A zinc sulfide-modified hydrogel with near-infrared light responsiveness and photothermal effect, and its preparation method and application

Abstract

The present invention relates to the field of medical technology, and provides a zinc sulfide modified hydrogel with near-infrared light responsiveness-photothermal effect, and a preparation method and application thereof. The zinc sulfide modified hydrogel provided by the present invention comprises a methacrylated gelatin hydrogel solution and zinc sulfide nanoparticles dispersed in the methacrylated gelatin (GelMA) hydrogel solution. The present invention utilizes the biological principle that low-frequency-low-heat stimulation can promote the osteogenic differentiation ability of BMSCs, introduces zinc sulfide nanoparticles into the GelMA hydrogel, and the resulting modified hydrogel has good biocompatibility, near-infrared responsiveness and photothermal effect, can promote the proliferation ability, ATP energy production ability, cell motility and osteogenic differentiation ability of bone marrow stem cells, and is expected to be applied to bone tissue damage sites under clinical conditions, providing a potentially available biological gel preparation for promoting bone tissue damage, and expanding the application of photothermal effect in bone tissue engineering.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a zinc sulfide-modified hydrogel with near-infrared light responsiveness-photothermal effect, and a preparation method and application thereof. Background Art

[0002] Bone defects are a common clinical problem, caused by various reasons such as trauma, tumor resection, and congenital diseases. Currently, bone defect repair remains a major challenge in the field of orthopedics.

[0003] It is well known that appropriate thermal therapy during the healing period of patients with bone injuries, such as fractures, can promote bone healing. However, bone tissue damage is generally located deep within the tissue, making it difficult for conventional external thermal therapy to effectively target deep tissue damage. In recent years, photothermal / photodynamic therapy (PTT / PDT) has garnered increasing attention and is emerging as an effective, non-invasive strategy for tissue repair and regeneration.

[0004] As a polymer material with a three-dimensional network structure, hydrogel has good biocompatibility, adjustable physicochemical properties and a structure similar to the extracellular matrix, and has received widespread attention in the field of bone tissue engineering. Among them, methacryloylated gelatin (GelMA) hydrogel is a synthetic hydrogel based on the natural biomacromolecule gelatin. It inherits the biological activity of gelatin and at the same time gives it the property of photocrosslinking through methacryloylation, which can quickly form a three-dimensional network structure with a certain mechanical strength under mild conditions. However, the function of pure GelMA hydrogel in promoting bone defect repair is limited, and the current GelMA hydrogel has no photothermal effect and cannot be used in photothermal / photodynamic therapy. Summary of the Invention

[0005] In light of this, the present invention provides a zinc sulfide-modified hydrogel with near-infrared light responsiveness and photothermal effect, as well as its preparation method and application. The zinc sulfide-modified hydrogel provided by the present invention combines good biocompatibility, near-infrared responsiveness, and photothermal effect, and has broad application prospects in bone defect repair.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] A zinc sulfide modified hydrogel with near-infrared light responsiveness and photothermal effect comprises a methacrylated gelatin hydrogel solution and zinc sulfide nanoparticles dispersed in the methacrylated gelatin hydrogel solution.

[0008] Preferably, the content of zinc sulfide nanoparticles in the zinc sulfide-modified hydrogel with near-infrared light responsiveness and photothermal effect is 50 to 5000 μg / mL.

[0009] Preferably, the components of the methacrylated gelatin hydrogel solution include a photoinitiator, methacrylated gelatin and water; the concentration of methacrylated gelatin in the methacrylated gelatin hydrogel solution is 5 to 30% (w / v).

[0010] The present invention also provides a method for preparing the zinc sulfide-modified hydrogel with near-infrared light responsiveness and photothermal effect described in the above scheme, comprising the following steps:

[0011] The zinc sulfide nanoparticles and the methacrylated gelatin hydrogel solution are mixed to obtain the zinc sulfide modified hydrogel with near-infrared light responsiveness and photothermal effect.

[0012] Preferably, the preparation method of the zinc sulfide nanoparticles comprises: mixing a thiourea solution and a soluble zinc salt solution to perform a precipitation reaction to obtain zinc sulfide nanoparticles; the molar ratio of thiourea in the thiourea solution to zinc ions in the soluble zinc salt solution is 1:1 to 1.5.

[0013] Preferably, the concentration of the thiourea solution is 10-20 mg / mL, and the pH value is 7.35-7.45;

[0014] The soluble zinc salt includes one or more of zinc sulfate, zinc nitrate and zinc chloride; the concentration of the soluble zinc salt solution is 0.3-0.4 mol / L.

