3D scaffold for repairing defective lung injury and construction method and application thereof

Abstract

This invention discloses a 3D scaffold for repairing defective lung injury, its construction method, and its applications, belonging to the field of biomedical materials and tissue engineering technology. This invention uses cationic polysaccharide chitosan and polymer γ-polyglutamic acid as raw materials, utilizing their electrostatic interaction to achieve cross-linking without cross-linking agents, and combines this with freeze-drying technology to prepare a three-dimensional spherical γ-PGA / CS composite scaffold. The above composite scaffold is combined with bone marrow stem cell exosomes (MSC-Exos) and type II alveolar epithelial cells (RLE-6TN). By loading exosomes onto the scaffold and implanting lung cells, local enrichment and targeted delivery are achieved, solving the problems of poor targeting of bone marrow stem cell exosomes and easy loss during transplantation of individual cells. This functional scaffold can accelerate lung function recovery by regulating the inflammatory microenvironment at the site of lung injury in SD rats, promoting lung cell regeneration, and reducing secondary lung tissue damage. It provides a novel implantable, minimally invasive treatment for defective lung injury-related diseases, possessing good clinical translational potential and application prospects in organ damage repair.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials and tissue engineering technology, specifically to a 3D scaffold for repairing lung defects, its construction method, and its application. Technical Background As vital gas exchange organs, the lungs are susceptible to various factors, including internal physiological processes and the external environment, which can lead to lung diseases. Partial lung resection is an important intervention in modern medicine. However, the body's own recovery capacity after lung resection is limited, and traditional chemotherapy often results in various adverse reactions, further increasing the risk of postoperative death. In recent years, cell transplantation and stem cell-derived exosome (SC-Exos) therapy have been widely researched and applied in various diseases.

[0002] Cell transplantation is a core therapeutic technology in regenerative medicine. It refers to the introduction of functional cells, which have been isolated, cultured, expanded, or modified in vitro, into damaged tissues or organs through methods such as local injection, scaffold mounting, or intravenous infusion. This novel treatment aims to replenish deficient cells, repair tissue structure, and restore physiological function. Its core logic is to utilize the proliferative and differentiation potential and paracrine function of cells to replace damaged or apoptotic cells, regulate the local microenvironment, and ultimately achieve tissue regeneration and functional recovery.

[0003] Exosomes are double-membrane vesicles with a diameter of 30-150 nm. They can be secreted by almost all cells and are widely distributed in bodily fluids such as blood, urine, and cell culture media. Their value lies in the functional components they encapsulate, including miRNA, mRNA, proteins, lipids, and cytokines. These components act as intercellular messengers and can be taken up by recipient cells through membrane fusion or endocytosis, regulating physiological processes such as cell proliferation, differentiation, and apoptosis, and participating in tissue repair and inflammation regulation.

[0004] However, when cells are transplanted alone, the retention and survival rates in vivo are often unsatisfactory, making it difficult for them to survive stably at the site of injury and exert their effects. On the other hand, relying solely on stem cell exosome therapy suffers from insufficient targeting, making it difficult to effectively accumulate cells locally at the lesion site for precise repair. At the same time, clinical practice places increasingly stringent requirements on medical biomaterials, demanding not only excellent biocompatibility but also core conditions such as controllable degradation rates and low immunogenicity.

[0005] Based on the current state of research, developing a composite scaffold system that can provide a stable microenvironment for cell colonization and efficiently enrich bioactive factors has become an urgent direction for breakthroughs in the field of lung tissue engineering. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a method for constructing a 3D scaffold for the repair of defective lung injury. The scaffold is a three-dimensional spherical γ-polyglutamic acid / chitosan (γ-PGA / CS) composite scaffold with dual loading function, which can simultaneously load rat alveolar type II epithelial cells (RLE-6TN) and mesenchymal stem cell exosomes (MSC-Exos), thereby effectively overcoming the limitations of single-cell or exosome therapy. Through structural support, the active factor regulation is achieved after cell colonization, which can effectively promote the repair process after lung resection.

[0007] The present invention further provides a bone marrow stem cell exosome 3D scaffold prepared using the above-described construction method.

[0008] Another objective of this invention is to provide the application of the above-mentioned bone marrow stem cell exosome 3D scaffold in the preparation of materials for repairing defective lung injuries.

[0009] The technical solution adopted by the present invention to achieve the above objectives is as follows: This invention provides a method for constructing a 3D scaffold for repairing lung defects, comprising the following steps: S1: Add the γ-polyglutamic acid solution dropwise to the chitosan solution, stir at a constant speed until the mixture is uniform, quickly mold the mixture, freeze dry to obtain a 3D γ-PGA / CS scaffold; S2: Exosomes of bone marrow stem cells and RLE-6TN alveolar epithelial cells were extracted and co-incubated with γ-PGA / CS scaffolds to obtain γ-PGA / CS / MSC-Exos and γ-PGA / CS / MSC-RLE-6TN. or S3: RLE-6TN alveolar epithelial cells were seeded onto a γ-PGA / CS / MSC-Exos scaffold to obtain γ-PGA / CS / MSC-Exos / RLE-6TN.

