Visualizing captured stem cells in hydrogel scaffolds and methods of making and using the same

Abstract

The application relates to the technical field of hydrogel scaffolds, and particularly discloses a visualized hydrogel scaffold for capturing stem cells and a preparation method and application thereof. A multifunctional hydrogel scaffold based on biological orthogonal chemistry, ultrasonic visualization and ultrasonic response regulation is constructed, which is used for solving the problems of low homing rate, difficulty in real-time monitoring and incapability of spatiotemporal regulation of functions in stem cell treatment of bone defects. The hydrogel scaffold provided by the application can efficiently capture mesenchymal stem cells under ultrasonic real-time monitoring, and can open the osteogenic differentiation function under ultrasonic regulation, thereby promoting efficient bone repair.

Description

Technical Field

[0001] This application relates to the field of hydrogel scaffold technology, and in particular to a hydrogel scaffold for visually capturing stem cells, its preparation method, and its application. Background Technology

[0002] Bone defects caused by traffic accidents, bone tumors, or specific diseases (such as chronic inflammation and necrosis) cause severe physical and psychological harm to patients. Currently, clinical methods for repairing bone defects include autologous bone grafting and allogeneic bone grafting. However, the former can lead to secondary trauma to the bone harvesting site and potential complications, while the latter carries drawbacks such as immune rejection and the risk of infection. Therefore, the reconstruction and repair of bone defects remains challenging, especially in cases of irregularly shaped, deep bone defects.

[0003] Against this backdrop, bone tissue engineering (BTE) has gradually emerged as a novel approach to address this challenge, and mesenchymal stem cells (MSCs) have become an ideal cell source for BTE due to their inherent repair and differentiation potential. Multiple studies have shown that MSC transplantation promotes bone regeneration in preclinical models. Among various MSC types, bone marrow-derived MSCs (BMSCs) can not only participate in bone regeneration through osteogenic differentiation but also promote bone repair by supporting the local microenvironment, such as regulating angiogenesis. Furthermore, in spinal cord injury models, transplanted bone marrow MSCs can recruit resident pericytes and promote vascular maturation. Therefore, the appropriate introduction of exogenous bone marrow MSCs is of great significance for the repair of bone defects.

[0004] However, due to the lack of appropriate cell surface receptors for adhesion and homing, bone marrow mesenchymal stem cells (BMSCs) have a low homing rate from the circulatory system or peripheral tissues to bone defect sites. Currently, there is a lack of real-time visualization imaging tools for BMSCs, making it impossible to monitor cell homing trajectories and numbers in real time, and difficult to monitor the activity of stem cells after in vivo injection. After exogenous stem cells enter the bone defect site, there is a lack of suitable methods to regulate stem cell function at specific times and locations, enabling them to undergo osteogenic differentiation when needed. Summary of the Invention

[0005] The purpose of this application is to overcome the shortcomings of the prior art by providing a hydrogel scaffold for visually capturing bone marrow mesenchymal stem cells, its preparation method, and its applications. This hydrogel scaffold achieves real-time ultrasound tracking and stem cell function modulation. This application not only provides a novel strategy for visually capturing stem cells at bone defect sites but also provides clear evidence that ultrasound can modulate bone repair in real time. Given the crucial role of stem cells in bone regeneration, this efficient, traceable, and controllable method has great potential for treating a wide range of bone defect-related diseases and advancing the clinical translation of bone marrow mesenchymal stem cell-based therapies.

[0006] To achieve the above objectives, the technical solution adopted in this application is as follows: This application provides a method for preparing a hydrogel scaffold for visually capturing stem cells, comprising the following steps: S1. Extract bio-bubbles, disperse the bio-bubbles in buffer solution, add DBCO-NHS ester dropwise to react, dialyze, centrifuge, and obtain the DBCO-modified bio-bubbles; S2. Extract mesenchymal stem cells, label mesenchymal stem cells with azide groups, incubate azide-labeled mesenchymal stem cells with DBCO-modified biovesicles obtained in step S1, and obtain mesenchymal stem cells loaded with TCO groups and biovesicles by adding N3-PEG2000-TCO. S3. Add methacrylic anhydride to the gelatin solution and react, then freeze-dry to obtain the GelMA precursor; dissolve the GelMA precursor in buffer solution, add Methyltetrazine-NHS ester, dialyze to obtain the Gel-Tz precursor; S4. Crosslink the photoinitiator and the Gel-Tz precursor to obtain a Gel-Tz hydrogel. The Gel-Tz hydrogel captures mesenchymal stem cells loaded with TCO groups and biological vesicles to obtain a hydrogel scaffold for visually capturing stem cells.

[0007] This application constructs a multifunctional hydrogel scaffold based on bioorthogonal chemistry, ultrasound visualization, and ultrasound response regulation to address the challenges of low homing rate, difficulty in real-time monitoring, and inability to control function in stem cell therapy for bone defects.

