Photo-thermal response bFGF hydrogel and application thereof

Abstract

The invention relates to the technical field of biology, in particular to photo-thermal response bFGF hydrogel and application thereof. The hydrogel provided by the invention is a multi-strategy hydrogel capable of realizing intelligent photo-thermal response and programmed adjustment of multiple biological functions. The hydrogel system is based on carboxymethyl chitosan and gelatin, and bFGF is loaded on a BP nanosheet coated with PDA. Under the action of light photothermal therapy, BP induces macrophages to be polarized to an anti-inflammatory M2 phenotype, the immunoregulation ability of the macrophages is recovered, a favorable immune microenvironment is created for bone repair, meanwhile, migration of osteoblasts is promoted, and differentiation of the osteoblasts from an adipogenic phenotype to an osteogenic phenotype is guided. The bFGF can promote reconstruction of a bone and blood vessel network, improve local blood circulation and convey oxygen and nutrient substances to a fracture area. BP can respond to near-infrared light to realize precise and controllable release of bFGF, synergistically exert the effects of reconstructing a vascular network and promoting osteogenic differentiation, guide bone regeneration and provide a new treatment strategy for promoting diabetic fracture healing.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a photothermal response bFGF hydrogel and application thereof. BACKGROUND

[0002] Diabetes and its complications have potential risk of disability and death, which can cause damage to multiple organs and systems such as bone, nerve and blood vessels, and seriously affect the quality of life of patients. Due to abnormal insulin level and long-term high blood sugar environment, the bone metabolism balance of diabetic patients is out of balance and the function of bone cells is impaired, which further causes osteoporosis, the characteristics of which are bone mass loss and destruction of bone tissue microstructure, eventually leading to increased bone fragility and increased risk of fracture. According to statistics, about 20% to 40% of diabetic patients will experience fracture. The factors affecting fracture healing include blood supply of fracture site, differentiation and proliferation of osteoblasts, formation of bone matrix proteins and deposition and mineralization of inorganic substances. Under the condition of diabetes, immune cell dysfunction and osteoblast function damage can lead to decreased bone density and damaged bone vascular network, which further causes delayed fracture healing or nonunion, making the difficulty of fracture repair in diabetic patients higher than that in normal population. Delayed fracture healing can cause long-term pain, joint stiffness, local infection and nerve damage, etc. Therefore, it is very important to propose a treatment method for promoting fracture healing under the condition of diabetes.

[0003] Diabetic fracture healing is a complex physiological process involving multi-stage regulation, including recruitment of mesenchymal stem cells, differentiation and maturation of osteoblasts, callus formation and remodeling, and finally tissue mineralization, which is also a major difficulty and challenge in the medical field. In the field of bone tissue engineering, various biomaterials have been developed to promote bone regeneration.

[0004] Fibroblast growth factor (FGF) family members play an important regulatory role in the growth, proliferation, differentiation and regeneration of cells. As a kind of multifunctional cytokine, FGF family has a key position in bone development, bone metabolism and bone repair. In the early stage of bone regeneration, the level of basic fibroblast growth factor (bFGF) increases significantly, and is expressed in fibroblasts, immature osteoblasts and periosteum near cartilage-like tissue. Activation of ERK 1 / 2 increases cell proliferation and promotes bone regeneration. In mature cells, bFGF regulates the expression of Runx 2 by activating ERK 1 / 2, promoting the differentiation of osteoblasts. bFGF also regulates bone regeneration through BMP signaling. In osteoblasts, it promotes the nuclear localization of Runx 2 and Smad 1 / 5 / 8 protein phosphorylation, stimulates BMP 2 gene expression, and thus promotes osteoblast function. In addition to the above signals, bFGF can interact with Wnt, parathyroid hormone and other pathways to produce two key transcription factors Runx 2 and Osx, forming a complex regulatory network to synergistically control gene expression at various stages of bone regeneration. Therefore, basic fibroblast growth factor (bFGF) has application prospects for promoting bone regeneration.

[0005] Hydrogel is widely selected as an exogenous bone graft biomaterial because it can simulate the physical and chemical properties of the natural extracellular matrix, and can be used as a drug delivery carrier to construct an in vivo bone regeneration microenvironment conducive to cell growth and differentiation. However, traditional single-function hydrogels can only provide limited support, and have poor stability in drug delivery, showing limitations in the dynamic and complex in vivo microenvironment of diabetes. Repairing bone vascular networks and bone fracture regeneration still face many challenges. SUMMARY

[0006] The purpose of the present application is to overcome the shortcomings and deficiencies of the prior art, and to provide a photothermal response bFGF hydrogel and its application.

[0007] The technical scheme adopted by the present application is as follows: a photothermal response bFGF hydrogel, comprising a hydrogel matrix composed of carboxymethyl chitosan and gelatin, and polydopamine modified black phosphorus nanosheets loaded with basic fibroblast growth factor dispersed in the hydrogel matrix.

[0008] Preferably, the mass percentage of the polydopamine modified black phosphorus nanosheets loaded with basic fibroblast growth factor in the system is 0.2%-1% w / w.

[0009] Preferably, the mass percentage of the polydopamine modified black phosphorus nanosheets loaded with basic fibroblast growth factor in the system is 0.5% w / w.

[0010] The preparation method of the photothermal responsive bFGF hydrogel described above involves mixing a carboxymethyl chitosan solution with a gelatin solution to obtain a carboxymethyl chitosan / gelatin solution, then adding polydopamine-modified black phosphorus nanosheets loaded with basic fibroblast growth factor and a crosslinking agent, and mixing and homogenizing to form a hydrogel.

[0011] Preferably, polydopamine is coated onto black phosphorus nanosheets by oxidative self-polymerization under alkaline conditions to obtain polydopamine-modified black phosphorus nanosheets; the polydopamine-modified black phosphorus nanosheets are co-incubated with alkaline fibroblast growth factor to obtain polydopamine-modified black phosphorus nanosheets loaded with alkaline fibroblast growth factor.

[0012] Preferably, the preparation process of the black phosphorus nanosheets includes the following steps: breaking the block black phosphorus into sheets and electrochemically exfoliating the sheet black phosphorus.

[0013] Preferably, the preparation process of the black phosphorus nanosheets includes the following steps: the nanosheets obtained by electrochemical exfoliation are ultrasonically treated in an ice bath until they are uniformly dispersed in anhydrous acetone, then the supernatant is separated by centrifugation at 1500 rpm for 10 min, and the precipitate is separated by centrifugation at 5000 rpm for 10 min to obtain black phosphorus nanosheets.

[0014] Preferably, the preparation process of the polydopamine-modified black phosphorus nanosheets includes the following steps: suspending black phosphorus nanosheets in ethanol, adding dopamine hydrochloride and sodium hydroxide, and stirring in the dark.

[0015] The photothermal responsive bFGF hydrogel described above is used in the preparation of bone repair materials.

[0016] The above-mentioned photothermal responsive bFGF hydrogel is used in products for the treatment of osteoporosis or bone defects caused by diabetes.