[0010] Furthermore, in S1, the concentration of the γ-polyglutamic acid solution is 5 g / L, and the solvent is a 0.2 mol / L NaOH solution; the concentration of the chitosan solution is 20 g / L, and a 0.2 mol / L acetic acid solution is used as a co-solvent; the volume ratio of the γ-polyglutamic acid solution to the chitosan solution is 1:5.

[0011] Furthermore, the γ-polyglutamic acid has a molecular weight of 1.1 million; the chitosan has a degree of deacetylation ≥95%, a viscosity of 20-500 mPa.s, and a pH value of 6.5-8.5.

[0012] Furthermore, in S2, the concentration of the bone marrow stem cell exosomes is 1×10⁻⁶. 9 ~12×10 9Particles / mL; the ratio of bone marrow stem cell exosomes to γ-PGA / CS scaffold is: 1 mL of 1×10⁻⁶ granules per 1 mg γ-PGA / CS scaffold. 9 ~12×10 9 Bone marrow stem cell exosomes per granule / mL.

[0013] Furthermore, when bone marrow stem cell exosomes are used, the co-incubation conditions are 37°C for 4 hours; when alveolar epithelial cells RLE-6TN are used, the co-incubation is performed by static culture in a 37°C CO2 incubator for 3 hours, followed by transfer to a cell culture dish, supplementing with 5 mL of culture medium, and continuing culture for 1-5 days; the culture medium is changed every day.

[0014] Further, in S3, 1 mg of γ-PGA / CS / MSC-Exos scaffold was weighed and added to 2 mL of 2.0~3.0×10 5 The cell suspension was 1 cell / mL LRLE-6TN; the co-incubation was performed by statically culturing the cells in a 37℃ CO2 incubator for 3 hours, then transferring them to a cell culture dish, adding 5 mL of culture medium, and continuing the culture for 1-5 days; the culture medium was changed every 1 day.

[0015] The present invention also provides a 3D scaffold prepared using the above-described construction method.

[0016] Another object of the present invention is to provide the application of the above-mentioned 3D scaffold in the preparation of materials for repairing lung defects.

[0017] In the preparation process of this invention, γ-polyglutamic acid solution and chitosan solution are magnetically stirred at 37°C and 500r / min until they are evenly mixed. The mixture is then transferred into a small spray bottle and liquid nitrogen is sprayed in to rapidly form the solution.

[0018] This invention involves implanting the prepared 3D scaffold γ-PGA / CS / MSC-Exos / RLE-6TN into the lung resection incision site. The specific process is as follows: First, a partial lung resection model is established. SD rats are anesthetized by intraperitoneal injection of a mixture of acetaminophen 50 (20 mg / kg) and celeratin (5 mg / kg). Hair in the chest cavity is removed using an electric shaver, and the rats are placed in a supine position on the operating table and secured with rubber bands. The area is disinfected three times with povidone-iodine and draped. The chest wall muscles are separated, and the chest cavity is opened layer by layer to expose the lung lobes. The root of the right middle lobe is gently located and clamped, and 1 / 4 of the lung lobe is removed. The γ-PGA / CS / MSC-Exos / RLE-6TN is implanted into the incision site. The chest wall muscles and skin are sutured layer by layer with 6-0 absorbable sutures, the pleural cavity is squeezed to expel pleural effusion, and the surgical site is disinfected.

[0019] This invention utilizes cationic polysaccharide chitosan and polymer γ-polyglutamic acid as raw materials, leveraging their electrostatic interaction to achieve cross-linking without cross-linking agents, and then employs freeze-drying technology to prepare a three-dimensional spherical γ-PGA / CS composite scaffold. This composite scaffold is combined with bone marrow stem cell exosomes (MSC-Exos) and type II alveolar epithelial cells (RLE-6TN). By loading exosomes onto the scaffold and implanting lung cells, local enrichment and targeted delivery can be achieved, solving the problems of poor targeting of bone marrow stem cell exosomes and easy loss during transplantation of individual cells. This functional scaffold can accelerate lung function recovery by regulating the inflammatory microenvironment at the site of lung injury in SD rats, promoting lung cell regeneration, and reducing secondary lung tissue damage. It provides a novel implantable, minimally invasive treatment for defective lung injury-related diseases, possessing good clinical translational potential and application prospects in organ damage repair.