[0008] This application achieves the specific capture of mesenchymal stem cells (MSCs) through the inverse electron demand Diels-Alder reaction (IEDDA). First, an azide group (N3) is introduced onto the surface of bone marrow mesenchymal stem cells (BMSCs) using a cell metabolism labeling technique (Ac4ManNAz). Simultaneously, a trans-cyclooctene (TCO) group is modified onto the surface of stem cells carrying vesicles (GVs) via a linker arm (forming a BMSCs-GV-TCO complex, i.e., mesenchymal stem cells loaded with TCO groups and vesicles). On the other hand, a hydrogel scaffold (GelMA) is modified with a tetrazine group (Tz), forming a Gel-Tz scaffold. When the TCO-loaded and vesicle-loaded MSCs come into contact with the Gel-Tz, a rapid, specific, and biocompatible bioorthogonal click chemistry reaction occurs between the TCO and Tz, thereby firmly capturing the MSCs on the scaffold.

[0009] The hydrogel scaffold provided in this application also enables visual monitoring by utilizing gas-filled gas vesicles (GVs) as an ultrasound contrast agent. GVs are protein nanostructures filled with gas, whose acoustic impedance differs significantly from that of surrounding tissues, strongly reflecting ultrasound signals. After modifying the surface of mesenchymal stem cells with GVs, the mesenchymal stem cells themselves become ultrasound contrast agents, making it possible to track the migration, homing, and accumulation of mesenchymal stem cells on the scaffold in real time, non-invasively, and dynamically using clinically common contrast-enhanced ultrasound (CEUS) technology.

[0010] The hydrogel scaffold provided in this application can also achieve functional modulation by triggering GV rupture using the mechanical effect of ultrasound. When low-intensity pulsed ultrasound (LIPUS) is applied to the target area, its mechanical waves can cause GVs to collapse.

[0011] The rupture of GVs can achieve two regulatory functions: a) restoring stem cell migration function; b) inducing differentiation: restoring the osteogenic differentiation function of mesenchymal stem cells.

[0012] In some specific embodiments, the mesenchymal stem cells include bone marrow mesenchymal stem cells.

[0013] In a preferred embodiment of the method for preparing the hydrogel scaffold for visually capturing stem cells described in this application, the pH value of the buffer solution in step S1 is 7.5~8.5. biological bubble concentration OD 500 It is 1.5~2.0.

[0014] In a preferred embodiment of the method for preparing the hydrogel scaffold for visually capturing stem cells described in this application, in step S1, the mass concentration of DBCO-NHS ester is 1~5 mM, preferably 1 mM.

[0015] In this application, DBCO-NHS ester is used to react with the primary amino groups on the surface of GVs to form stable amide bonds.

[0016] In a preferred embodiment of the method for preparing the hydrogel scaffold for visually capturing stem cells described in this application, the source of the azide group in step S2 includes azide-modified mannose.

[0017] In some specific embodiments, the concentration of azide-modified mannose is 20-50 µM, and the labeling treatment time is 24-48 h.

[0018] This application incorporates azide-modified mannose, which can effectively label mesenchymal stem cells (BMSCs). The concentration and labeling time of the azide-modified mannose determined the labeling density of the azide group (N3) on the surface of BMSCs, directly affecting the subsequent ligation efficiency with DBCO-GVs. Insufficient concentration or labeling time of the azide-modified mannose will lead to underlabeling, while excessive concentration may cause cytotoxicity.

[0019] In a preferred embodiment of the method for preparing the hydrogel scaffold for visually capturing stem cells described in this application, in step S2, the concentration OD of the DBCO-modified biovapors... 500 The value is 1~1.5, and the incubation time is 6~12 hours.

[0020] The DBCO-modified biovesicles of this application, at the above concentration, increase the loading of biovesicles on the surface of mesenchymal stem cells, thereby affecting the signal intensity of ultrasound imaging and the sensitivity of subsequent ultrasound modulation. Among them, OD... 500 It is a key optical parameter for measuring GV concentration.

[0021] In a preferred embodiment of the method for preparing the hydrogel scaffold for visually capturing stem cells described in this application, the mass concentration of N3-PEG2000-TCO in step S2 is 100 µM.

[0022] In the technical solution of this application, N3-PEG2000-TCO is added, wherein DBCO and N3 in azide-labeled mesenchymal stem cells undergo a rapid bioorthogonal reaction, thereby linking BMSCs and GVs.

[0023] The addition of N3-PEG2000-TCO introduces TCO groups, further introducing TCO groups onto the cells that connect to the biovaposomes; the TCO groups can undergo a bioorthogonal reaction with the Tetrazine groups on the surface of the hydrogel, enabling the mesenchymal stem cells loaded with biovaposomes to connect rapidly with the hydrogel.