[0017] The beneficial effects of this invention are as follows: 1. In the hydrogel system constructed in this invention, black phosphorus (BP) can serve as an excellent antioxidant and regulate the microenvironment. It possesses excellent free radical scavenging ability and biocompatibility, effectively alleviating oxidative stress. Simultaneously, BP can degrade into phosphate ions under physiological conditions, which not only accelerates cell proliferation, differentiation, and signal transduction but also spontaneously promotes bone regeneration and participates in bone metabolism. Near-infrared irradiation accelerates the degradation process of black phosphorus, promotes the biomineralization of phosphate and calcium ions, promotes new bone formation, and improves the mineralization of bone tissue, playing a crucial role in bone regeneration.

[0018] 2. Black phosphorus (BP) in the hydrogel system constructed in this invention possesses photoresponsive nanotechnology, endowing the hydrogel with photoresponsive intelligent drug release capabilities. As a drug delivery carrier, black phosphorus's high specific surface area facilitates efficient drug loading, while its strong absorption of near-infrared light allows it to penetrate biological tissues and exert a photothermal response. This controllable photothermal effect enhances targeted drug release and improves therapeutic efficiency. Furthermore, black phosphorus's high photothermal conversion efficiency can also generate a mild thermotherapy effect, promoting local blood circulation and accelerating the repair process of damaged tissues.

[0019] 3. Black phosphorus (BP) also acts as a mechanical strength enhancer in the hydrogel system constructed in this invention. The mechanical properties of hydrogels can affect the mechanosensory and response of cells. This mechanical stimulation is transmitted through the cytoskeleton and related signaling pathways. Hydrogels with higher mechanical strength can promote osteoblast differentiation. Furthermore, to better mimic the strength of natural bone, it is necessary to match the mechanical properties of bone tissue. This invention has found that introducing black phosphorus nanomaterials into the carboxymethyl chitosan (CCS) / gelatin (Gel) hydrogel (CS) system can regulate the mechanical strength of the hydrogel and improve its application effect in bone tissue processes. After incorporating black phosphorus into the hydrogel, compression tests showed that the mechanical properties of the hydrogel were significantly improved, maintaining stability during biological applications and closely approximating the mechanical strength of natural bone.

[0020] 4. BP is prone to degradation, limiting its in vivo application. Therefore, this invention further utilizes polydopamine (PDA) to modify the surface of BP nanosheets. The polyphenolic structure of PDA can form bonds with the hydroxide groups on the BP surface, creating a uniform and stable protective layer and allowing for the continuous release of phosphate ions, which is significant for bone tissue mineralization. Furthermore, the catechol structure of polydopamine endows it with antioxidant capabilities, further coordinating the protection of tissues from oxidative stress damage.

[0021] 5. This invention utilizes basic fibroblast growth factor (bFGF), loaded onto PDA-modified BP nanosheets. On one hand, it stimulates the growth and proliferation of osteoblasts, promoting their differentiation into mature osteoblasts. On the other hand, it also exhibits a strong pro-angiogenic effect, promoting the formation of new blood vessels, accelerating bone vascular network repair, and speeding up the bone healing process. Simultaneously, black phosphorus and basic fibroblast growth factor synergistically protect osteoblasts under oxidative stress, promoting their recruitment and differentiation. The modified BP@bFGF composite material reactivates mesenchymal stem cells (hBMSCs) under oxidative stress and inflammation, exogenously promoting osteoblast migration and guiding osteoblast differentiation from an adipogenic phenotype to an osteogenic phenotype, thereby promoting bone repair under diabetic conditions.

[0022] In summary, this invention constructs a near-infrared intelligent responsive hydrogel system based on carboxymethyl chitosan and gelatin, combined with PDA-modified BP nanosheets loaded with bFGF. Through mild photothermal therapy, it continuously reverses oxidative stress and induces macrophage transformation from the M1 phenotype to the M2 phenotype, restoring their immunomodulatory capacity and creating a favorable immune microenvironment for bone repair. Under a suitable bone microenvironment, it plays a role in reconstructing the vascular network and promoting osteogenic differentiation, guiding bone regeneration. This research provides a new therapeutic strategy for promoting the treatment of diabetic osteoporosis and the healing of diabetic fractures. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, obtaining other drawings based on these drawings without creative effort still falls within the scope of the present invention.

[0024] Figure 1 Schematic diagram of the design strategy and bone regeneration mechanism of photothermal responsive CG / BPb hydrogel; a) Preparation process of CG / BPb hydrogel; b) Schematic diagram of in vivo drug delivery process of CG / BPb hydrogel in skull defect model; c) Application of CG / BPb hydrogel in bone regeneration of diabetes and its potential biological mechanism. Figure 2 Modification of black phosphorus nanosheets; Figure 3 Zeta potential plots for BP, bFGF, and BP@bFGF; Figure 4 The injectability of CCG / BPb hydrogel; Figure 5 Mechanical tests for each group of hydrogels: a) Compression stress-strain curves for each group of hydrogels; b) Stress diagrams for each group of hydrogels at compressive fracture. Figure 6 The in vitro degradation behavior of each group of hydrogels; a) the amount of hydrogels remaining after degradation during the degradation process; b) the in vitro biodegradation rate of each group of hydrogels in collagenase solution; Figure 7 SEM image of CCG / Bb hydrogel; Figure 8 For the photothermal performance testing of CCG / Bb hydrogel: a) Infrared thermal images of the hydrogel under 808 nm NIR irradiation at different powers; b) Photothermal heating curves of the hydrogel under NIR irradiation at different powers; and c) Photothermal stability test. Figure 9For in vivo photothermal performance testing of CCG / Bb hydrogel: a) Infrared thermographs of hydrogel in vivo under 808 nm NIR irradiation at different power densities; b) Photothermal heating curves of hydrogel under NIR irradiation at different power densities; and c) Photothermal stability test. Figure 10 The cumulative release curve of bFGF from CCG / Bb hydrogel; Figure 11 For the evaluation of cell compatibility of photothermal responsive hydrogels; a, b) live and dead staining and quantitative statistics of MC3T3-E1 cells; c, d) live and dead staining and quantitative statistics of hBMSC cells; Figure 12 For observation of cell proliferation and morphology in photothermal responsive hydrogels; a) EdU staining and cytoskeleton staining of MC3T3-E1 cells; b) EdU staining and cytoskeleton staining of hBMSC cells; Figure 13 Evaluation of the antioxidant stress of photothermal responsive hydrogels; a) Reactive oxygen species fluorescence image; b, d) Relative mRNA expression of antioxidant enzymes Gpx1, Cat, and Sod1; Figure 14 Cell viability in an in vitro oxidative stress model; a) live / dead staining under H2O2 stimulation; b) Annexin V-FIT / PI apoptosis flow cytometry under H2O2 stimulation; Figure 15 To evaluate the anti-inflammatory effects of photothermal responsive hydrogels, immunofluorescence staining was performed using inflammatory markers iNOS and CD206. Figure 16 To evaluate the osteoclast function of photothermal responsive hydrogels: a) oxidative stress; b) quantitative ROS analysis; c) quantitative statistics of osteoclast count; d) quantitative statistics of filamentous actin ring area. Figure 17 For MAPK signaling pathway; Figure 18 To evaluate the angiogenesis-promoting ability of photothermal responsive hydrogels: a) Scratch assay, transwell assay, and tube formation assay of HUVECS cells; b, d) Quantitative statistical graphs of the scratch assay, transwell assay, and tube formation assay. Figure 19 To evaluate the release of pro-angiogenic factors from photothermal responsive hydrogels: a) Immunofluorescence staining images of HUVECS after different treatments of eNOS; b) Immunofluorescence staining images of HUVECS after different treatments of CD31. Figure 20To evaluate the osteogenic differentiation capacity of photothermal responsive hydrogels: a) ALP, ARS, and Von Kossa staining of MC3T3-E1 cells; b) ALP, ARS, and Von Kossa staining of hBMSC cells. Figure 21 The relative mRNA expression of osteogenic markers (including ALP, Col-1, Runx 2, OPN and OCN) in MC3T3-E1 cells after different treatments; Figure 22 Histological staining of each group of hydrogels, including HE staining and Masson staining; Figure 23 In vivo evaluation of the antioxidant stress, anti-inflammatory and angiogenic effects of each group of hydrogels; Figure 24 For bone tissue morphological recovery; a) BV / TV value; b) BMD value; Figure 25 To evaluate the effect of photothermal-responsive hydrogels on bone tissue regeneration in vivo; a) fluorescence staining of TNF-α, IL-10, CD31 and Runx2; b, c) quantitative statistics of TNF-α and IL-10; d, e) quantitative statistics of fluorescence intensity of CD31 and Runx2. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings.