[0020] The beneficial effects of the present invention are: (1) The present invention prepares a 3D spherical γ-PGA / CS scaffold. The scaffold has excellent biocompatibility, no obvious cytotoxicity, does not cause excessive inflammatory response after implantation in vivo, and maintains local microenvironment stability during degradation, thus avoiding interference with repair.

[0021] (2) The 3D spherical γ-PGA / CS scaffold used in this invention provides a stable microenvironment for cell colonization with its three-dimensional porous structure, avoiding the defect of easy loss of free cells; at the same time, it efficiently loads bone marrow stem cell exosomes through electrostatic interaction, realizes the local directional enrichment of exosomes, and solves the problem of poor targeting of exosomes and easy dilution and clearance by body fluids.

[0022] (3) By constructing a γ-PGA / CS / MSC-Exos / RLE-6TN complex system, the present invention can directly supplement the functional cells missing after surgery by type II alveolar epithelial cells, which can proliferate and differentiate into type I alveolar epithelial cells and quickly rebuild the integrity of the alveolar barrier; bone marrow stem cell exosomes can further promote the proliferation of transplanted cells and inhibit apoptosis by delivering bioactive molecules such as miRNA and protein, while enhancing cell migration ability and regulating the inflammatory microenvironment of the damaged site, reducing the risk of fibrosis; the synergistic repair effect of the two is significantly better than single cell transplantation or exosome therapy. Attached Figure Description

[0023] Figure 1 The image shows the morphology of the 3D spherical γ-PGA / CS scaffold using scanning electron microscopy (SEM). Figure 2The results of confocal microscopy observation of RLE-6NT live / dead cell staining in each group are shown in the figures: (A) 0h fluorescence observation, (B) 24h fluorescence observation, and (C) 48h fluorescence observation (ag values ​​are PBS, 0.0625, 0.1250, 0.2500, 0.5000, 0.7500, and 1.0000 mg / mL scaffold extract, respectively; scale bar is 50 μm). Figure 3 The results of serum IL-6 and IL-10 protein levels were obtained by ELISA after in vivo implantation of a 3D spherical γ-PGA / CS scaffold. Figure 4 The degradation performance test results of the γ-PGA / CS scaffold are shown in Figure 1. (a) is the curve of the change in the remaining weight of the 3D spherical γ-PGA / CS scaffold during the degradation performance test, and (b) is the curve of the change in the solution pH value of the 3D spherical γ-PGA / CS scaffold during the degradation performance test. Figure 5 (a) Zeta potentials of γ-PGA, CS, γ-PGA / CS stents and MSC-Exos. Figure 5 (b) MSC-Exos loaded on a γ-PGA / CS stent; Figure 6 To observe the seeding results of RLE-6TN cells on a 3D spherical γ-PGA / CS scaffold using confocal microscopy; Figure 7 The fluorescence spectrum of the γ-PGA / CS / MSC-Exos / RLE-6TN composite system prepared in Example 7; Figure 8 (a) HE staining results of lung tissue from rats in each group (scale bar is 20 μm. Experimental groups A, B, and C represent the model + γ-PGA / CS scaffold group, the model + γ-PGA / CS / RLE-6TN group, and the γ-PGA / CS / RLE-6TN / MSC-Exos group, respectively). Figure 8 (b) shows the dynamic changes in AECIIs cell density in the lung tissue of rats in each group at each time point. Specific implementation methods To make the objectives, technical means, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be noted that the scope of this invention is not limited to the embodiments described.

[0024] Example 1 (1) Add 1g of chitosan to 50mL of 0.2M acetic acid solution and vortex for 10min until completely dissolved to obtain chitosan solution; add 0.25g of γ-polyglutamic acid to 50mL of 0.2M NaOH solution and sonicate for 30min until completely dissolved to obtain γ-polyglutamic acid solution; add 10mL of γ-polyglutamic acid solution dropwise to 50mL of chitosan solution at 60℃ and stir magnetically at 37℃ and 500r / min until homogeneous to obtain chitosan / γ-polyglutamic acid mixture; place liquid nitrogen in a stainless steel insulated container, transfer the chitosan / γ-polyglutamic acid mixture into a 100mL spray bottle, spray it into liquid nitrogen for rapid cooling and shaping; after shaping, quickly place it in a freeze dryer for 36h, and then screen it with a 50-mesh (0.355mm) sieve to obtain 3D spherical γ-PGA / CS scaffold with a diameter of less than 355μm.

[0025] (2) Observe the surface morphology of the support using scanning electron microscopy. The 3D spherical γ-PGA / CS scaffold was evenly adhered to the surface of the SEM sample stage, which was pre-prepared with conductive adhesive. Then, gold was sputtered onto the scaffold surface using a vacuum ion sputtering coating system to enhance conductivity. Next, the sample stage was placed in a scanning electron microscope to observe and photograph the scaffold's morphology. Throughout the imaging process, the accelerating voltage of the SEM was maintained at 10 kV.

