytometry. Its positive expression rates of CD105 and CD90 were ≥90%, and its negative expression of CD34 was ≥98%, consistent with the stem characteristics of mesenchymal stem cells. After 21 days of osteogenic induction, alizarin red staining revealed obvious calcium nodule formation, verifying that GMSCs possess stable osteogenic differentiation potential. Flow cytometry and osteogenic differentiation were used to screen cell purity and verify osteogenic function, thereby obtaining gingival mesenchymal stem cells with a clear origin and stable stemness, providing a highly functional cell source for the subsequent preparation of GMSC-EXO. Contamination by hematopoietic stem cells and other contaminating cells was excluded to ensure cell purity and avoid interference from exosomes secreted by contaminating cells in subsequent treatment effects, also ensuring that GMSC-EXO possesses the osteogenic activity required for periodontal tissue repair.

[0038] c. Passage the second product and take the fourth generation of cells with 80-90% confluence and culture them in serum-free medium for 36 hours. Collect the supernatant. This generation is in a stable proliferation phase with strong cell viability and uniform biological characteristics, which can ensure the yield and quality of exosomes. Starving the cells in serum-free medium for 36 hours can stimulate the secretion level of exosomes and further increase the yield.

[0039] d. The supernatant was subjected to a gradient centrifugation process: 800 rpm for 10 min, 8000 rpm for 50 min, and 110000 rpm for 2 h. The precipitate was collected and then resuspended in PBS to obtain the third product. The first centrifugation removes large particulate impurities such as cell debris and apoptotic bodies, preventing co-precipitation of impurities with exosomes during subsequent ultracentrifugation. The second centrifugation removes microvesicles and protein aggregates, further purifying the exosome supernatant. The third ultracentrifugation precisely captures exosome vesicles, ensuring complete precipitation of exosomes.

[0040] e. The third product was purified by ultrafiltration using a 100 kDa ultrafiltration membrane, and the resulting product was stored at -80℃ to obtain an exosome sample (lyophilized powder). The molecular sieving effect of the 100 kDa ultrafiltration membrane further retained GMSC-EXO and removed impurities such as small molecule proteins and free nucleic acids, thereby further improving the purity.

[0041] Transmission electron microscopy (TEM) revealed that the extracted exosomes exhibited a typical cup-shaped structure with no obvious membrane rupture or aggregation. NTA analysis showed that the GMSC-EXO particles were concentrated in the 100-200 nm range, with an average particle size of 191.1 nm and a concentration ≥1×10⁻⁶. 10 particles / mL; BCA detection showed a protein concentration ≥50 μg / mL; Western blot analysis showed positive expression of CD9, CD81, and TSG101 markers.

[0042] S2. Weigh 2g of chitosan and add it to 100mL of 1% acetic acid solution. Stir to dissolve and then sterilize using a 0.22μm sterile filter membrane to obtain a 2% (w / v) chitosan precursor solution. The chitosan molecular chain contains amino groups, which can be protonated in acetic acid solution to form a positively charged acidic precursor solution.

[0043] S3. Reconstitute the exosome sample (lyophilized powder) in PBS to a concentration of 5 × 10⁻⁶. 10 The exosome solution was prepared by mixing particles / mL of exosomes, and then adding the exosome solution to the chitosan precursor solution at a volume ratio of 1:1. The mixture was gently stirred at 4°C for 30 min to obtain an exosome-chitosan mixture. The exosome sample (usually lyophilized powder) was reconstituted in PBS to restore the exosome biological activity and disperse it in the exosome solution. The exosome solution was then mixed with the chitosan precursor solution. The membrane surface of GMSC-EXO is rich in acidic lipids such as phosphatidylserine, which are negatively charged. After mixing, a stable "exosome-chitosan" complex can be formed through electrostatic adsorption, which improves the loading efficiency and also allows the exosomes to be uniformly dispersed in the precursor solution, avoiding uneven distribution or leakage.