[0024] The mass concentration of N3-PEG2000-TCO affects the grafting rate of TCO and is one of the core factors determining the capture reaction efficiency of BMSCs-GV-TCO and Gel-Tz scaffolds.

[0025] In a preferred embodiment of the method for preparing the hydrogel scaffold for visually capturing stem cells described in this application, in step S3, the ratio of methacrylic anhydride to porcine gelatin is (2~4ml):10g.

[0026] Preferably, the reaction time in step S3 is 1 hour.

[0027] In the technical solution of this application, the methacrylic anhydride and gelatin are used in the above ratio and reaction time, which can improve the degree of methacrylation of GelMA, resulting in better mechanical properties (such as stiffness and elasticity) and degradation rate of the hydrogel.

[0028] In a preferred embodiment of the method for preparing the hydrogel scaffold for visually capturing stem cells described in this application, in step S3, the mass ratio of Methyltetrazine-NHS ester to GelMA precursor is (1~4) mg: 180 mg.

[0029] This application incorporates Methyltetrazine-NHS ester, which can react with the primary amino group in the GelMA (methacrylated gelatin) chain. Its main function is to covalently link the methyltetrazine functional group to the GelMA molecule, as the NHS ester reacts with the primary amino group to form a stable amide bond.

[0030] The Methyltetrazine-NHS ester and GelMA precursor in this application are used in the above-mentioned mass ratio, resulting in a better density of Tz groups in Gel-Tz. Too low a density will lead to low capture efficiency; too high a density may affect the gelling properties of the hydrogel or cause non-specific reactions.

[0031] In a preferred embodiment of the method for preparing the hydrogel scaffold for visually capturing stem cells described in this application, in step S4, the photoinitiator includes lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid.

[0032] This application also provides the above-mentioned method for preparing a hydrogel scaffold for visually capturing stem cells, resulting in a hydrogel scaffold for visually capturing stem cells.

[0033] The hydrogel scaffold provided in this application can efficiently capture mesenchymal stem cells under real-time ultrasound monitoring and activate osteogenic differentiation under ultrasound regulation, thereby promoting efficient bone repair.

[0034] This application also provides the application of the above-mentioned visualized stem cell-capturing hydrogel scaffold in the preparation of bone defect repair products.

[0035] Compared with the prior art, this application has the following beneficial effects: This application provides a hydrogel scaffold for visually capturing bone marrow mesenchymal stem cells, its preparation method, and its applications. The hydrogel scaffold of this application possesses highly efficient and active stem cell homing and anchoring capabilities. Through a click chemistry strategy, TCO groups are modified on the surface of mesenchymal stem cells, and their complementary ligand Tz groups are modified on the hydrogel scaffold. When mesenchymal stem cells reach the vicinity of the hydrogel scaffold, TCO and Tz can undergo a rapid bioorthogonal reaction, achieving active capture of stem cells. This significantly improves the retention rate and utilization rate of stem cells at the lesion site, laying a solid foundation for subsequent repair. The hydrogel scaffold of this application has non-invasive, real-time, and dynamic in vivo tracking and monitoring capabilities. Natural "biological vesicles" (GVs) are loaded onto stem cells as ultrasound contrast agents. GVs are sensitive to ultrasound and can generate strong contrast enhancement signals. Using clinically common ultrasound imaging equipment, the injection path of stem cells, their migration trajectory to the defect site, and their capture and enrichment on the scaffold can be visualized non-invasively, in real-time, and repeatedly during and after surgery. This achieves "visualized" monitoring of the treatment process, providing intuitive evidence for efficacy evaluation and treatment plan optimization. Using low-intensity pulsed ultrasound (LIPUS) as an external trigger source, ultrasound can break down GVs on the surface of stem cells, restore the osteogenic differentiation and proliferation and migration functions of stem cells, effectively realize remote ultrasound regulation of stem cells, and prevent side effects such as ectopic proliferation and differentiation of stem cells. Attached Figure Description

[0036] Figure 1 Transmission electron micrographs of GVs and DBCO-GVs; Figure 2 The ultraviolet and infrared spectra of GVs and DBCO-GVs are shown. Figure 3 Fluorescence imaging of the BMSCs-N3 and BMSCs-GV-TCO complex; Figure 4 The synthesis diagram of Gel-Tz hydrogel and the characterization diagram of its components are shown. Figure 5 Figure showing the cell adhesion assessment results of Gel-Tz hydrogels at different concentrations; Figure 6 For GVs, DBCO-GVs, GVs-PEG 2000 - Ultrasonic imaging of TCO nanobubbles; Figure 7 Image of the BMSCs-GV-TCO complex; Figure 8 GVs-PEG 2000 -TCO Target Assessment Map; Figure 9 A target evaluation diagram of the BMSCs-GV-TCO complex; Figure 10Image showing ultrasound-triggered GV rupture; Figure 11 This is a map showing the regulation of osteogenic differentiation in BMSCs-GV-TCO mediated by ultrasound. Figure 12 A map showing the ultrasound-mediated migration regulation of BMSCs-GV-TCO. Figure 13 In vivo ultrasound tracking image of BMSCs-GV-TCO captured by Gel-Tz hydrogel at the skull defect site; Figure 14 In vivo fluorescence tracking image of BMSCs-GV-TCO captured by Gel-Tz hydrogel at the skull defect site; Figure 15 A diagram illustrating the therapeutic effects of hydrogel scaffolds that capture stem cells in repairing skull defects in rats. Detailed Implementation