[0026] Experimental results are as follows Figure 1 As shown, the 3D γ-PGA / CS composite scaffold exhibits a regular spherical morphology with a diameter of approximately 300 μm. It displays a distinct porous network characteristic, possessing large pore sizes and high porosity. The porous structure on the scaffold surface and the interconnected cavities within form continuous channels, providing support for cell migration to the scaffold's core region, facilitating cell adhesion and proliferation, and ensuring nutrient infiltration and metabolic waste removal. This provides the necessary structural basis and feasibility guarantee for subsequent cell seeding.

[0027] Example 2: Detection of scaffold cytotoxicity using live / dead cell staining method 1. Preparation of extract of 3D spherical γ-PGA / CS scaffold Weigh the 3D spherical γ-PGA / CS scaffolds, soak them in anhydrous ethanol for 30 min, centrifuge, and discard the supernatant. Wash the scaffolds three times with PBS buffer, and then sterilize them under UV light for 30 min. Prepare scaffold microsphere suspensions using complete culture medium (F12 (Ham's F-12) basal medium + 10% fetal bovine serum (FBS) + 1% penicillin / spatial antibody (P / S) + 1% ITS + 5 ng / mL EGF) as the matrix, with concentrations of 0.0625, 0.1250, 0.2500, 0.5000, 0.7500, and 1.0000 mg / mL. Incubate the suspensions in a 37℃ constant temperature shaking incubator for 12 h, centrifuge, and collect the supernatant to obtain the scaffold extract.

[0028] 2. Cell resuscitation and passage RLE-6TN cells were removed from liquid nitrogen and rapidly transferred to a 37°C water bath, where they were quickly thawed by agitation. The cells were then centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cell pellet was resuspended in complete culture medium. After observation and adjustment of cell density under an inverted microscope, the cells were transferred to a 37°C, 5% CO2 incubator. The culture medium was changed every 2-3 days. When the cell density reached 70%-90%, the cells were passaged: the old culture medium was discarded, the cells were washed twice with PBS buffer, and a suitable amount of trypsin was added for digestion for 1-2 minutes. Digestion was then stopped with serum-containing complete culture medium, and the cells were gently pipetted to disperse them evenly. After centrifugation at 1000 rpm for 5 minutes, the cells were resuspended in fresh culture medium and aliquoted at a 1:2 ratio into new culture flasks. The cells were then continued to be cultured in a 37°C, 5% CO2 incubator.

[0029] 3. Live / dead cell staining to detect cell compatibility RLE-6NT cells cultured to the logarithmic growth phase were digested and their concentration adjusted to 2.0–3.0 × 10⁻⁶. 5 Cells were seeded at a density of 200 μL / mL into 96-well plates. After static incubation for 24 h to allow cell adhesion, the old culture medium was aspirated, and the cells were washed twice with PBS. The control group received 200 μL of complete culture medium, while the experimental groups received 200 μL of different concentrations of γ-PGA / CS scaffold extract. Each group had six replicates, and cells were incubated at 37°C for 0, 24, and 48 h. After each time point, cells were washed three times with PBS buffer. Staining working solutions were prepared using serum-free complete culture medium (Calcein diluted 1:1000, PI diluted 1:5000), and 1 mL of working solution was added to each well to cover the bottom. Cells were incubated in the dark for 10-15 min. Cells were then washed three times with PBS, and fluorescence was observed using a confocal microscope to analyze cell viability.

[0030] The results are as follows Figure 2As shown in (A), (B), and (C), compared with the control group (containing only complete culture medium), after treatment with different concentrations of γ-PGA / CS scaffold extract for 24 h and 48 h, there were no significant differences in the intensity and distribution of green fluorescence (Calcein staining, marking live cells) in RLE-6NT cells. The green fluorescence signal was uniform and bright, indicating that the γ-PGA / CS scaffold extract treatment did not significantly affect cell viability, and the number of live cells remained at a high level. Simultaneously, no large concentrated areas of red fluorescence (PI staining, marking dead cells) were observed in the field of view of any of the extract treatment groups; only occasional individual red fluorescent spots were seen, suggesting that the scaffold extract did not induce large-scale cell death. These findings demonstrate that the γ-PGA / CS scaffold has good cell compatibility.