[0015] Preferably, the precipitation reaction time is 20 to 30 minutes.

[0016] The present invention also provides a zinc sulfide-modified hydrogel cured product, which is obtained by photocuring the zinc sulfide-modified hydrogel with near-infrared light responsiveness and photothermal effect described in the above scheme or the zinc sulfide-modified hydrogel with near-infrared light responsiveness and photothermal effect prepared by the preparation method described in the above scheme.

[0017] Preferably, the light source used for the light curing is ultraviolet light, and the irradiation time is 10 to 30 seconds.

[0018] The present invention also provides the use of the zinc sulfide-modified hydrogel with near-infrared light responsiveness and photothermal effect described in the above scheme, the zinc sulfide-modified hydrogel with near-infrared light responsiveness and photothermal effect prepared by the preparation method described in the above scheme, or the zinc sulfide-modified hydrogel solidified material described in the above scheme in the preparation of products for repairing bone defects.

[0019] The present invention provides a zinc sulfide-modified hydrogel with near-infrared light responsiveness and a photothermal effect, comprising a methacrylated gelatin hydrogel solution and zinc sulfide nanoparticles dispersed within the methacrylated gelatin hydrogel solution. Zinc sulfide (ZnS) is a semiconductor nanomaterial with unique photoelectric properties, including good chemical stability, biocompatibility, and low toxicity. Furthermore, zinc sulfide nanoparticles have a certain light absorption capacity in the near-infrared region, capable of producing a photothermal effect. The inventors have found that the osteogenic differentiation ability of BMSCs can be promoted by low-frequency-low-heat stimulation. Utilizing this biological principle, the present invention introduces ZnS nanoparticles into GelMA hydrogel, and provides a zinc sulfide-modified hydrogel (abbreviated as ZnS@GelMA) with near-infrared light responsiveness-photothermal effect. The zinc sulfide-modified hydrogel has good biocompatibility, near-infrared responsiveness and photothermal effect. Its design ideas and principles are as follows: utilizing the good tissue penetrability of near-infrared light, based on the potential photoresponsiveness of the unique molecular structure of zinc sulfide, under near-infrared illumination conditions, the light energy absorbed by zinc sulfide nanoparticles intensifies the molecular or atomic motion inside it, resulting in a proper increase in the local temperature of the gel and surrounding tissues. The warm effect generated under such conditions can promote the proliferation ability, ATP energy production ability, cell motility and osteogenic differentiation ability of bone marrow stem cells. The zinc sulfide-modified hydrogel provided by the present invention is expected to be applied to bone tissue damage sites under clinical conditions, providing a potentially available biological gel preparation for promoting bone tissue damage, and expanding the application of photothermal effect in bone tissue engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The results of the photothermal effect test of the zinc sulfide modified hydrogel in Example 2 are shown. The upper figure shows the thermal imaging device detection structure, and the lower figure shows the temperature change with photothermal time.

[0021] Figure 2 The results of the hydrogel curing ability test in Example 3 are as follows;

[0022] Figure 3 The results of the CCK-8 assay for cell proliferation in Example 4 are as follows;

[0023] Figure 4 The results of the fluorescence method for detecting cell proliferation in Example 4 are as follows;

[0024] Figure 5 The cell viability test results in Example 5;

[0025] Figure 6 The alkaline phosphatase ALP activity test results in Example 6;

[0026] Figure 7 The results of the cell mineralization ability test in Example 7 are as follows;

[0027] Figure 8 is Ca in Example 8 2+ Concentration test results;

[0028] Figure 9 This is the result of the osteogenesis-specific gene detection in Example 9. DETAILED DESCRIPTION

[0029] The invention provides a zinc sulfide modified hydrogel with near-infrared light responsiveness and photothermal effect, comprising a methacrylated gelatin hydrogel solution and zinc sulfide nanoparticles dispersed in the methacrylated gelatin hydrogel solution.

[0030] In the present invention, the content of zinc sulfide nanoparticles in the zinc sulfide-modified hydrogel with near-infrared light responsiveness-photothermal effect is preferably 50 to 5000 μg / mL, specifically 50 μg / mL, 100 μg / mL, 200 μg / mL, 500 μg / mL, 1000 μg / mL, 2000 μg / mL, 3000 μg / mL or 5000 μg / mL, preferably 100 μg / mL.