[0044] S4. Weigh 0.56g β-GP, dissolve it in 10mL of double-distilled water, and sonicate it for 30min to obtain a 56% (w / v) β-glycerophosphate sodium aqueous solution. Then, slowly add the β-GP aqueous solution dropwise to the exosome-chitosan mixture at a volume ratio of 7:3, and stir magnetically at 300rpm for 30min to obtain a preliminary gel. Mixing β-GP with the "exosome-chitosan" complex triggers the thermosensitive gelation of chitosan to produce the characteristics of "low-temperature flow and body temperature gelation", which not only facilitates injection administration but also facilitates adhesion to lesions and avoids the diffusion and loss of exosomes. The slow dropwise addition under magnetic stirring allows β-GP to be evenly dispersed in the mixture. During the gelation process, chitosan molecules slowly cross-link to form a three-dimensional network, embedding GMSC-EXO in the pores.

[0045] S5. The initial gel is placed in a sterile mold and subjected to three physical cross-linking treatments. The physical cross-linking treatment involves first freezing at -20℃ for 12 hours, and then thawing at room temperature for 6 hours. Low-temperature freezing can cause water in the gel to crystallize, pushing the chitosan molecular chains closer together to form intermolecular hydrogen bonds and hydrophobic interactions. Natural thawing can cause the ice crystals in the initial gel to sublimate, but the chitosan molecular chains will retain the three-dimensional network structure formed by cross-linking. By repeating this freeze-thaw process three times, the gel can be solidified. The freezing temperature of -20℃ and the freezing time of 12 hours can control the ice crystal growth size, so that the hydrogel forms a porous structure of 50-200 μm to match the particle size of GMSC-EXO and facilitate GMSC infiltration and nutrient exchange.

[0046] S6. The product obtained in step S5 is placed in a 37°C constant temperature water bath for incubation to obtain a chitosan hydrogel loaded with exosomes derived from gingival mesenchymal stem cells; gelation is completed by triggering the β-GP thermosensitive effect at 37°C body temperature to stably embed GMSC-EXO in the hydrogel network.

[0047] On the other hand, this embodiment also provides a chitosan hydrogel loaded with exosomes derived from gingival mesenchymal stem cells, which is prepared by the above-described preparation method. Example 2

[0048] The difference between this embodiment and Example 1 lies in the concentration of each component and the preparation conditions; the rest of the preparation methods are the same as in Example 1. In step S1 of this embodiment, after mincing the gingival tissue, a mixed solution of 0.25% trypsin and 0.1% collagenase I is added, followed by digestion at 37°C for 40 min. After centrifuging the first product, the precipitate is inoculated into α-MEM medium containing 10% fetal bovine serum and cultured according to the aforementioned method until the cell confluence reaches 80-90%. The second product is passaged, and P3 cells with a confluence of 80-90% are cultured in bloodless medium for 24 h. The supernatant is collected and subjected to gradient centrifugation at 600 rpm for 8 min, 6000 rpm for 30 min, and 100000 rpm for 1.5 h. In step S3, the mixture is gently stirred at 4°C for 20 min to obtain an exosome-chitosan mixture. In step S4, 0.5 g of β-GP was weighed and dissolved in 10 mL of double-distilled water to obtain a 50% (w / v) β-GP aqueous solution. This solution was then mixed with an exosome-chitosan mixture and magnetically stirred at 200 rpm for 20 min to obtain a preliminary gel. In step S5, the preliminary gel underwent two physical cross-linking treatments: first, it was frozen at -15℃ for 10 h, and then thawed at room temperature for 5 h. Example 3

[0049] The difference between this embodiment and Example 1 lies in the concentration of each component and the preparation conditions; the rest of the preparation methods are the same as in Example 1. In step S1 of this embodiment, after mincing the gingival tissue, a mixed solution of 0.25% trypsin and 0.1% collagenase I is added, followed by digestion at 37°C for 80 min. After centrifuging the first product, the precipitate is inoculated into α-MEM medium containing 20% ​​fetal bovine serum and cultured according to the aforementioned method until the cell confluence reaches 80-90%. The second product is passaged, and P5 cells with a confluence of 80-90% are cultured in bloodless medium for 48 h. The supernatant is collected and subjected to gradient centrifugation at 900 rpm for 15 min, 10000 rpm for 60 min, and 120000 rpm for 2.5 h. In step S3, the mixture is gently stirred at 4°C for 40 min to obtain an exosome-chitosan mixture. In step S4, 0.6 g of β-GP was weighed and dissolved in 10 mL of double-distilled water to obtain a 60% (w / v) β-GP aqueous solution. This solution was then mixed with an exosome-chitosan mixture and magnetically stirred at 400 rpm for 40 min to obtain a preliminary gel. In step S5, the preliminary gel underwent four physical cross-linking treatments: first, it was frozen at -25°C for 14 h, and then thawed at room temperature for 7 h.