[0031] Example 3: Tissue compatibility testing of γ-PGA / CS scaffold 1. A rat lung resection model was established using intraperitoneal anesthesia. A mixed anesthetic solution was prepared at a dose of 20 mg / kg of acetaminophen and 5 mg / kg of celazine, and administered to the rats via intraperitoneal injection. After the anesthesia took effect, the hair in the chest area was shaved with an electric shaver. The rats were then fixed in a supine position on the operating table (limbs secured with rubber bands). The surgical area was disinfected three times with povidone-iodine and draped with sterile surgical towels. During the surgery, the chest wall muscles were dissected layer by layer, the chest cavity was opened to expose the lung lobes, the root of the right middle lobe was gently dissected and double-ligated with silk sutures, and then one-quarter of the lung lobe was removed. Under aseptic conditions, a γ-PGA / CS scaffold was precisely placed at the lung tissue section, and the chest cavity and skin wound were sutured layer by layer. Postoperatively, the rats were managed with routine feeding and care. Seven days later, the rats were euthanized using an overdose anesthesia method, and serum samples were collected.

[0032] 2. The expression levels of two inflammatory factors, IL-6 and IL-10, in serum were detected by ELISA, and the intensity of the inflammatory response in the body after γ-PGA / CS stent implantation was evaluated by this indicator. The ELISA procedure was strictly followed according to the instructions of the corresponding kits: Before detection, the IL-6 and IL-10 ELISA kits were removed from the -80℃ ultra-low temperature freezer and allowed to equilibrate to room temperature for 20 minutes. 100 μL of standard or serum sample was added to each well of the ELISA plate. After addition, the plate was sealed with sealing film and incubated at 37℃ for 90 minutes. After incubation, the waste liquid was discarded, and 100 μL of biotinylated antibody working solution was added to each well. Incubation continued at 37℃ for 60 minutes. The waste liquid was then discarded, and the plate was washed three times. 100 μL of HRP conjugate working solution was added to each well, and incubation was continued at 37℃ in the dark for 30 minutes. After incubation, the waste liquid was discarded, and the plate was washed five times. 90 μL of TMB substrate solution was added to each well, and color development was performed at 37℃ in the dark for 20 minutes. Finally, 50 μL of stop solution was added to each well to terminate the reaction. The ELISA plate was immediately placed in a microplate reader, and the absorbance of each well was measured at 450 nm. The calc software analyzes the test data and calculates the relative protein content of inflammatory factors.

[0033] Experimental results are as follows Figure 3 As shown, compared with the sham surgery group that only underwent surgery but did not remove lung tissue, the serum IL-6 protein expression level of rats in the lung resection model group was significantly increased, while the IL-10 protein level was significantly decreased. This result indicates that lung resection surgery has triggered a local inflammatory response in the body.

[0034] Further comparison revealed that the expression levels of IL-6 and IL-10 inflammatory factors were not statistically different between the experimental group and the model group implanted with the γ-PGA / CS scaffold. This indicates that the γ-PGA / CS scaffold has good tissue safety and will not induce additional immune inflammatory responses after implantation, which meets the requirements for in vivo application of biomedical materials.

[0035] Example 4: Degradability determination of 3D γ-PGA / CS scaffold Accurately weigh a certain mass of γ-PGA / CS scaffold sample and record the initial weight as W0. Immerse the sample in PBS buffer and place it in a 37℃ constant temperature shaking incubator, shaking at a frequency of 30 times / min. Remove the sample at preset time points, measure the pH value of the solution, and plot the pH change curve. Then gently rinse the sample twice with ultrapure water, centrifuge, freeze-dry, and weigh it again, recording the weight as W1. The degradation rate is calculated using the following formula: Degradation rate = (W0 - W1) / W0 × 100%. Plot the γ-PGA / CS scaffold degradation rate curve. (The average value of three parallel samples in each group is taken.)

[0036] Experimental results are as follows Figure 4 As shown in (a) and (b), the experimental results indicate that under the normal physiological environment simulated by PBS, the remaining mass of the scaffold decreased slowly and steadily over time, demonstrating good in vitro degradation behavior. Simultaneously, the pH of the solution used to soak the scaffold gradually decreased from an initial 7.4 to around 7.0, with small overall fluctuations and mild volatility, without drastic acid-base fluctuations. These results demonstrate that the γ-PGA / CS composite scaffold releases degradation products gently and gradually during degradation, maintaining stable pH levels. It avoids drastic changes in the local microenvironment caused by the rapid accumulation of acidic or alkaline products, effectively maintaining a cell growth microenvironment close to the body's normal physiological state, and preventing adverse effects on cell adhesion, proliferation, differentiation, and tissue regeneration processes. This further proves that the scaffold possesses excellent biocompatibility and potential for in vivo application.

[0037] Example 5: Zeta potential detection and preparation of γ-PGA / CS / MSC-Exos 1. Extraction of MSC-Exos MSC cells were cultured using standard methods. To minimize interference from impurities during exosome extraction, the culture medium was replaced with one free of fetal bovine serum (FBS) 24 hours before the cells reached the logarithmic growth phase. The cell culture supernatant was collected and transferred to 50 mL centrifuge tubes. Gradient centrifugation was performed at 4°C using a high-speed refrigerated centrifuge: first, centrifugation at 500 g for 10 min to remove suspended cells; then, centrifugation at 2000 g for 40 min to remove dead cells and cell debris; next, centrifugation at 10000 g for 40 min; and finally, ultracentrifugation at 100000 g for 90 min.