[0026] like Figure 1 As shown, this invention selects a carboxymethyl chitosan (CCS) / gelatin (Gel) hydrogel (CS) system as a model matrix for skull defect repair, used to reinforce damaged bone tissue and deliver therapeutic drugs via a carrier. Gelatin, as a natural polymer material, has good biocompatibility and degradability, and low immune rejection, effectively promoting cell adhesion, proliferation, and differentiation. Carboxymethyl chitosan significantly enhances the mechanical strength of the hydrogel and has good hydrophilicity. Both are used to simulate key components in the natural extracellular matrix—collagen and glycosaminoglycans. When gelatin and carboxymethyl chitosan are combined to construct a hydrogel, the overall performance of the material is significantly improved, showing a wider range of application potential. The CS hydrogel formed by carboxymethyl chitosan (CCS) / gelatin (Gel) maintains good physiological stability and has low immune rejection. The BP / PDA / bFGF nanocomposite material is encapsulated in the pores of the CS hydrogel network, providing a relatively stable microenvironment for the drug, delaying the release rate of growth factors and black phosphorus, and ensuring their continuous and stable release into the surrounding environment. At the same time, the drug can be precisely regulated by external near-red light irradiation, providing a regenerative microenvironment that meets the sequential requirements of bone tissue regeneration.

[0027] Example 1: Preparation of CCS / Gel / BP / PDA / bFGF hydrogel: This embodiment prepares a CCS / Gel / BP / PDA / bFGF hydrogel, such as Figure 1 As shown in figure a, bulk black phosphorus (BP) was first electrochemically exfoliated layer by layer into nanosheets. Then, under alkaline conditions, the oxidative self-polymerization properties of polydopamine (PDA) were utilized to coat the surface of the black phosphorus nanosheets. Afterward, an alkaline fibroblast growth factor (bFGF) solution was added and stirred to obtain a BPb nanocomposite material. PDA-modified BP and bFGF were then added to a CG hydrogel to successfully prepare a hydrogel scaffold with photothermal response properties.

[0028] The specific preparation process in this embodiment is as follows: Preparation of black phosphorus nanosheets BP nanosheets were prepared using an electrochemical-ultrasonic synergistic method. First, bulk BP was broken into flakes, and approximately 100 mg of well-formed BP flakes were selected for electrochemical exfoliation. In a two-electrode system, BP crystals served as the cathode, and a platinum sheet was used as the anode (voltage -5V). The nanosheets were immersed in a 0.01 M tetrabutylammonium hexafluorophosphate (TBA·PF6) solution in propylene carbonate (PC), and under the influence of an electric field, BP was exfoliated layer by layer into nanosheets (approximately 2 h). To enhance dispersibility, the exfoliated BP flakes were ultrasonically treated in an ice bath for 1 h until uniformly dispersed in anhydrous acetone. Large particles and incompletely exfoliated flakes were separated by centrifugation at 1500 rpm for 10 min, followed by further removal of electrolytes by centrifugation at 5000 rpm for 10 min, yielding purified BP nanosheets. The final product was stored in a brown glass bottle in a cool, dark place for future use in polydopamine (PDA) modification and bFGF loading.

[0029] Modification of black phosphorus nanosheets PDA was coated onto BP nanosheets via oxidative self-polymerization under alkaline conditions. Ultrasonic cleaning removed impurities from the BP nanosheet surface to ensure purity and facilitate subsequent modification. One mg of BP nanosheets was suspended in 1 ml of ethanol, and dopamine hydrochloride (50 mg / ml, 100 μL) and sodium hydroxide (20 mg / ml, 150 μL) were added. The mixture was stirred in the dark for 5 h to allow dopamine to oxidatively polymerize and form a PDA film covering the BP surface, enhancing its stability and introducing new functions. After centrifugation at 5000 rpm for 10 min, pure BP@PDA nanosheets were obtained and incubated with an appropriate amount of bFGF solution for 12 h to obtain bFGF-loaded BP@PDA nanosheets for further research.

[0030] Preparation of CCS / Gel / BP / PDA / bFGF hydrogel A 2% (w / w) carboxymethyl chitosan solution and a 2% (w / w) gelatin solution were mixed at a 1:1 ratio and stirred for 10 min. BP@PDA nanosheets loaded with bFGF (0.2% w / w, 0.5% w / w, 1% w / w) were added to 10 ml of the carboxymethyl chitosan / gelatin solution. Finally, 1 mM genipin crosslinking agent was added and stirred until homogeneous. The mixture was homogenized using a vortex mixer and stored at room temperature for 1 h to form a CCS / Gel / BP / PDA / bFGF composite hydrogel (the three hydrogel contents are designated as CGG / Bb-L, CGG / Bb-M, and CGG / Bb-H, respectively; CGG / Bb-M is also referred to as CG / PBb below).

[0031] Example 2: Structural and property analysis of CCS / Gel / BP / PDA / bFGF composite hydrogel: 1. Characterization of black phosphorus nanosheets Blocky black phosphorus was broken into small flakes, followed by electrochemical layer-by-layer exfoliation. The exfoliated black phosphorus flakes were then ultrasonically treated to obtain finer and more uniform BP nanosheets. After ultrasonic treatment, these nanosheets were further dispersed and became more uniform. The morphology of the exfoliated BP nanosheets was characterized by transmission electron microscopy (TEM), such as... Figure 2 As shown, it can be clearly observed that the prepared nanosheets have been successfully exfoliated into sheet-like structures, exhibiting transparent sheet-like characteristics.

[0032] After loading bFGF onto the exfoliated BP nanosheets, TEM revealed a uniformly distributed granular coating on the surface of the BP nanosheets, forming a typical 2D multilayer structure. This structure not only ensures the effective loading of bFGF but also provides a reliable nanoplatform for subsequent therapeutic applications.