[0050] Comparative Example 1

[0051] The difference between this comparative example and Example 1 is that the physical crosslinking step of freezing-thawing is omitted and replaced with ionic crosslinking. All other preparation methods and component concentrations are the same as in Example 1. In this comparative example, the initial gel obtained in step S4 is placed in a sterile mold. 0.5 g of sodium tripolyphosphate (TPP) is weighed and dissolved in 10 mL of double-distilled water to prepare a 5% (w / v) TPP crosslinking solution. The TPP crosslinking solution is slowly and evenly dripped onto the surface of the initial gel using a syringe until it is completely covered. Crosslinking is allowed to occur at 4°C for 12 h. After crosslinking is complete, the gel surface is gently rinsed with deionized water to remove unreacted TPP. The ionicly crosslinked gel is then placed in a 37°C constant temperature water bath for incubation to obtain the final hydrogel.

[0052] Comparative Example 2

[0053] The difference between this comparative example and Example 1 is that only the cryo-crosslinking technique was used to construct the gel system; the rest of the preparation methods and component concentrations were the same as in Example 1. In this comparative example, the exosome-chitosan mixture obtained in step S3 was directly placed in a sterile mold, and the mixture underwent three freeze-thaw physical crosslinking treatments.

[0054] Comparative Example 3

[0055] The only difference between this comparative example and Example 1 is the number of physical cross-linking treatments; the other preparation methods and component concentrations are the same as in Example 1. In step S5 of this comparative example, the initial gel is placed in a sterile mold and subjected to one physical cross-linking treatment. The physical cross-linking treatment conditions are: first, freezing at -20°C for 12 h, and then thawing at room temperature for 6 h.

[0056] Comparative Example 4

[0057] The only difference between this comparative example and Example 1 is the number of physical cross-linking treatments; the other preparation methods and component concentrations are the same as in Example 1. In step S5 of this comparative example, the initial gel was placed in a sterile mold and subjected to 5 physical cross-linking treatments. The physical cross-linking treatment conditions were: first, freezing at -20°C for 12 h, and then thawing at room temperature for 6 h.

[0058] Comparative Example 5

[0059] The only difference between this comparative example and Example 1 is the physical cross-linking treatment conditions; the other preparation methods and component concentrations are the same as in Example 1. In step S5 of this comparative example, the initial gel was placed in a sterile mold and subjected to three physical cross-linking treatments. The physical cross-linking treatment conditions were: first, freezing at -30°C for 14 hours, and then thawing at room temperature for 7 hours.

[0060] Comparative Example 6

[0061] The only difference between this comparative example and Example 1 is the physical cross-linking treatment conditions; the other preparation methods and component concentrations are the same as in Example 1. In step S5 of this comparative example, the initial gel was placed in a sterile mold and subjected to three physical cross-linking treatments. The physical cross-linking treatment conditions were: first, freezing at -10°C for 10 hours, and then thawing at room temperature for 5 hours.

[0062] The products prepared in the above examples and comparative examples were subjected to the following performance tests, and the specific test methods are as follows:

[0063] (1) Gelation time: The time required from the start of cross-linking to the point where the tube no longer flows after inversion was recorded by the inverted test tube method;

[0064] (2) Microstructure: After freeze-drying, the internal pore structure of the hydrogel was observed by scanning electron microscopy, and the pore size and porosity were statistically analyzed;

[0065] (3) Haze and transparency: The transmittance of the hydrogel was detected under visible light by means of the Tyndall effect;

[0066] (4) Loading efficiency: The total amount of exosome protein encapsulated in the hydrogel was detected by BCA protein quantification method, and the loading efficiency was calculated by comparing it with the initial amount of exosome protein added.

[0067] (5) In vitro release: The hydrogel was immersed in 2 mL of PBS buffer, and the mixture was shaken at 37 °C. The supernatant was collected periodically and the exosome protein concentration was detected.