[0038] The above-mentioned complete culture medium for RLE-6TN cells without fetal bovine serum (FBS) consists of F12 (Ham's F-12) basal medium + 1% penicillin-dipase (P / S) + 1% ITS + 5 ng / mL EGF.

[0039] After centrifugation, the supernatant was gently discarded, and a small amount of milky white precipitate was visible at the bottom of the tube, which was the desired exosome. To further remove impurities, the precipitate was resuspended in PBS and then centrifuged again at 100,000 g for 90 min to obtain the purified exosome precipitate. The exosome precipitate was resuspended in 200 μL of PBS, aliquoted into EP tubes, and stored at -80°C for later use.

[0040] 2. Zeta potential detection of γ-PGA / CS stent and MSC-Exos The potential of the samples was detected using a Zeta potential analyzer. γ-PGA, CS, γ-PGA / CS scaffold, and MSC-Exos were diluted 10-fold with ddH2O. Each group of samples was then added to the sample cell using a 1000 μL pipette, and the samples were analyzed to generate Zeta potential maps. Three replicates were performed for each group. Results are expressed as mean ± standard deviation.

[0041] 3. Preparation of γ-PGA / CS / MSC-Exos Before the experiment, MSC-Exos were labeled and traced using the fluorescent dye Dio. First, 1 μL of Dio dye was diluted with 1 mL of PBS buffer to prepare a staining working solution. The exosome precipitate was resuspended in this working solution and incubated at 37°C in the dark for 20 min to complete the fluorescent labeling of the exosomes. After labeling, unbound free dye was thoroughly removed by ultracentrifugation to obtain purified fluorescently labeled MSC-Exos.

[0042] 1 mL of the above fluorescently labeled 10×10 9 Bone marrow stem cell exosomes at a density of 1 / mL were co-incubated with 1 mg of γ-PGA / CS composite scaffold for 4 h to allow MSC-Exos to fully interact with the γ-PGA / CS scaffold. After incubation, the scaffold was washed multiple times with PBS buffer to remove unbound free exosomes. The bound γ-PGA / CS / MSC-Exos were observed under a laser confocal microscope. The distribution and intensity of the green fluorescence signal on the scaffold surface and in the internal pores were used to determine the attachment status and enrichment degree of MSC-Exos on the scaffold, thereby quantitatively and qualitatively evaluating the binding capacity, loading efficiency, and spatial distribution characteristics between exosomes and the scaffold.

[0043] Experimental results are as follows Figure 5 As shown in (a)(b). Figure 5(a) The test results showed that the zeta potential of CS was +58.6 mV, indicating a positive surface charge; the potential of γ-polyglutamic acid was -25.8 mV, indicating a negative surface charge; while the potential of the prepared γ-PGA / CS composite scaffold was +15.4 mV, exhibiting a weak positive charge overall. On the one hand, the potential characteristics of the three materials corroborated each other, confirming from an electrochemical perspective that γ-PGA and CS were successfully combined, and the weak positive potential of the γ-PGA / CS scaffold was more suitable for the cell growth microenvironment; on the other hand, the potential of MSC-Exos was -17.4 mV. The weakly negatively charged MSC-Exos and the weakly positively charged γ-PGA / CS composite scaffold could form a stable cation-anion interaction through electrostatic attraction. This charge-complementary affinity could effectively drive the enrichment, adsorption, and fixation of exosomes on the scaffold surface and in the internal pores, preventing their diffusion and loss in the physiological environment, and achieving efficient loading and local retention of exosomes on the scaffold. Figure 5 (b) The detection results showed that Dio dye stained MSC-Exos green, and the surface and interior of the γ-PGA / CS scaffold showed clear green fluorescence signals, with the fluorescence distribution concentrated on the scaffold, revealing that MSC-Exos and the γ-PGA / CS scaffold were combined due to electrostatic interaction, and γ-PGA / CS / MSC-Exos was successfully constructed.

[0044] Example 6: RLE-6NT cells seeded on a γ-PGA / CS scaffold 1. Construction of composite systems After sterilizing the 3D spherical γ-PGA / CS scaffold with UV irradiation, accurately weigh 1 mg and place it at the bottom of a 15 mL centrifuge tube. Add 2 mL of RLE-6NT cell suspension to the tube, loosen the cap, and place it vertically on a tube rack. Incubate statically in a 37°C CO2 incubator for 3 h. Then, gently transfer the γ-PGA / CS / RLE-6NT to a 10 cm cell culture dish, add 5 mL of complete culture medium, and continue culturing. Replace the medium with fresh medium every day thereafter.