[0037] To better illustrate the purpose, technical solution, and advantages of this application, the following description will be provided in conjunction with the accompanying drawings and specific embodiments.

[0038] In the following examples and comparative examples, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified. Furthermore, the raw materials used in each parallel experiment are the same.

[0039] Example 1: A hydrogel scaffold for visually capturing bone marrow mesenchymal stem cells and its preparation method. This embodiment provides a method for preparing a hydrogel scaffold for visually capturing bone marrow mesenchymal stem cells, including the following steps: S1. Obtaining DBCO-modified bio-bubbles: Halo bacteria were cultured in ATCC medium at 42°C and 120 rpm for 14 days to obtain a culture medium. The culture medium was transferred to a separatory funnel and allowed to stand for one week to allow Halo to produce gaseous villi (GVs) and float to the top. After floating, Halo was separated from the culture medium, lysed with TMC buffer, and centrifuged at 300g for 4 hours at 4°C for a total of three centrifugations to obtain milky white GVs, which were stored at 4°C. The GVs were then dispersed in PBS (pH=8.5, OD500). 500 =2), DBCO-NHS ester (1 mM) was slowly added dropwise; after 6 hours of reaction, the mixture was dialyzed for 48 hours using a dialysis tube (MWCO = 5000); then, DBCO-GVs (DBCO-modified biovesicles) were collected by flotation centrifugation (300 g, 4 h, 4 ℃). The structures of GVs and DBCO-GVs were subsequently analyzed using transmission electron microscopy (TEM). Figure 1Simultaneously, ultraviolet-visible spectrophotometry and infrared spectroscopy were used to confirm that the DBCO group was successfully attached to the surface of GVs. Figure 2 ).

[0040] S2. Obtain mesenchymal stem cells loaded with TCO groups and biological vesicles: BMSCs were extracted from the bone marrow of two-week-old male SD rats. Specifically, the femur and tibia were removed under aseptic conditions and cut off at both ends. The bone marrow was washed with α-medium medium supplemented with 10% FBS and 1% penicillin-streptomycin, and then the cells were incubated under standard cell culture conditions (5% CO2 concentration). The medium was changed every three days. When the cell confluence reached 80%-90%, the BMSCs were passaged. After 3-4 passages of cell expansion, the cells were used for other experiments. To label the cells with azide groups, the BMSCs were treated with Ac4ManNAz (50 µM) for 24 hours to obtain BMSCs-N3. Subsequently, the BMSCs-N3 were washed with PBS and further treated with DBCO-GVs (OD2000). 500 =1) Incubate for 6 hours. After washing BMSCs twice with PBS, add N3-PEG2000-TCO (100 µM) to obtain mesenchymal stem cells loaded with TCO groups and biological vesicles (BMSCs-GV-TCO complex).

[0041] The presence of the N3 group on BMSCs-N3 was confirmed by DBCO-Cy5 staining. Furthermore, DBCO-GVs were labeled with DiL (10 µM) and then co-incubated with BMSCs-N3. The nucleus and cytoskeleton were stained with Hoechst 33342 and FITC-phalloidin, respectively, and imaged using confocal fluorescence microscopy to verify that DBCO-GVs successfully reacted with and linked to the N3 group on the BMSCs surface. The morphology and subcellular localization of GVs in BMSCs-GV were then determined using TEM. The presence of the TCO group on BMSCs-GV-TCO was further verified by Tetrazine-Cy5.5 staining. Figure 3 ).

[0042] S3. Obtain the Gel-Tz precursor containing the Tetrazine group: Solution A was prepared by dissolving 10 g of porcine gelatin in 50 mL of PBS in a 50 °C water bath. Methacrylic anhydride (4 mL) was added to solution A at a rate of 0.2 mL / min using a syringe pump. The reaction was stirred for 1 hour in the dark, and then terminated by adding 200 mL of PBS at 40 °C. The solution was dialyzed against a membrane (3600 Da) for 2 weeks, and then frozen at -80 °C for 2 days. Finally, the solution was freeze-dried for 2 days to obtain the GelMA precursor. The GelMA precursor (180 mg) was dissolved in 1 mL of borate buffer (pH=8.5). Then, 2 mg of Methyltetrazine-NHS ester dissolved in 100 µL DMSO was added dropwise. The reaction was stirred overnight at 50 °C. The solution was dialyzed against a membrane (3600 Da) for 2 weeks, and then frozen at -80 °C for 2 days. Finally, the solution was freeze-dried for 2 days to obtain the Gel-Tz precursor. The GelMA and Gel-Tz precursors were characterized by 1H-NMR, UV spectrophotometry, and infrared spectroscopy. Figure 4 ).