[0033] 2. Zeta potential of black phosphorus nanosheets modified with bFGF The successful synthesis of BP@bFGF nanomaterials was further verified by zeta potential measurement experiments. Figure 3 As shown, after loading bFGF onto BP nanosheets, the surface potential of the original BP nanosheets changed significantly, increasing from -29.6 mV to -19 mV. This phenomenon can be explained by the successful assembly of bFGF onto the BP nanosheet surface through a series of non-covalent interactions. Specifically, the positively charged bFGF is fixed onto the negatively charged BP nanosheets through non-covalent forces, thus leading to the change in surface potential. This result is consistent with the transmission electron microscopy results, confirming the successful synthesis of BP@bFGF nanomaterials.

[0034] 3. Injectability of photothermal responsive hydrogels To verify the broad application of the prepared hydrogel in bone regeneration, its injectability was further evaluated. Figure 4 As shown, the hydrogel can be shaped into the letters "WMU" using a syringe, demonstrating its injectability, which shows unique advantages in local applications.

[0035] 4. Mechanical property testing of photothermal responsive hydrogels The mechanical properties of hydrogels play a crucial role in maintaining structural stability and supporting cell growth during the dynamic process of bone defect repair. Therefore, the effect of different concentrations of BP@FGF nanosheets on the mechanical properties of the hydrogel was characterized by testing its compression. Figure 5 As shown, the concentration of BP@FGF nanosheets in the CCG / Bb-L hydrogel is 0.2%, in the CCG / Bb-M hydrogel it is 0.5%, and in the CCG / Bb-H hydrogel it is 1%. Figure 5 As shown in Figure a, the compressive strength of pure CCG hydrogel at 55% strain is 10 kPa. The mechanical properties of hydrogels incorporating nanosheets are significantly improved, with CCG / Bb hydrogel exhibiting greater mechanical strength. The mechanical strength of the hydrogel increases with increasing nanosheet concentration. Specifically, at 55% strain, the compressive strength of CCG / Bb-L hydrogel reaches 12 kPa, CCG / Bb-M hydrogel reaches 17 kPa, and CCG / Bb-H hydrogel reaches 25 kPa. CCG / Bb-M hydrogel most closely approximates the mechanical strength of natural cancellous bone.

[0036] 5. In vitro degradation behavior of photothermal responsive hydrogels To ensure the structural stability of the hydrogel after implantation in vivo, its swelling and degradability were further tested. First, the biodegradation behavior of the CCG / Bb hydrogel was evaluated in vitro to simulate the in vivo environment and preliminarily assess its degradation efficiency in practical applications. Results are as follows: Figure 6 As shown in Figure a, after 27 days of enzymatic degradation, all hydrogels gradually degraded with increasing soaking time, and the degradation rate decreased with increasing concentration of BP@FGf nanosheets. This trend may be related to the additional cross-linking sites and mechanical support provided by BP nanosheets in the hydrogel, thereby delaying the degradation process. Similarly, as... Figure 6 As shown in b, the swelling behavior of the hydrogel also exhibits a similar trend. This indicates that the introduction of BP@bFGF nanosheets significantly increases the crosslinking density of the hydrogel, delaying the swelling and deformation process and making the hydrogel structure more stable. In summary, CCG / Bb hydrogels possess a low swelling ratio and a slow biodegradation rate, which is particularly important for bone regeneration applications, as materials need to maintain their shape and structural integrity for extended periods during bone repair.

[0037] 6. SEM morphology observation of photothermal responsive hydrogels Images were displayed using a scanning electron microscope, such as Figure 7 As shown, the cross-sections of all hydrogels exhibit a highly interconnected porous structure with relatively smooth pore walls, mimicking the structure of natural bone and providing cells with similar structural support and biological functions. Furthermore, no significant BP@bFGF was observed on the pore walls of the hydrogels, indicating that the nanomaterials may be uniformly distributed throughout the hydrogel network, facilitating the uniform release of growth factors and enabling more effective regulation.

[0038] 7. Photothermal performance testing of photothermal responsive hydrogels When using different power levels (0, 0.5, 1, 1.5 W / cm) 2 The infrared thermogram of the hydrogel under 808nm near-infrared light irradiation is as follows: Figure 8 As shown in Figure a, the temperature of the hydrogel increases continuously with increasing power density and irradiation time. When using 1 W / cm²... 2 Under high-power NIR irradiation, the hydrogel heated to 42±1℃ and stabilized after 5 min. This not only avoids side effects on normal tissues and regulates the microenvironment at the site of injury, but also regulates the slow release of bFGF through photothermal effect.

[0039] To further investigate the photothermal stability of the hydrogel, different power intensities (0, 0.5, 1, 1.5 W / cm²) were used. 2 The hydrogel was cyclically irradiated with near-infrared light. After five cycles of laser on-off testing, the final performance parameters of the hydrogel were recorded, such as... Figure 8 As shown in Figure c, compared with the initial measurement results, the CCG / Bb hydrogel did not exhibit significant temperature decay after five laser irradiation cycles. This result strongly demonstrates that the hydrogel has good photothermal reversibility and excellent photostability, which is crucial for the long-term stability of materials, especially for bone repair processes that require repeated photothermal therapy and photocontrolled drug release.

[0040] 8. In vivo photothermal performance testing of photothermal responsive hydrogels To further verify the actual effect of photothermal response in vivo and evaluate its tissue penetration depth, photothermal stimulation was applied after implanting hydrogels in rats. Figure 9 As shown in figure a, a stable photothermal response can still be observed in vivo, exhibiting a similar heating rate to the in vitro photothermal response. After NIR irradiation, 5 min later, 0 W / cm²... 2 No significant temperature change was observed in the group, 0.5 W / cm 2 The group temperature was 35℃, 1 W / cm 2The group's temperature was 42℃, and the concentration was 1.5 W / cm². 2 The temperature of the group was 48℃, with almost no heat loss compared to direct in vitro NIR irradiation, demonstrating high photothermal conversion efficiency in a living environment. Similarly, after five laser irradiation cycles, the hydrogel in the animal body also exhibited the same photothermal stability, with no significant temperature decay, showing the potential of near-infrared controlled photothermal therapy. Exploring the optimal time and power is crucial for subsequent experiments; therefore, based on the results of in vitro and in vivo photothermal response experiments, a power of 1 W / cm² was selected. 2 The experimental condition of irradiating with near-infrared laser for 5 minutes was used for subsequent experiments.

[0041] 9. Drug sustained-release experiment of photothermal responsive hydrogels In the complex process of bone repair, on-demand drug delivery via photothermal responsive stimulation has significant clinical implications for promoting angiogenesis and new bone formation. Studies have shown that the pH of healthy tissues is 7.3-7.4, while after bone injury, it becomes weakly acidic (pH≈6.5). To further investigate the drug delivery mechanism in vivo, the release of bFGF from CCG / Bb hydrogel was tested. Under healthy and injured pH conditions, NIR irradiation was applied at a fixed frequency. The results showed that the CCG / Bb hydrogel exhibited a significant dual NIR / pH response release behavior, both of which significantly accelerated the release of bFGF. Under photothermal irradiation, the drug exhibited a significant burst release in the initial stage, especially at pH=6.5, where the cumulative release rate of bFGF reached 60% within the first 20 minutes. This indicates that photothermal stimulation significantly accelerated the initial release of the drug. Subsequently, the release proceeded at a lower and more stable rate. This behavior of promoting bFGF release in a weakly acidic environment may be due to the pH sensitivity of the PDA layer and the protonation of the amino groups. This confirms that bFGF can achieve precise responsive release in the damaged environment under NIR irradiation, providing an effective strategy for on-demand drug delivery.