[0045] The complete culture medium is RLE-6TN cell complete culture medium, which consists of F12 (Ham's F-12) basal medium + 10% fetal bovine serum (FBS) + 1% penicillin / spatial antibody (P / S) + 1% ITS + 5 ng / mL EGF.

[0046] 2. Characterization of composite systems The scaffolds / cells were harvested from the culture dishes at days 1, 3, and 5, and the cells were labeled with live / dead cell dyes. Cell proliferation in the scaffolds was observed under a two-photon laser confocal microscope.

[0047] Experimental results are as follows Figure 6As shown, at 1 day, a small amount of green fluorescence was distributed around the scaffold, which was due to the initial construction of γ-PGA / CS / RLE-6NT. At 3 days, the fluorescence increased, with strong green fluorescence signal predominating in the field of view and only a very small amount of red fluorescence signal, indicating that the cells proliferated on the scaffold. At 5 days, the fluorescence was widely distributed on the surface and inside of the scaffold, indicating that the cells could proliferate in large quantities on the surface and in the pores inside the scaffold and were in good growth condition.

[0048] Example 7 γ-PGA / CS / MSC-Exos / RLE-6TN Add 2 mL of 2.0 × 10 5 RLE-6TN alveolar epithelial cells per mL were seeded onto a 1 mg γ-PGA / CS / MSC-Exos scaffold (prepared in Example 5), statically cultured for 3 h in a 37 °C CO2 incubator, then transferred to a cell culture dish, supplemented with 5 mL of culture medium, and cultured for 1-5 days; the culture medium was changed every day.

[0049] The culture medium is RLE-6TN complete cell culture medium, which consists of F12 (Ham's F-12) basal medium + 10% fetal bovine serum (FBS) + 1% penicillin / spatial antibody (P / S) + 1% ITS + 5 ng / mL EGF.

[0050] MSC-Exos were labeled with Dio dye using the same method as in Example 5, and the location of exosomes was determined by observing green fluorescence using confocal microscopy. Cells were also labeled with DAPI dye, and their location was determined by observing blue fluorescence. The results are shown in the figure below. Figure 7 It can be clearly seen that exosomes (MSC-Exos) are stained green and cell nuclei (RLE-6TN) are stained blue. Both green and blue fluorescence are distributed on the surface and inside the γ-PGA / CS scaffold, and MSC-Exos are distributed around the RLE-6NT cells, indicating the successful construction of the composite system γ-PGA / CS / MSC-Exos / RLE-6TN.

[0051] Example 8: Analysis of the Repair Effect of 3D Scaffold Based on Bone Marrow Stem Cell Exosomes / Lung Cells A lung resection animal model was established in SD rats. The sham-operated group, model group, model + γ-PGA / CS scaffold group, model + γ-PGA / CS / RLE-6TN group, and γ-PGA / CS / RLE-6TN / Exos group were implanted into the lung resection site, respectively. Rats in each group were sacrificed using overdose anesthesia on postoperative days 3, 7, 14, and 28. After complete removal of lung tissue, it was fixed in 4% paraformaldehyde solution for 24 hours. Subsequently, the lung tissue underwent gradient dehydration with ethanol, xylene clearing, and routine paraffin embedding to prepare paraffin blocks. The paraffin blocks were serially sectioned, attached to glass slides, and dried and fixed. After dewaxing and rehydration, the sections were stained with hematoxylin for 5-10 min, rinsed with running water to remove excess stain, differentiated with 1% hydrochloric acid alcohol, bluing, and then stained with eosin for 2-5 min. Afterward, the sections underwent gradient dehydration with ethanol, xylene clearing, and finally mounted with neutral resin. The pathological morphology of lung tissue was observed and images were acquired under an inverted microscope, and the number of type II alveolar epithelial cells was statistically analyzed.

[0052] Experimental results are as follows Figure 8 As shown in (a)(b), experimental groups A, B, and C represent the model + γ-PGA / CS scaffold group, the model + γ-PGA / CS / RLE-6TN group, and the model + γ-PGA / CS / RLE-6TN / Exos group, respectively. Figure 8 (a) The H&E results showed that the lung tissue of the sham-operated rats was structurally intact, morphologically normal, with clear alveolar boundaries and wide cavities, exhibiting a typical honeycomb-like normal tissue structure. The model group and experimental groups A, B, and C all showed significant acute lung tissue injury after surgery: significant thickening of the alveolar septa, accompanied by obvious inflammatory infiltration and fibrosis, and disordered alveolar cavity morphology and structural collapse. Compared with the model group, the pathological morphology of the lung tissue in experimental group A was not significantly improved; experimental group B showed some tissue repair, with a reduction in the degree of lung injury; experimental group C showed a more prominent repair effect, exhibiting a clear time dependence, with the lung tissue structure gradually recovering and the repair effect becoming more significant as the observation time increased (3, 7, 14, 28 days).