[0043] S4. Under light-protected conditions, 1 mL of phenyl-2,4,6-trimethylbenzoyl lithium phosphinate (0.25%, w / v) was used to mix Gel-Tz precursor (100 mg). After irradiation with light (405 nm) for 30 s, Gel-Tz hydrogel was successfully obtained. Gel-Tz hydrogel captured mesenchymal stem cells loaded with TCO groups and biological vesicles, and a hydrogel scaffold for visually capturing stem cells was obtained.

[0044] In this process, the GelMA precursor is covalently linked to Methyltetrazine-NHS ester (methyltetrazine-benzene-NHS ester) via an amide reaction, yielding tetrazine-functionalized GelMA (Gel-Tz), which enables it to bioorthogonally capture TCO groups. Figure 4 (A). After adding the photoinitiator LAP and irradiating with 405 nm light for 30 seconds, both GelMA and Gel-Tz hydrogels were successfully synthesized. Figure 4 B, Figure 4 (C). Ultraviolet spectroscopy first confirmed the presence of the tetrazine group in Gel-Tz, evidenced by a characteristic absorption peak at 500-550 nm. Figure 4 (D). Subsequently, the disappearance of this characteristic peak after the addition of N3-PEG2000-TCO verified its successful bioorthogonal reaction with TCO. Figure 4 (E). In addition, the hydrogen nuclear magnetic resonance spectrum of Gel-Tz ( Figure 4The results (F) show two characteristic peaks at 8.4 and 7.6 ppm attributed to tetraazine aromatic protons, while peaks corresponding to vinyl protons of methacrylate groups are also retained at 5.3 and 5.6 ppm. To verify the bioorthogonal reactivity of Gel-Tz, we co-incubated GelMA and Gel-Tz hydrogels with TCO-Cy5.5, respectively. Figure 4 As shown in Figure G, Gel-Tz exhibited a strong fluorescent signal due to the rapid tetrazine-TCO cycloaddition reaction, while GelMA showed no detected signal. Notably, the Gel-Tz solution also rapidly changed from pink to colorless and transparent after reacting with TCO-Cy5.5. Swelling analysis over 12 hours indicated that the introduction of the tetrazine group slightly increased the water absorption of the hydrogel. Figure 4 (H). In summary, these results confirm the successful synthesis of Gel-Tz hydrogels, which retain photocrosslinking ability while maintaining robust bioorthogonal reactivity with TCO groups.

[0045] Example 2: A hydrogel scaffold for visually capturing bone marrow mesenchymal stem cells and its preparation method. This embodiment provides a method for preparing a hydrogel scaffold for visually capturing bone marrow mesenchymal stem cells, including the following steps: S1. Obtaining DBCO-modified bio-bubbles: Halo bacteria were cultured in ATCC medium at 42°C and 120 rpm for 14 days to obtain a culture medium. The culture medium was transferred to a separatory funnel and allowed to stand for one week to allow Halo to produce gaseous villi (GVs) and float to the top. After floating, Halo was separated from the culture medium, lysed with TMC buffer, and centrifuged at 300 g for 4 hours at 4°C for a total of three centrifugations to obtain milky white GVs, which were stored at 4°C. The GVs were then dispersed in PBS (pH = 7.5, OD500). 500 DBCO-NHS ester (5 mM) was slowly added dropwise to the solution (MWCO = 1.5); after reacting for 6 hours, the solution was dialyzed for 48 hours using a dialysis tube (MWCO = 5000); then, DBCO-GVs (DBCO-modified biovesicles) were collected by flotation centrifugation (300 g, 4 h, 4 °C).

[0046] S2. Obtain mesenchymal stem cells loaded with TCO groups and biological vesicles: BMSCs were extracted from the bone marrow of two-week-old male SD rats. Specifically, the femur and tibia were removed under aseptic conditions and cut off at both ends. The bone marrow was washed with α-medium medium supplemented with 10% FBS and 1% penicillin-streptomycin, and then the cells were incubated under standard cell culture conditions (5% CO2 concentration). The medium was changed every three days. When the cell confluence reached 80%-90%, the BMSCs were passaged. After 3-4 passages of cell expansion, the cells were used for other experiments. To label the cells with azide groups, the BMSCs were treated with Ac4ManNAz (20 µM) for 48 hours to obtain BMSCs-N3. Subsequently, the BMSCs-N3 were washed with PBS and further treated with DBCO-GVs (OD2000). 500 =1.5) Incubate for 12 hours. After washing BMSCs twice with PBS, add N3-PEG2000-TCO (200 µM) to obtain mesenchymal stem cells loaded with TCO groups and biological vesicles (BMSCs-GV-TCO complex).