[0068] The specific test results are shown in the table below:

[0069]

[0070] in conclusion:

[0071] Examples 1-3 respectively selected the medium, low, and high values ​​of the concentration of each component and the preparation parameters in the preparation method of the present invention for preparation detection. Comparative Example 1 omits the physical cross-linking treatment step and uses only chemical ionic cross-linking technology. Comparative Example 2 is the opposite of Comparative Example 1, using only physical cross-linking technology. In Comparative Example 3, the number of physical cross-linking treatments is reduced to below the limit of the present invention. In Comparative Example 4, the opposite is true, the number of physical cross-linking treatments is increased to above the limit of the present invention. Comparative Example 5 keeps the number of physical cross-linking treatments constant and uses a lower freezing temperature and a longer freezing time. Comparative Example 6 uses a higher freezing temperature and a shorter freezing time.

[0072] In the test results of the three examples in the table above, the gelation time, pore size, and porosity of Examples 1-3 are all within a suitable range, indicating that the gelation process is relatively mild, which can ensure the activity of GMSC-EXO and the uniformity of the gel structure, and can also meet the needs of clinical operations such as injection. In the haze and transparency test based on the Tyndall effect, all three examples can show appropriate brightness transmission under visible light, indicating that the gel network structure is uniform and there is no obvious aggregation or impurities. Among them, Example 1 has the best haze and transparency. Due to the upper limit of freezing temperature, time and number of treatments, the gel structure of Example 3 is relatively dense, resulting in relatively high haze, relatively low transparency and general light transmittance, which is in line with expectations. In the test of loading efficiency and in vitro release rate, the crosslinking strength, microstructure, network density, etc. of the three examples are all balanced, with good loading efficiency, which can effectively retain exosomes. The cumulative release rate after 7 days also indicates that there is no burst release phenomenon and long-term sustained release can be achieved. Among them, the ratio and preparation conditions of Example 1 reach the best balance, and the structural stability and release controllability of its hydrogel reach the best level.

[0073] Based on the test results in the table above, Comparative Example 1, using only ionic crosslinking technology, had the fastest gelation time. This is because the ionic bonding between TPP and chitosan is a rapid chemical reaction, and it does not rely on ice crystal pore formation, resulting in a dense, non-porous structure controlled by ionic crosslinking points. Comparative Example 1, due to its high density and lack of large pores, formed a structure with poor homogeneity, strong light scattering, and opacity. While ionic crosslinking makes the gel network dense, its rapid formation process may hinder the uniform dispersion and embedding of exosomes, and the washing step may cause losses, resulting in moderate efficiency. Furthermore, the extremely dense gel network or small pores severely impede diffusion, leading to slow and incomplete release and a "lock-in" effect. The dense, non-porous structure (porosity <60%) formed by ionic crosslinking, while possessing high mechanical strength, severely hinders exosome release (42%) and tissue ingrowth, making it unsuitable for tissue repair scenarios requiring cell migration and active ingredient delivery. The dense, non-porous sheet structure (<10 μm) almost completely blocks cell infiltration and nutrient exchange, severely violating the basic principles of tissue engineering scaffolds. This prevents exosomes from effectively exerting their biological activity. Their stable structure, formed by rapid chemical cross-linking, may have an excessively long degradation cycle and is prone to encapsulation by foreign matter.

[0074] Comparative Example 2 failed to gel due to the use of simple freeze-crosslinking technology without the addition of β-GP aqueous solution. The role of β-GP is to neutralize the charge of chitosan, disrupting its hydration layer, thereby initiating a temperature-sensitive sol-gel transition (thermosensitive gelation). Its main purpose and result is to endow the system with "injectability" and "body temperature curing" properties, representing a special, mild ion-triggered thermal response process. In contrast, the comparative example, lacking β-GP to neutralize the charge, may not have formed a gel of sufficient strength, or the gelation process may have been uncontrollable. This demonstrates that β-GP-induced charge neutralization and hydrophobic interactions are prerequisites for thermosensitive gelation of chitosan in this system; pure physical freezing cannot induce gelation. It also proves that the initial network initiated by β-GP is necessary for the successful implementation of subsequent physical crosslinking, and the two have a synergistic effect.