[0042] Example 3: In vitro functional evaluation of CCS / Gel / BP / PDA / bFGF composite hydrogel: 1. Biocompatibility evaluation of photothermal responsive hydrogels Cell contact assays were used to evaluate the adhesion, morphological changes, and viability of osteoblasts (MC3T3-E1) and mesenchymal stem cells (hBMSCs) on the surface of CG / BPb hydrogels. These cells are the primary cellular source for bone formation and repair. In the experiments, CG, CG / BPb-L, CG / BPb-M, and CG / BPb-H hydrogels were placed in 12-well cell culture plates, with cells cultured at a density of 1 × 10⁶ cells per well. 5Cells were seeded at a density of 1000 mcg and cultured in culture plates for 72 h. Blank cell culture plates served as the control group. First, the culture medium was removed and the cells were washed with PBS. Then, the cells were stained with Calcein-AM / PI staining kit and incubated in the dark for 30 min. Fluorescence images of each group were then captured using a fluorescence microscope, and the fluorescence intensity was statistically analyzed.

[0043] like Figure 11 As shown in a and c, MC3T3-E1 and hBMSC cells adhered well to the hydrogel surface and grew well. Most cells showed good viability and were in a proliferative state, with few red dead cells. This result indicates that the photothermal-responsive hydrogel has good biocompatibility and can support cell survival and growth. Furthermore, the cell viability was quantitatively analyzed after co-culturing for 1, 2, and 3 days, and the results are as follows: Figure 11 As shown in b and d, CG, CG / BPb-L, CG / BPb-M and CG / BPb-H hydrogels all exhibit excellent cell compatibility with MC3T3-E1 and hBMSC, meeting the essential biocompatibility requirements for biomaterials. Among them, CG / BPb-M shows the best cell viability.

[0044] Osteoblast proliferation is fundamental for subsequent growth and osteogenic differentiation. EdU staining was performed on cells. Following the manufacturer's kit instructions, 50 μM EdU was added to the culture medium, and after 2 h of incubation, cells were fixed, stained with Apollo, permeabilized, and stained nuclearly. EdU-positive cells were then observed under a fluorescence microscope to assess cell proliferation. Compared to the control and GC groups, the number of EdU-positive cells in the CG / BPb-L, CG / BPb-M, and CG / BPb-H groups was significantly increased, significantly promoting cell proliferation. Cell actin staining was performed to observe cell proliferation and spreading morphology after 3 days of co-culture with hydrogel. Figure 12 As shown in Figure a, compared with the control group, MC3T3-E1 cells in the CG / Bb-M group exhibited a more stretched morphology, with a well-developed cytoskeleton and cell clusters, while extending numerous branched pseudopodia that were tightly connected to each other, showing good growth. Figure 12 As shown in b, hBMSC cells also exhibited a similar cell state. In the CG group, only a small number of cells spread on the hydrogel, while the cell density attached to the surface of the CG / Bb hydrogel increased and grew well, which was more prominent and obvious in the CG / BPb-M group.

[0045] 2. Antioxidant stress capacity of photothermal responsive hydrogels In the early stages of bone defects, oxidative stress can promote osteoblast differentiation and mineralization by activating the inflammatory response, thereby accelerating bone tissue formation. However, excessive oxidative stress can impair the proliferation and differentiation capacity of osteoblasts and mesenchymal stem cells, and inhibit angiogenesis and the expression of osteogenic-related factors, thus hindering bone tissue regeneration.

[0046] To investigate the effect of CG / BPb hydrogel on oxidative stress, an in vitro oxidative stress model was constructed, and the changes in ROS levels in different hydrogel treatment groups under the action of hydrogen peroxide (H2O2) were compared. Figure 13 Fluorescence images showed that in the Control group without H2O2 treatment, ROS levels remained low, while in the Control and GC groups treated with H2O2, ROS levels significantly increased, indicating that H2O2 successfully induced oxidative stress. In contrast, ROS levels gradually decreased in the CG / Bb and CG / BPb+NIR groups. This is because under oxidative stress, BP nanosheets can induce the expression of intracellular antioxidant enzymes, effectively clearing excess ROS, while activating intracellular signaling pathways, protecting the proliferation and differentiation capacity of osteoblast-related cells, and accelerating bone repair. Furthermore, bFGF enhances the expression of intracellular antioxidant enzymes, reduces ROS damage to cells, and further promotes bone tissue repair and regeneration. Notably, the ROS level in the CG / BPb+NIR group was close to that of the control group, which may be mainly due to the regulatory effect of the mild photothermal stimulation of NIR on bFGF and the synergistic effect of BP nanosheets.

[0047] Glutathione peroxidase 1 (GPX1), catalase (Cat), and superoxide dismutase 1 (SOD1) are important intracellular antioxidant enzymes that play a crucial role in antioxidant processes. The expression levels of these enzymes are often used to assess cellular antioxidant capacity and reflect the cell's response to oxidative stress. Figure 13 The real-time polymerase chain reaction (qRT-PCR) results shown in diagram bd indicate that the CG / BPb hydrogel significantly upregulated the expression of antioxidant enzyme-related genes GPX1, Cat, and SOD1, especially after NIR irradiation, where the expression levels of these genes further increased. In summary, the CG / BPb hydrogel effectively mitigates oxidative stress-induced cell damage by upregulating the expression of intracellular antioxidant enzymes, and this protective effect is particularly pronounced after NIR irradiation. This suggests that the CG / BPb hydrogel may provide better protection for cells under oxidative stress conditions, promoting tissue repair and regeneration.

[0048] Oxidative stress can induce premature apoptosis in osteoblasts. To analyze the cell rescuing effect of CG / Bb hydrogel on apoptosis under oxidative stress, MC3T3-E1 cells and hBMSC cells were seeded on the surface of each hydrogel group. After culturing for 24 h, hydrogen peroxide (H2O2) stimulation was applied, followed by cell viability detection. The culture medium was removed, and live and dead cells were labeled using Calcein-AM / PI staining. Figure 14 As shown in Figure a, the H2O2+Control group exhibited a large number of dead cells due to oxidative stress, while almost no dead cells were observed in the CG / BPb and CG / Bb+NIR groups. To quantitatively analyze cell apoptosis in different treatment groups, Annexin V-FITC / PI flow cytometry was used. After H2O2-induced stimulation, cells were digested with trypsin, centrifuged, washed with PBS, centrifuged again to remove the supernatant, gently resuspended in 195 μl of Annexin V-FITC binding buffer, and then incubated with 5 μl of Annexin V-FITC and 10 μl of PI for 20 min at room temperature before immediate flow cytometry analysis. Figure 14 As shown in b, after treatment with the oxidative stress inducer H2O2, the number of Annexin V-FITC positive and PI negative cells significantly increased, indicating a significant increase in the proportion of early apoptotic cells, accounting for 27.04% of the total cells. In contrast, early apoptotic cells were significantly reduced in the CG / Bb group and the CG / BPb+NIR group, accounting for 9.47% and 2.67% of the total cells, respectively. In particular, the apoptosis rate in the CG / BPb+NIR group was almost the same as that in the control group. These results indicate that the CG / Bb+NIR group can support the survival of bone-related cells under oxidative stress and alleviate oxidative stress-induced apoptosis. This is mainly attributed to the antioxidant properties of BP, which can scavenge excess reactive oxygen species, while NIR enhances the photothermal properties of BP, improving its antioxidant capacity, inhibiting oxidative stress-induced apoptosis, and protecting bone repair-related cells.