[0047] S3. Obtain the Gel-Tz precursor containing the Tetrazine group: Solution A was prepared by dissolving 10 g of porcine gelatin in 50 mL of PBS in a 50 °C water bath. Methacrylic anhydride (2 mL) was added to solution A at a rate of 0.2 mL / min using a syringe pump. The reaction was stirred for 1 hour in the dark, and then terminated by adding 200 mL of PBS at 40 °C. The solution was dialyzed against a membrane (3600 Da) for 2 weeks, and then frozen at -80 °C for 2 days. Finally, the solution was freeze-dried for 2 days to obtain the GelMA precursor. The GelMA precursor (180 mg) was dissolved in 1 mL of borate buffer (pH=8.5). Then, 4 mg of Methyltetrazine-NHS ester dissolved in 100 µL DMSO was added dropwise. The reaction was stirred overnight at 50 °C. The solution was dialyzed against a membrane (3600 Da) for 2 weeks, and then frozen at -80 °C for 2 days. Finally, the solution was freeze-dried for 2 days to obtain the Gel-Tz precursor.

[0048] S4. Under light-protected conditions, 1 mL of phenyl-2,4,6-trimethylbenzoyl lithium phosphinate (0.25%, w / v) was used to mix Gel-Tz precursor (100 mg). After irradiation with light (405 nm) for 30 s, Gel-Tz hydrogel was successfully obtained. Gel-Tz hydrogel captured mesenchymal stem cells loaded with TCO groups and biological vesicles, and a hydrogel scaffold for visually capturing stem cells was obtained.

[0049] The test results of the hydrogel scaffold for visually capturing stem cells provided in this embodiment are similar to those in Example 1.

[0050] Comparative Example 1 Comparative Example 1 provides a method for preparing GVs-PEG2000-TCO, including the following steps: GVs were dispersed in PBS (pH = 8.5, OD500 = 2), and DBCO-NHS ester (1 mM) was slowly added dropwise. After reacting for 6 hours, the mixture was dialyzed for 48 hours using a dialysis tube (MWCO = 5000). Then, DBCO-GVs were collected by flotation centrifugation (300 g, 4 h, 4 °C). DBCO-GVs (OD500 = 1) were incubated with N3-PEG2000-TCO (100 µM) for 6 hours to obtain GVs-PEG2000-TCO.

[0051] Comparative Example 2 Comparative Example 2 provides a method for preparing a GelMA hydrogel, comprising: Solution A was prepared by dissolving 10 g of porcine gelatin in 50 mL of PBS in a 50 °C water bath. Methacrylic anhydride (4 mL) was added to solution A at a rate of 0.2 mL / min using a syringe pump. The reaction was stirred for 1 hour in the dark, and then terminated by adding 200 mL of PBS at 40 °C. The solution was dialyzed against a membrane (3600 Da) for 2 weeks, and then frozen at -80 °C for 2 days. Finally, the solution was freeze-dried for 2 days to obtain the GelMA precursor (GelMA hydrogel).

[0052] Experimental Example 1: Assessment of Cell Adhesion 1. Cell adhesion assessment on Gel-Tz hydrogel: 200 µL of Gel-Tz precursor solution (10% w / v) was pipette into a confocal culture dish, followed by 405 nm photocrosslinking (30 s) to form a hydrogel. After conditioning with culture medium (500 µL, 30 min, 37°C / 5% CO2) and washing with PBS, BMSCs (5 × 10⁶ cells / year) were seeded. 4 After culturing for 24 hours, cells were fixed, stained with FITC-phalloidin / DAPI, and imaged by confocal microscopy to assess adhesion morphology.

[0053] 2. Blood compatibility assessment of different concentrations of Gel-Tz hydrogel: Fresh mouse blood was collected in heparinized tubes, centrifuged (3000 rpm, 10 min) to separate red blood cells, and then washed twice with PBS. The purified red blood cells were diluted to 10% (v / v) in PBS, and 200 μL aliquots were mixed with 1 mL of test solution (distilled water as a positive control, PBS as a negative control, and different concentrations (1.25%, 2.5%, 5%, 10%, 20%) of Gel-Tz hydrogel (Example 1) as experimental groups). After incubation at 37°C for 5 hours, the samples were centrifuged (3000 rpm, 10 min), and 100 μL of the supernatant was transferred to a 96-well plate. The absorbance was measured at 541 nm using a microplate reader.