[0075] Comparative Example 3, due to fewer freezing cycles, resulted in insufficient cross-linking, slow or incomplete gelation, insufficient accumulation of intermolecular forces, a fragile network, and large, irregular pores. Furthermore, because the single-cycle cross-linking network was incomplete, a large amount of exosomes were lost during subsequent processing or washing, resulting in the lowest efficiency. Simultaneously, the loose and porous network allowed for almost free diffusion of exosomes, leading to rapid burst release. The loose and fragile structure of Comparative Example 3 easily disintegrated in the complex wet mechanical environment of the periodontium, failing to provide a stable three-dimensional space. Almost all exosomes were rapidly lost within a few days (burst release >96%), failing to achieve sustained pharmacological effects and potentially causing unknown effects due to excessively high local concentrations.

[0076] Comparative Example 4, due to its higher number of cycles, higher degree of cross-linking, and lower temperature, forms a network that resists flow, thus requiring a shorter gelation time. However, each cycle reinforces cross-linking and may damage existing pores, leading to excessive structural shrinkage and densification. Furthermore, excessive cross-linking results in overly slow release (56%), potentially affecting bioefficacy. Excessive cross-linking causes pore collapse and structural rigidity. While the sustained release is prolonged, the dense network severely hinders cell migration and tissue ingrowth, potentially causing the hydrogel to be encapsulated by fibers rather than integrated with newly formed tissue. Overly slow release may also miss the critical window of cell proliferation and differentiation.

[0077] Comparative Example 5, due to its high supercooling, has numerous ice crystal nucleation sites and a short growth time, resulting in many small ice crystals and ultimately a dense structure with small, uniform pore size. Comparative Example 6, due to its low supercooling, has fewer ice crystal nucleation sites and a longer growth time, resulting in fewer, larger ice crystals and ultimately a structure with large, uneven pore size. The pore size distribution issues in Comparative Examples 5 and 6 (too small and dense or too large and uneven) lead to enhanced scattering and increased haze. Comparative Example 5, due to its low temperature, forms small, uniform pores (30-80 μm), exhibiting good sustained-release performance (65%). This homogeneous and dense structure may be valuable in certain biomanufacturing applications where high material uniformity is required. However, in periodontal regeneration, its pore size is at the critical lower limit for cell migration, potentially allowing only a small number of cells or pseudopodia to penetrate, which is detrimental to large-scale cell community migration and rapid vascularization. In contrast, Comparative Example 6, while its large and uneven pores (200-500 μm) may allow cell entry, suffers from a coarse and uneven network that results in weak mechanical properties and poor exosome embedding (rapid release, 90%). In vivo, this structure may lead to uneven growth of repaired tissue, with some areas showing good cell ingrowth while others fail to repair due to premature material degradation or collapse.

[0078] In summary, the test results of Examples 1-3 demonstrate the effectiveness of the preparation method of the present invention; Examples 1 and Comparative Examples 1 and 2 demonstrate the effectiveness of the synergistic effect of β-GP ionic crosslinking and freeze-thaw cycle physical crosslinking compared to simple ionic crosslinking or simple physical crosslinking; Examples 1 and Comparative Examples 3 and 4 demonstrate the effectiveness of the number of physical crosslinking treatments described in the present invention; Examples 1 and Comparative Examples 5 and 6 demonstrate the effectiveness of the freeze-thaw treatment conditions described in the present invention. The combined test results of Examples 1-3 and Comparative Examples 1-6 indicate that the preparation method of the present invention, which combines β-GP ionic crosslinking with freeze-thaw cycle physical crosslinking, can stabilize the network structure and control the micropore size under mild conditions without damaging exosome activity. This avoids the disadvantages of poor mechanical strength and structural instability of simple thermosensitive gels, and also avoids the toxicity and complex processes associated with chemical crosslinking agents. It ensures the drug loading and sustained-release properties of the hydrogel, prevents initial burst release, and ensures the residence time and duration of action at the lesion site. Furthermore, the entire preparation process is gentle and easy to operate, reducing preparation costs and ensuring preparation efficiency, thus providing a foundation for large-scale and standardized production.