[0049] 3. Evaluation of the osteoclast-promoting function of photothermal responsive hydrogels Osteophytes, located near osteoblasts, play a crucial role in osteoogenesis by clearing apoptotic osteoblasts and promoting mineral deposition in osteoblasts. Osteoclasts, mainly formed by the fusion of medullary progenitor cells, primarily clear mineralized bone matrix by producing lysosomal enzymes, including tartrate-resistant acid phosphatase (TRAP) and cathepsin K (CTSK). Therefore, considering the roles of macrophages and osteoclasts in bone remodeling, an in vitro co-culture model was constructed to investigate the role of CG / BPb hydrogel in bone regeneration. First, primary macrophages and primary osteoblasts were extracted from mouse tissues and cultured in appropriate culture media at 37°C and 5% CO2. An in vitro oxidative stress model was established by stimulating MC3T3-E1 cells with hydrogen peroxide, and an in vitro inflammation model was established by stimulating Raw264.7 cells with LPS. Once both in vitro models stabilized, a co-culture system was established.

[0050] Macrophages play a prominent role in tissue regeneration, and excessive inflammation after bone injury has an adverse effect on bone regeneration. To construct an inflammation model and induce macrophage polarization towards the M1 phenotype, RAW 264.7 cells were selected as the macrophage model and stimulated with LPS (100 ng / mL) for 48 h to simulate an in vitro inflammatory environment. Subsequently, macrophages were co-cultured with different hydrogel groups, and then validated by immunofluorescence staining and real-time quantitative polymerase chain reaction. Numerous studies have shown that M1 macrophages exacerbate inflammation, while M2 macrophages exert anti-inflammatory effects and improve the inflammatory environment. First, the M1 and M2 phenotypes of macrophages were labeled with iNOS and CD206, respectively, and the inflammatory status was assessed by immunofluorescence staining. The results showed that in the unstimulated control group, both iNOS and CD206 were lowly expressed. In the LPS-stimulated control group, iNOS was induced to be highly expressed, accompanied by a decrease in CD206 expression, indicating that LPS successfully induced macrophage polarization towards the M1 phenotype. Compared with the control group, iNOS was significantly downregulated in the CG / BPb group and the CG / BPb+NIR group. Notably, after periodic NIR irradiation, iNOS expression levels decreased to the lowest, while CD206 expression was the highest, effectively regulating the polarization of M1 macrophages to M2 type. This confirms that CG / BPb combined with mild heat stimulation is more effective than CG or CG / BPb in inducing macrophage polarization to M2 type, exerting anti-inflammatory capabilities, and shortening the inflammatory period. Furthermore, cytokines released by M1 macrophages, including TNF-α, IL-1β, iNOS, and CD86, can promote osteoclast bone resorption activity. Cytokines released by M2 macrophages, including IL-4, IL-10, CD206, and Arg-1, can inhibit the active nuclear proliferation of osteoclasts by inhibiting activated T cell nuclear factor 1 (NFATc1).

[0051] like Figure 16As shown in Figure a, the highest ROS expression was observed in the LPS-stimulated control group, indicating successful induction of cellular oxidative stress. Simultaneously, both the CG / BPb and CG / BPb+NIR groups effectively inhibited LPS-induced ROS levels. In particular, the CG / BPb+NIR group exhibited very low DCF fluorescence intensity, indicating a protective effect against oxidative stress-induced cell damage and mitigating excessive oxidative stress induced by inflammation. This study demonstrates that BP possesses strong ROS scavenging capabilities, and PDA can also effectively remove intracellular ROS. The mild photothermal effect induced by near-infrared light accelerates the release of the nanocomposite material and enhances the free radical scavenging ability of the hydrogel.

[0052] Following bone homeostasis disruption, osteoclasts dominate bone resorption. Osteoclasts secrete TRAP, and co-cultured cells are stained using a TRAP staining kit. Positively stained osteoclasts are observed under a microscope, allowing for semi-quantitative analysis of osteoclast activity. Both the Control and CG groups stimulated with LPS showed significant purplish-red precipitates, with a significantly increased number compared to the control group, indicating the presence of active osteoclasts. However, the number of purplish-red precipitates was significantly reduced in the CG / BP and CG / BPb+NIR groups, indicating decreased osteoclast activity.

[0053] F-actin rings provide essential support for cell migration. During osteoclast resorption on the bone surface, they form a special structure called a closed zone, surrounded by F-actin rings. The presence of F-actin rings helps osteoclasts adhere tightly to the bone surface, creating a relatively closed microenvironment that allows them to secrete acidic substances and proteases, effectively dissolving and degrading bone. Figure 16 As shown in figure a, morphological changes in RAW264.7 cells were studied by staining the cytoskeleton. Under near-infrared radiation, the area of ​​F-actin rings on CG / BPb+NIR hydrogel was significantly reduced. These experimental results collectively demonstrate that CG / BPb+NIR hydrogel can regulate macrophages, inhibit osteoclast activity, and promote bone remodeling and regeneration.

[0054] The MAPK signaling pathway plays a crucial role in cell growth, apoptosis, and stress response, including signaling pathways such as JNK, ERK, and p38. Figure 17As shown, the expression levels of p-JNK, p-ERK, and p-p38 were elevated in the control group, indicating that the MAPK signaling pathway was overactivated. NFATC1 is associated with osteoclast function and promotes osteoclast formation and bone resorption. Its high expression usually indicates enhanced osteoclast differentiation and activation, while increased CTSK expression means increased osteoclast bone resorption activity. In the CG / BP and CG / BPb+NIR groups, the MAPK signaling pathway was attenuated, reducing NFATC1 expression, which in turn reduced CTSK secretion, ultimately leading to decreased bone resorption. The reduced osteoclast activity resulted in bone formation exceeding bone resorption, which is conducive to new bone formation.

[0055] 4. In vitro angiogenesis ability of photothermal responsive hydrogels To comprehensively evaluate the promoting effect of CG / BPb hydrogel on endothelial cell chemotaxis, scratch assays and Transwell assays were performed. Figure 18 As shown in Figure a, within 24 hours, all treatment groups showed a tendency for cells to migrate to the blank area, but the migration tendency was particularly significant in the CG / BPb group and the CG / BPb+NIR group, especially in the CG / BPb+NIR group where the scratch was almost completely closed. Quantitative analysis further showed that the healing rate of the CG / BPb+NIR group reached 85% within 24 hours, demonstrating a high scratch healing ability. Similarly, in the Transwell assay, compared with the control group and the CG group, both the CG / BPb group and the CG / BPb+NIR group exhibited significant chemotaxis, resulting in more intracellular cells migrating to the lower surface of the upper chamber. Especially under NIR irradiation, the migration ability of the CG / BPb hydrogel was significantly enhanced. This is mainly attributed to the periodic NIR irradiation triggering the release of phosphate and bFGF, thereby enhancing cell chemotaxis and migration ability. Therefore, CG+BPb combined with on-demand NIR irradiation can improve the in vitro recruitment capacity of osteoblasts, which is of great significance for new bone formation and bone defect repair.