[0054] The formula for calculating the percentage of hemolysis is: [(OD sample - OD negative) / (OD positive - OD negative)] × 100%. Results are as follows: Figure 5 As shown.

[0055] Experiment 2: GVs, DBCO-GVs, GVs-PEG 2000 Contrast-enhanced ultrasound (CEUS) of TCO and BMSCs-GV-TCO Imaging 1. For nanobubble imaging ( Figure 6 ), to different concentrations (OD) 500 GVs, DBCO-GVs, and GVs-PEG (values ​​= 0.25, 0.5, 0.7, 1.0, and 2.0) 2000 -TCO suspensions (500 μL each) were embedded in agarose phantoms. CEUS was performed using a Vevo F2 system with the following parameters: linear array transducer (frequency 15-29 MHz), imaging depth 20 mm, transmit power 3%, dynamic range 30 dB, and contrast gain set to 0 dB.

[0056] 2. For complex imaging ( Figure 7 BMSCs-GV-TCO complexes were collected from culture plates and suspended at different cell densities (2.5 × 10⁻⁶). 5 5×10 5 10×10 5 20×10 5 and 40×10 5 BMSCs of the same density were used as a control in agarose gels containing cells / mL. CEUS was performed using a Vevo F2 system with the following parameters: linear array transducer (frequency 15-29 MHz), imaging depth 20 mm, emission power 3%, dynamic range 30 dB, and contrast gain set to 0 dB.

[0057] Experiment 3: Gel-Tz on GVs-PEG 2000 In vitro assessment of TCO and BMSCs-GV-TCO capture capacity 1. GVs-PEG 2000-TCO targeting evaluation: The GVs-PEG target was evaluated by incubation with DiL (10 µM) for 12 h in the dark. 2000 -TCO(OD 500 =1) Fluorescent labeling was performed, followed by purification by flotation centrifugation (300 rpm, 4 h). Gel-Tz hydrogel (100 µL / well) was photocrosslinked in a 96-well plate. Fluorescently labeled GV-PEG was then... 2000 A suspension of TCO was added to each well, allowing them to interact for different times (1, 3, 5, and 10 minutes). After washing with PBS, fluorescence images were acquired using an IVIS imaging system (Perkin Elmer, USA). Figure 8 ).

[0058] 2. Targeted Evaluation of BMSCs-GV-TCO Complex: For cell-targeted studies, Gel-Tz hydrogels (300 µL / well) were prepared in 24-well plates. BMSCs-GV-TCO complexes (10 × 10⁻⁶) collected from the culture plates were then analyzed. 5 Cells / mL were added to the surface of the hydrogel. After specified incubation times (0.5, 1, 3, 5, and 10 minutes), unbound complexes were removed by three washes with PBS. Immediately after incubation, fluorescence microscopy imaging and quantitative analysis were performed. Figure 9 ).

[0059] Experiment 4: Regulation of ultrasound-mediated osteogenic differentiation and migration of BMSCs-GV-TCO 1. Ultrasonic-triggered GVs rupture: The GVs suspension (OD) 500 = 1) Exposure to low-intensity ultrasound at a power of 0.1W for 1 second. Changes were recorded by photographing before and after ultrasound treatment. In addition, morphological changes in GVs induced by ultrasound were examined by TEM.

[0060] 2. Assessment of osteogenic differentiation of BMSCs: BMSCs from different treatment groups were cultured in osteogenic induction medium (α-MEM medium supplemented with 10% fetal bovine serum, 100 nmol / L dexamethasone, 10 mmol / L sodium β-glycerophosphate, and 50 μg / ml vitamin C) for 7 or 14 days, washed with PBS, and fixed with 4% paraformaldehyde for 20 minutes. Subsequently, cells were stained with ALP (7 days) and ARS (14 days) kits for 30 minutes, respectively, and then washed three times with ultrapure water. Representative images of early and late osteogenic differentiation were taken using an inverted microscope. Figure 11 ).

[0061] 3. Scratch Healing Assay: Linear scratches were created on confluent cell monolayers in 6-well plates using a 200 μL pipette tip. After washing three times with PBS to remove detached cells, the sonicated group received a single 0.1W low-intensity pulsed ultrasound (LIPUS) exposure for 1 second. Cell migration was recorded by phase-contrast microscopy at 0 h (baseline) and 20 h post-scratch. The relative wound closure area was quantified by measuring the reduction in scratch width using ImageJ software. Figure 12 ).

[0062] Experiment 5: Verification of ultrasound tracing and scaffold repair effects in animals 1. Animal Model Establishment: Make a 1-1.5 cm longitudinal incision on the skull of an adult SD rat (200 g). Laterally fold the skin and periosteum to expose the parietal bone. Under continuous saline irrigation, use an electric drill with a 5 mm diameter trephine to symmetrically create two full-thickness circular defects on the parietal bone, at least 3-5 mm apart, avoiding the major cranial sutures. Verify the size and symmetry of the defects with calipers, and suture the wound in layers after hemostasis.