[0053] Figure 8(b) Counting results showed no significant difference in AECII cell density at each time point in the sham surgery group, suggesting that the number of type II alveolar epithelial cells in the lungs can remain stable without surgical trauma. In contrast, the number of AECII cells with mitotic phase increased to varying degrees in all lung resection model groups. The AECII density in the model group was only slightly higher than normal 3 days post-operation and remained at a relatively low proliferation level throughout the subsequent observation period, suggesting that the proliferation and differentiation rate of the body's own AECII cells is slow after simple lung resection, and the endogenous repair capacity is limited. There was no statistically significant difference in the number of AECII cells in experimental group A at each time point compared with the model group, indicating that implantation of γ-PGA / CS scaffold alone did not significantly promote the proliferation of AECII cells after lung injury, and the repair effect was not significant. The AECII density in experimental group B was consistently higher than that in experimental group A from 3 to 28 days post-operation, indicating that the scaffold combined with exogenous lung cell transplantation can effectively increase the number of local AECII cells, directly compensate for the lack of cells in the defect area, and thus accelerate lung tissue repair. In experimental group C, the density of AECIIs reached 547 cells / mm² 3 days after surgery, which was significantly higher than that of the other groups and remained at a high level thereafter. This suggests that bone marrow stem cell exosomes can significantly promote the proliferation and survival of AECIIs in the early postoperative period. With the synergistic effect of exogenous lung cells, its proliferative effect was further enhanced, providing an important cellular basis for the rapid reconstruction of the lung epithelial barrier and the restoration of its structure and function.

Claims

1. A method for constructing a 3D scaffold for repairing lung defects, characterized in that, Includes the following steps: S1: Add the γ-polyglutamic acid solution dropwise to the chitosan solution, stir at a constant speed until the mixture is uniform, quickly mold the mixture, freeze dry to obtain a 3D γ-PGA / CS scaffold; S2: Exosomes of bone marrow stem cells and RLE-6TN alveolar epithelial cells were extracted and co-incubated with γ-PGA / CS scaffolds to obtain γ-PGA / CS / MSC-Exos and γ-PGA / CS / MSC-RLE-6TN. or S3: Alveolar epithelial cells RLE-6TN were seeded on a γ-PGA / CS / MSC-Exos scaffold and co-incubated to obtain γ-PGA / CS / MSC-Exos / RLE-6TN.

2. The construction method according to claim 1, characterized in that, In S1, the concentration of the γ-polyglutamic acid solution is 5 g / L, and the solvent is a 0.2 mol / L NaOH solution; the concentration of the chitosan solution is 20 g / L, and a 0.2 mol / L acetic acid solution is used as a co-solvent; the volume ratio of the γ-polyglutamic acid solution to the chitosan solution is 1:

5.

3. The construction method according to claim 2, characterized in that, The γ-polyglutamic acid has a molecular weight of 1.1 million; the chitosan has a degree of deacetylation ≥95%, a viscosity of 20-500 mPa.s, and a pH value of 6.5-8.

5.

4. The construction method according to any one of claims 1-3, characterized in that, In S2, the concentration of the bone marrow stem cell exosomes is 1×10⁻⁶. 9 ~12×10 9 Particles / mL; the ratio of bone marrow stem cell exosomes to γ-PGA / CS scaffold is: 1 mL of 1×10⁻⁶ granules per 1 mg γ-PGA / CS scaffold. 9 ~12×10 9 Bone marrow stem cell exosomes per granule / mL.

5. The construction method according to claim 4, characterized in that, In S2, when bone marrow stem cell exosomes are used, the co-incubation conditions are 37°C for 4 hours; when alveolar epithelial cells RLE-6TN are used, the co-incubation is performed by statically culturing in a 37°C CO2 incubator for 3 hours, then transferring the cells to a cell culture dish, adding 5 mL of culture medium, and continuing the culture for 1-5 days; the culture medium is changed every day.

6. The construction method according to any one of claims 1-3, characterized in that, In S3, weigh 1 mg of γ-PGA / CS / MSC-Exos scaffold and add 2 mL of 2.0~3.0×10 5 RLE-6TN cell suspension per mL; the co-incubation was performed by statically culturing the cells in a 37°C CO2 incubator for 3 hours, then transferring them to a cell culture dish, adding 5 mL of culture medium, and continuing the culture for 1-5 days; the culture medium was changed every day.

7. A 3D scaffold prepared using the construction method according to any one of claims 1-6.

8. The application of the 3D scaffold as described in claim 7 in the preparation of materials for repairing lung defects.

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