[0056] The angiogenesis capacity of CG / BPb hydrogel was explored through angiogenesis experiments, such as... Figure 18 As shown in Figure a, tubular structures were observed in the control group, while in the Control group subjected to in vitro simulated oxidative stress, this pro-angiogenic capacity was disrupted, resulting in a small number of incomplete tubular networks. Compared to other groups, the CG / BPb+NIR group exhibited a more pronounced vascular network, accompanied by a more mature capillary-like network. Figure 18The quantitative results in b and c show that the total number of connections and the percentage of vessel area in the CG / BPb+NIR group were significantly increased, indicating that the combination of CG / BPb hydrogel and NIR can promote in vitro angiogenesis. This is mainly because bFGF is rapidly released from the hydrogel under NIR stimulation. bFGF is an important angiogenic factor that can stimulate the proliferation and migration of vascular endothelial cells, and its combination with photothermal therapy has the potential to significantly promote new angiogenesis.

[0057] eNOS and CD31 are biomarkers closely related to vascular function, and their expression levels are crucial for angiogenesis. HUVECS cells were seeded onto the surface of various hydrogels and co-cultured for 7 days. Immunofluorescence staining for eNOS and CD31 was then performed to further verify the angiogenesis-promoting effect of CG / BPb hydrogels. Figure 19 As shown in a and b, the fluorescence intensity of eNOS and CD31 in HUVECS of the CG / BPb+NIR group was significantly higher than that of other groups, indicating that the release of bFGF also helps to improve the expression level of eNOS and CD31. The fluorescence staining results are consistent with the tube formation experiment results.

[0058] 5. Osteogenic differentiation capacity of photothermal responsive hydrogels To evaluate the effect of hydrogel on osteogenic differentiation of MC3T3-E1 and hBMSC cells.

[0059] ALP levels can reflect the early differentiation of osteoblasts. ALP staining was followed by imaging and recording using an optical microscope, such as... Figure 20 As shown, the CG / BPb+NIR group exhibited the deepest ALP staining and significantly increased ALP activity, followed by the CG / BPb group. This confirms that the photothermal responsive CG / BPb hydrogel has the ability to promote early osteogenic differentiation of MC3T3-E1 cells.

[0060] Alizarin Red S (ARS) is an important indicator of late-stage osteogenic differentiation. Extracellular calcium mineral deposition was observed using Alizarin Red and Von Kossa staining methods. The results were consistent with ALP staining. The CG / BPb+NIR group showed the clearest and most prominent deep red color, with a gradual increase in bone mineralization nodules, indicating high osteogenic potential. Similarly, in Von Kossa staining, the CG / BPb+NIR group showed brownish-black calcium salt deposits, a marker of new bone formation.

[0061] The osteogenic differentiation and mineralization trends of hBMSCs are similar to those of MC3T3-E1 cells. BP nanosheets recruit osteoblasts and promote osteogenic activity by regulating the secretion of osteoblast cytokines. When used in combination with NIR, they can enhance the ability to promote osteoblast differentiation.

[0062] To investigate the effects of CG / BPb hydrogel on the expression of osteogenic differentiation-related genes in MC3T3-E1 cells, five major marker genes (ALP, Col-1, Runx2, OPN, and OCN) were detected by qRT-PCR. Figure 21 As shown, the expression of ALP, Col-1, Runx2, OPN, and OCN are key characteristics of osteoblast differentiation and mineralization, and can be used to assess the functional status of osteoblasts. After co-culturing with hydrogels of each group, osteogenic differentiation factors were significantly upregulated in the CG / BPb+NIR group, exhibiting the highest level of osteogenic-related gene expression, thereby accelerating MC3T3 cell differentiation. This indicates that the release of bFGF and phosphate triggered by BP and NIR has a synergistic effect on the expression of osteogenic-related factors. Photothermal therapy stimulates osteoblast proliferation and differentiation, regulates cytokines in the bone microenvironment, and accelerates new bone formation. Simultaneously, photothermal control of the release of bFGF and phosphate in the culture medium helps enhance osteogenic activity.

[0063] Example 4: In vivo functional evaluation of photothermal responsive hydrocoagulation: This embodiment further explores the practical application of CG / BPb hydrogels in vivo. During tissue repair and regeneration, acute inflammation often hinders bone growth. To investigate the regulatory ability of hydrogels on inflammation, different hydrogels were implanted subcutaneously in rats. After implantation, the CG / BPb+NIR group received 808nm NIR irradiation every two days for 5 minutes each time, with a power of 1 W / cm². 2 Ensure that the temperature of the damaged area is maintained at 42±1℃.

[0064] This embodiment establishes a diabetic rat bone defect model and conducts in vivo experiments on the CCS / Gel / BP / PDA / bFGF composite hydrogel prepared in Example 1. Figure 1 As shown in b, diabetic rats were randomly selected to construct a critical-sized skull defect model. After the model stabilized, CG / BPb hydrogel was precisely injected into the defect site. The filled area was then immediately irradiated with 808nm near-infrared light to evaluate the bone regeneration capacity of the CCS / Gel / BP / PDA / bFGF composite hydrogel.

[0065] The process of constructing a diabetic rat model of skull defects is as follows: Four-month-old male SPF-grade SD rats were randomly divided into experimental and control groups. The experimental group received a high-fat, high-sugar diet for four weeks, with 12 hours of water intake allowed daily. Streptozotocin (STZ) was dissolved in a 0.1 mmol / L sterile citrate-sodium citrate solution to prepare a 1% STZ solution. Note that the solution should be prepared fresh, stored on ice, and handled in the dark.

[0066] Before injection, rats were fasted and deprived of water for 12 hours. The experimental group received intraperitoneal injections of STZ solution daily at a dose of 40 mg / kg for one week. The control group received an equal volume of citrate buffer solution intraperitoneally and maintained a high-fat, high-sugar diet. Fasting plasma glucose (FPG) was measured after 48 hours. A FPG ≥ 11.1 mmol / L was considered a successful establishment of diabetes. Otherwise, STZ injections were repeated to establish a suitable diabetes model.

[0067] After establishing the diabetic rat model, a skull defect model needs to be established. The procedure is performed under sterile conditions. Rats are anesthetized by intraperitoneal injection of 2% pentobarbital. The rats' heads are shaved and disinfected. The scalp is incised to expose the skull, and a standard-sized defect is created on the skull using a grinder. Each experimental group of rats receives an injection of their respective hydrogel at the defect site, while the control group receives an equal volume of physiological saline. The incision is sutured layer by layer, and the rats are housed individually post-operatively. To prevent infection, penicillin is administered intraperitoneally daily for three days post-operatively. The rats' recovery is closely monitored post-operatively to ensure no infection, and a high-fat, high-sugar diet is maintained, with blood glucose levels monitored regularly.