[0063] 2. In vivo ultrasound tracking of BMSCs-GV-TCO captured by Gel-Tz hydrogel at the skull defect site: After establishing a bilateral rat skull defect model, before layered suturing closure, Gel-Tz hydrogel was dripped into the right side of the bilateral rat skull defect and irradiated with 405nm for 30s for in situ gelation; GelMA hydrogel was dripped into the left side and irradiated with 405nm for 30s for in situ gelation, serving as the control group.

[0064] Both hydrogels underwent in-situ photocrosslinking using 405 nm light irradiation for 30 seconds. DiD-labeled BMSCs-GV-TCO complexes (10 × 10⁻⁶) collected from culture plates were also used. 6 BMSCs / mL were slowly injected subcutaneously along the midline of the skull using a syringe. Real-time ultrasound and fluorescence imaging were then performed to monitor the migration trajectory of the BMSCs-GV-TCO complex and to assess its targeted capture efficiency at the defect site. Figure 13 , Figure 14 ).

[0065] 3. A skull defect model was established, and the defect sites in the experimental groups were subsequently filled with in situ photocrosslinked hydrogels (GelMA or Gel-Tz). Six hours after implantation, specific cell suspensions (BMSCs or BMSCs-GV-TCO) for each group were administered via subcutaneous injection along the midline of the skull. The ultrasound treatment group received transcranial LiPUS irradiation (0.1W, 5s) 12 hours after injection. Longitudinal in vivo micro-CT assessments were performed at 4, 6, and 8 weeks postoperatively to reconstruct and quantify new bone formation. Figure 15 ).

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit the scope of protection of this application. Although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this application without departing from the substance and scope of the technical solutions of this application.

Claims

1. A method for preparing a hydrogel scaffold for visually capturing stem cells, characterized in that, Includes the following steps: S1. Extract bio-bubbles, disperse the bio-bubbles in buffer solution, add DBCO-NHS ester dropwise to react, dialyze, centrifuge, and obtain the DBCO-modified bio-bubbles; S2. Extract mesenchymal stem cells, label mesenchymal stem cells with azide groups, incubate azide-labeled mesenchymal stem cells with DBCO-modified biovesicles obtained in step S1, and obtain mesenchymal stem cells loaded with TCO groups and biovesicles by adding N3-PEG2000-TCO. S3. Add methacrylic anhydride to the gelatin solution and react, then freeze-dry to obtain the GelMA precursor; dissolve the GelMA precursor in buffer solution, add Methyltetrazine-NHS ester, dialyze to obtain the Gel-Tz precursor; S4. Crosslink the photoinitiator and the Gel-Tz precursor to obtain a Gel-Tz hydrogel. The Gel-Tz hydrogel captures mesenchymal stem cells loaded with TCO groups and biological vesicles to obtain a hydrogel scaffold for visually capturing stem cells.

2. The method for preparing the hydrogel scaffold for visually capturing stem cells as described in claim 1, characterized in that, In step S1, the pH value of the buffer solution is 7.5~8.5; biological bubble concentration OD 500 It is 1.5~2.

0.

3. The method for preparing the hydrogel scaffold for visually capturing stem cells as described in claim 1, characterized in that, In step S1, the mass concentration of DBCO-NHS ester is 1~5 mM.

4. The method for preparing the hydrogel scaffold for visually capturing stem cells as described in claim 1, characterized in that, In step S2, the source of the azide group includes azide-modified mannose; And / or, the concentration of azide-modified mannose is 20–50 µM, and the labeling time is 24–48 h.

5. The method for preparing the hydrogel scaffold for visually capturing stem cells as described in claim 1, characterized in that, In step S2, the mass concentration of N3-PEG2000-TCO is 100~200 µM.

6. The method for preparing the hydrogel scaffold for visually capturing stem cells as described in claim 1, characterized in that, In step S2, the concentration OD of the DBCO-modified biovapor is... 500 The value is 1~1.5, and the incubation time is 6~12 hours.

7. The method for preparing the hydrogel scaffold for visually capturing stem cells as described in claim 1, characterized in that, In step S3, the ratio of methacrylic anhydride to gelatin is (2~4ml):10g.

8. The method for preparing the hydrogel scaffold for visually capturing stem cells as described in claim 1, characterized in that, In step S3, the mass ratio of Methyltetrazine-NHS ester to GelMA precursor is (1~4 mg): 180 mg.

9. The visually captured stem cell hydrogel scaffold prepared by the method of any one of claims 1 to 8.

10. The application of the hydrogel scaffold for visually capturing stem cells as described in claim 9 in the preparation of bone defect repair products.

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