[0031] In the present invention, the average particle size of the zinc sulfide nanoparticles is preferably 20 to 40 nm.

[0032] In the present invention, the components of the methacrylated gelatin hydrogel solution preferably include a photoinitiator, methacrylated gelatin and water; the photoinitiator is preferably lithium (phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP); the concentration of methacrylated gelatin in the methacrylated gelatin hydrogel solution is preferably 5-30% (w / v), preferably 8-20% (w / v), and more preferably 10% (w / v); the concentration of the photoinitiator in the methacrylated gelatin hydrogel solution is preferably 0.2-0.5% (w / v), preferably 0.25% (w / v); the molecular weight of the methacrylated gelatin is preferably 100-200 KDa, and the degree of amino substitution is preferably 25-95%. In a specific embodiment of the present invention, the methacrylated gelatin hydrogel solution is a commercially available product, model EFL-GM-60 ， The concentration of methacrylated gelatin was 10% (w / v), the concentration of photoinitiator (LAP) was 0.25% (w / v), and the degree of amino substitution was 60±5%, which were purchased from Suzhou Yongqinquan Intelligent Equipment Co., Ltd.

[0033] The present invention also provides a method for preparing the zinc sulfide-modified hydrogel with near-infrared light responsiveness and photothermal effect described in the above scheme, comprising the following steps:

[0034] The zinc sulfide nanoparticles and the methacrylated gelatin hydrogel solution are mixed to obtain the zinc sulfide modified hydrogel with near-infrared light responsiveness and photothermal effect.

[0035] In the present invention, the mixing is preferably carried out under stirring conditions, and the stirring speed is preferably less than 500 rpm, specifically 100 rpm, 200 rpm, 300 rpm or 400 rpm; the mixing temperature is preferably room temperature, and the mixing time is preferably 30 min.

[0036] In the present invention, the method for preparing the zinc sulfide nanoparticles preferably comprises: mixing a thiourea solution and a soluble zinc salt solution to perform a precipitation reaction to obtain zinc sulfide nanoparticles; the molar ratio of thiourea in the thiourea solution to zinc ions in the soluble zinc salt solution is preferably 1:1 to 1.5, more preferably 1:1.

[0037] In the present invention, the concentration of the thiourea solution is preferably 10 to 20 mg / mL, specifically 15 mg / mL, and the pH value of the thiourea solution is preferably 7.35 to 7.45, preferably 7.3 to 7.4. The thiourea solution is preferably prepared by dissolving thiourea in water and then adjusting the pH value to 7.35 to 7.45 with aqueous ammonia to obtain the thiourea solution. In a specific embodiment of the present invention, the thiourea solution is prepared and used immediately.

[0038] In the present invention, the soluble zinc salt preferably includes one or more of zinc sulfate, zinc nitrate and zinc chloride; the concentration of the soluble zinc salt solution is preferably 0.3-0.4 mol / L; and the volume ratio of the thiourea solution to the soluble zinc salt solution is preferably 10-15:10-15.

[0039] In the present invention, the precipitation reaction time is preferably 20 to 30 minutes, specifically 20 minutes, 25 minutes or 30 minutes; the precipitation reaction temperature is preferably room temperature; in a specific embodiment of the present invention, the soluble zinc salt solution is preferably added dropwise to the thiourea solution for precipitation reaction, and the precipitation reaction time starts after the dropwise addition of the soluble zinc salt solution is completed.

[0040] After the precipitation reaction is completed, the present invention preferably centrifuges the obtained product liquid, collects the solid product, washes and dries it, and obtains the zinc sulfide nanoparticles; the centrifugal speed is preferably 12000 rpm, and the time is preferably 10 min; the washing is preferably performed by alternating washing with deionized water and ethanol; the drying temperature is preferably 60°C, and the drying is preferably vacuum drying.

[0041] The present invention also provides a zinc sulfide-modified hydrogel cured product, characterized in that it is obtained by photocuring the zinc sulfide-modified hydrogel with near-infrared light responsiveness and photothermal effect described in the above scheme or the zinc sulfide-modified hydrogel with near-infrared light responsiveness and photothermal effect prepared by the preparation method described in the above scheme; the light source used for the photocuring is preferably ultraviolet light, the wavelength of the ultraviolet light is preferably 405 nm, and the irradiation time is preferably 10 to 30 seconds.

[0042] The zinc sulfide modified hydrogel provided by the present invention is liquid at room temperature, has good fluidity, and can be cured and shaped under ultraviolet light.