[0068] 1. Histological staining of photothermal responsive hydrogels Two weeks after implantation, experimental samples were collected and fixed in paraformaldehyde. The samples were then embedded in paraffin, sectioned, and stained with hematoxylin and eosin to detect morphological changes in the bone tissue after implantation. Figure 22 As shown, under an optical microscope, the CG group exhibited obvious cavities accompanied by mild inflammatory cell infiltration. In contrast, the CG / BPb group showed a large number of inflammatory cells evenly distributed within the hydrogel. Especially after NIR irradiation, the hydrogel degraded into fragments and fused with the tissue, a change that significantly promoted cell infiltration and penetration. Simultaneously, Masson staining revealed a more pronounced collagen fiber network in the CG / BPb group, with a gradual increase in the area of ​​mature bone tissue. Furthermore, the induction of immune cell migration to the wound site to adapt to the immune microenvironment resulted in a more pronounced and orderly repair trend, laying a good foundation for subsequent bone tissue repair.

[0069] The levels of reactive oxygen species (ROS) were detected using a DHE fluorescent probe to assess the effects of different treatment groups on ROS levels. The results showed that the CG group had obvious red fluorescence, indicating a high level of ROS. The red fluorescence intensity of the CG / BPb group decreased significantly, especially after NIR irradiation, when the red fluorescence almost disappeared, indicating that the ROS were basically cleared.

[0070] Further investigation using immunofluorescence staining revealed the regulatory effects of CG / BPb hydrogel on local inflammation and macrophage polarization. CD86, a marker of M1 macrophages, promotes the activation of the immune response, while CD206, another marker of M1 macrophages, inhibits excessive polarization. After establishing the bone defect model, a significant increase in CD86-positive cells was detected in the CG group, indicating a significant inflammatory state. CG / BPb hydrogel induced higher levels of CD206 expression and decreased CD86 expression, thus alleviating the inflammatory state. This change was more pronounced after NIR treatment, indicating the activation of M2 macrophages. These data suggest that the CG / BPb+NIR group plays an important role in immunomodulation, effectively reducing local inflammation during bone repair and regulating the immune microenvironment at the injury site.

[0071] To further verify the differences among different hydrogel groups in angiogenesis and the recruitment and migration of endogenous stem cells during the repair process at the defect site, immunofluorescence staining using the vascular marker CD31 and the endogenous stem cell marker CD90 was used for further verification. The results showed that the CG group exhibited almost no accumulation of stem cells, with only a small number of sporadic vascular endothelial cells observed. In contrast, the CG / BPb group showed significant recruitment of endogenous stem cells and infiltration of newly formed blood vessels into the damaged area. Particularly after NIR irradiation, a ring-shaped vascular system appeared, and the expression levels of CD90 and CD31 were significantly increased. This confirms that the CG / BPb hydrogel combined with mild photothermal stimulation can promote angiogenesis and endogenous stem cell recruitment, providing strong support for enhancing bone vascular network reconstruction and bone tissue repair.

[0072] 2. Study on in vivo bone regeneration using photothermal responsive hydrogels To conduct a detailed evaluation of the repair effect, quantitative analysis was performed on the defect area, including bone volume fraction (BV / TV) and bone mineral density (BMD). These two indicators are key factors in evaluating bone regeneration; higher values ​​indicate stronger bone regeneration. The CG / BPbM group showed significantly increased BV / TV and BMD values, confirming the bone repair potential of CG / BPb hydrogel in vivo and suggesting it as a promising candidate for bone repair materials.

[0073] 3. Fluorescent staining of photothermal-responsive hydrogels for in vivo bone regeneration To further evaluate the expression of inflammatory factors in bone tissue and their impact on bone repair, the expression levels of pro-inflammatory factor TNF-α and anti-inflammatory factor IL-10 in bone tissue were detected using immunocombinant chemistry (IHC) staining. Figure 25As shown, the staining intensity of TNF-α was high in the injury group, indicating a significant inflammatory response. In contrast, TNF-α levels were significantly decreased in the CG / BPbM group, while the expression of the anti-inflammatory factor IL-10 was significantly increased. These results suggest that CG / BPb-M hydrogel can effectively regulate the inflammatory microenvironment, inhibit the expression of pro-inflammatory factors, and promote the release of anti-inflammatory factors, thereby creating more favorable conditions for bone repair.

[0074] Immunofluorescence staining of CD31 and Runx2 was used to further assess angiogenesis and bone differentiation within the defect area. Figure 25 As shown in figure a, compared with other groups, the CG / BPbM group had the highest levels of CD31 and Runx2 expression, indicating that this group formed a large number of blood vessels in the bone defect area, accompanied by significant bone matrix deposition.

[0075] The above description discloses only preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. Therefore, equivalent variations made in accordance with the claims of the present invention are still within the scope of the present invention.

Claims

1. A photothermal responsive bFGF hydrogel, characterized in that: It comprises a hydrogel matrix composed of carboxymethyl chitosan and gelatin, wherein polydopamine-modified black phosphorus nanosheets loaded with basic fibroblast growth factor are dispersed in the hydrogel matrix.

2. The photothermal responsive bFGF hydrogel according to claim 1, characterized in that: The mass percentage of the polydopamine-modified black phosphorus nanosheets loaded with basic fibroblast growth factor in the system is 0.2% - 1% w / w.

3. The photothermal responsive bFGF hydrogel according to claim 2, characterized in that: The mass percentage of the polydopamine-modified black phosphorus nanosheets loaded with basic fibroblast growth factor in the system is 0.5% w / w.

4. The method for preparing photothermal responsive bFGF hydrogel according to any one of claims 1-3, characterized in that: The process involves mixing a carboxymethyl chitosan solution with a gelatin solution to obtain a carboxymethyl chitosan / gelatin solution. Then, polydopamine-modified black phosphorus nanosheets loaded with basic fibroblast growth factor and a crosslinking agent are added. After mixing and homogenization, a hydrogel is formed.

5. The method for preparing photothermal responsive bFGF hydrogel according to claim 4, characterized in that: Polydopamine was coated onto black phosphorus nanosheets via oxidative self-polymerization under alkaline conditions to obtain polydopamine-modified black phosphorus nanosheets; the polydopamine-modified black phosphorus nanosheets were then co-incubated with alkaline fibroblast growth factor to obtain polydopamine-modified black phosphorus nanosheets loaded with alkaline fibroblast growth factor.

6. The method for preparing photothermal responsive bFGF hydrogel according to claim 5, characterized in that: The preparation process of the black phosphorus nanosheets includes the following steps: breaking the block black phosphorus into sheets and electrochemically exfoliating the sheet black phosphorus.

7. The method for preparing photothermal responsive bFGF hydrogel according to claim 5, characterized in that: The preparation process of the black phosphorus nanosheets includes the following steps: the nanosheets obtained by electrochemical exfoliation are ultrasonically treated in an ice bath until they are uniformly dispersed in anhydrous acetone, then the supernatant is separated by centrifugation at 1500 rpm for 10 min, and the precipitate is separated by centrifugation at 5000 rpm for 10 min to obtain black phosphorus nanosheets.

8. The method for preparing photothermal responsive bFGF hydrogel according to claim 4, characterized in that: The preparation process of the polydopamine-modified black phosphorus nanosheets includes the following steps: suspending black phosphorus nanosheets in ethanol, adding dopamine hydrochloride and sodium hydroxide, and stirring in the dark.

9. The application of the photothermal responsive bFGF hydrogel as described in any one of claims 1-3 in the preparation of bone repair materials.

10. The use of the photothermal responsive bFGF hydrogel as described in any one of claims 1-3 for the treatment of osteoporosis or bone defects caused by diabetes.

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