[0079] The above embodiments are only for illustrating the technical concept and features of the present invention. Their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be used to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a chitosan hydrogel loaded with exosomes derived from gingival mesenchymal stem cells, characterized in that, include: S1. Collect and culture gingival mesenchymal stem cells, and extract exosomes from them to obtain exosome samples; S2. Add chitosan to the acetic acid solution and stir to dissolve, to obtain the chitosan precursor solution; S3. Mix the exosome sample with phosphate buffer to form an exosome solution, then add the exosome solution to the chitosan precursor solution and stir to mix to obtain an exosome-chitosan mixture. S4. Add the sodium β-glycerophosphate aqueous solution dropwise to the exosome-chitosan mixture and stir magnetically to obtain a preliminary gel. S5. Place the initial gel in a sterile mold and perform 2 to 4 physical cross-linking treatments; the physical cross-linking treatment includes: first freezing at -25℃ to -15℃ for 10 to 14 hours, and then thawing at room temperature for 5 to 7 hours; S6. The product obtained in step S5 is stored at a constant temperature of 37°C to obtain a chitosan hydrogel loaded with gingival mesenchymal stem cell exosomes.

2. The method for preparing chitosan hydrogel loaded with exosomes derived from gingival mesenchymal stem cells according to claim 1, characterized in that, In step S1, the methods for collecting and culturing gingival mesenchymal stem cells include: a. Take gingival tissue from healthy individuals, rinse repeatedly with phosphate buffer, and mince to 1 mm. 3 Next, a mixed solution of 0.25% trypsin and 0.1% collagenase I was added, and then digested at 37°C for 40-80 min to obtain the first product; wherein, the volume ratio of trypsin to collagenase I in the mixed solution was 1:

1. b. Centrifuge the first product, inoculate the precipitate into α-MEM medium containing fetal bovine serum, and incubate the medium in an incubator. Change the medium every 3 days until the cell confluence reaches 80-90% to obtain the second product. The volume fraction of fetal bovine serum is 10-20%. The incubator environment is 37°C and 5% CO2.

3. The method for preparing chitosan hydrogel loaded with exosomes derived from gingival mesenchymal stem cells according to claim 2, characterized in that, In step S1, the method for extracting exosomes includes: a. Passage the second product, and take cells with a cell confluence of 80-90% and culture them in serum-free medium for 24-48 hours, and collect the supernatant; wherein, the cells are P3-P5. b. The supernatant is subjected to gradient centrifugation, the precipitate is collected, and then the precipitate is resuspended in phosphate buffer to obtain the third product; wherein the gradient centrifugation conditions are as follows: first centrifuge at 600-900 rpm for 8-10 min, then centrifuge at 6000-10000 rpm for 30-60 min, and then centrifuge at 100000-120000 rpm for 1.5-2.5 h; c. The third product is purified by ultrafiltration using an ultrafiltration membrane, and the resulting product is stored at -80°C to obtain the exosome sample.

4. The method for preparing chitosan hydrogel loaded with exosomes derived from gingival mesenchymal stem cells according to claim 1, characterized in that: In step S2, the acetic acid solution has a mass-volume concentration of 1%, and the chitosan precursor solution has a mass-volume concentration of 2%.

5. The method for preparing chitosan hydrogel loaded with exosomes derived from gingival mesenchymal stem cells according to claim 1, characterized in that: In step S2, after stirring and dissolving, the solution is sterilized using a 0.22μm sterile filter membrane to obtain the chitosan precursor solution.

6. The method for preparing chitosan hydrogel loaded with exosomes derived from gingival mesenchymal stem cells according to claim 1, characterized in that, In step S3, the concentration of the exosome solution is 5 × 10⁻⁶. 10 particles / mL; and the volume ratio of the exosome solution to the chitosan precursor solution is 1:

1.

7. The method for preparing chitosan hydrogel loaded with exosomes derived from gingival mesenchymal stem cells according to claim 1, characterized in that, In step S3, the mixing conditions are 4°C and stirring for 20-40 minutes.

8. The method for preparing chitosan hydrogel loaded with exosomes derived from gingival mesenchymal stem cells according to claim 1, characterized in that, In step S4, the mass-volume concentration of the sodium β-glycerophosphate aqueous solution is 50-60%; and the volume ratio of the sodium β-glycerophosphate aqueous solution to the exosome-chitosan mixture is 7:

3.

9. The method for preparing chitosan hydrogel loaded with exosomes derived from gingival mesenchymal stem cells according to claim 1, characterized in that, In step S4, the magnetic stirring conditions are 200~400 rpm for 20~40 min.

10. A chitosan hydrogel loaded with exosomes derived from gingival mesenchymal stem cells, characterized in that: It is prepared by any one of the preparation methods according to claims 1 to 9.

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