[0043] The present invention also provides the use of the zinc sulfide-modified hydrogel with near-infrared light responsiveness and photothermal effect described in the above scheme, the zinc sulfide-modified hydrogel with near-infrared light responsiveness and photothermal effect prepared by the preparation method described in the above scheme, or the cured zinc sulfide-modified hydrogel described in the above scheme in preparing a product for repairing bone defects. In the present invention, the product for repairing bone defects can specifically be a biological gel preparation.

[0044] The zinc sulfide modified hydrogel with near-infrared light responsiveness and photothermal effect and its cured product provided by the present invention can produce photothermal effect under near-infrared light irradiation, which can promote the proliferation ability, ATP energy production ability, cell motility and osteogenic differentiation ability of bone marrow stem cells, and at the same time have good biocompatibility and have broad application prospects in the field of bone defect repair. In a specific embodiment of the present invention, the wavelength of the near-infrared light irradiation light source is preferably 808nm, and the power is preferably 1.5W / cm 2 .

[0045] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0046] Example 1

[0047] Eleven drops of 7 wt % ammonia water were added to 10 mL of a 15 mg / mL thiourea solution to obtain a weakly alkaline thiourea solution with a pH of 7.4. A 0.3 mol / L zinc sulfate solution was prepared and slowly added dropwise to the weakly alkaline thiourea solution using a pipette, with the molar ratio of thiourea to zinc ions being controlled at 1:1. After the addition was complete, the mixture was magnetically stirred at room temperature for 30 minutes. After the room temperature reaction was completed, the complex product, zinc sulfide, was collected by centrifugation (12,000 rpm for 10 minutes). The zinc sulfide product was washed alternately with deionized water and ethanol until the supernatant was colorless, and then dried under vacuum at 60°C to obtain zinc sulfide nanoparticles.

[0048] The collected zinc sulfide nanoparticles were added to the finished methacrylated gel (GelMA) hydrogel solution (EFL-GM-60) in a sterile operating table, placed in a magnetic stirrer, and stirred at 300 rpm / min at room temperature for 30 minutes to thoroughly mix to obtain zinc sulfide modified hydrogel (ZnS@GelMA). By controlling the amount of zinc sulfide nanoparticles added, ZnS@GelMA hydrogels with zinc sulfide nanoparticle contents of 50 μg / mL, 100 μg / mL, 200 μg / mL, 500 μg / mL, 1000 μg / mL, 2000 μg / mL, 3000 μg / mL and 5000 μg / mL were obtained, respectively.

[0049] ZnS@GelMA hydrogel usage conditions: This hydrogel is liquid at room temperature and has good fluidity, making it easy to apply to plates, spread, and perform other common cell culture operations. After completing these operations, if curing and shaping are required, use a 405nm light source for 20 seconds to achieve gelation.

[0050] Example 2 Detection of the thermal effect of zinc sulfide modified hydrogel under light irradiation

[0051] Cell viability was analyzed using cellular ATP energy production. The specific method is as follows: the ZnS@GelMA hydrogels of different concentrations prepared in Example 1 were used as raw materials. GelMA hydrogel and ZnS@GelMA hydrogels of different concentrations were added to a 1.5mL EP tube, with the addition amount of 1000μL each, and irradiated with 405nm ultraviolet light for 1min for curing and crosslinking. A near-infrared light source of 808nm (power of 1.5W / cm 2 During the irradiation period, a thermal imager was used to detect and record the near-infrared light heating effect of each group of hydrogels at 1-minute intervals.

[0052] The test results are as follows Figure 1 As shown, Figure 1The results in the literature show that the ZnS@GelMA hydrogel of the present invention has a low NIR heat generation effect, and the heat generation effect of the ZnS@GelMA hydrogel with an addition amount of zinc sulfide of 100 μg / mL is obvious and stable.

[0053] Example 3 Hydrogel curing ability test

[0054] The curing ability of the hydrogel was tested using the principle of UV cross-linking and curing hydrogels. The specific method is: add 200μL of GelMA hydrogel or ZnS@GelMA hydrogel (zinc sulfide content is 100μg / mL) to a 1.5mL EP tube. Use 405nm UV light to irradiate for 0, 5, 10, 30, and 60s respectively for curing and cross-linking. Observe and record the curing and gelation of the hydrogel. At the same time, set up a GelMA hydrogel without UV light as a control group.

[0055] The test results are as follows Figure 2 As shown, Figure 2 The results show that the GelMA hydrogel without UV irradiation (LAP-) is in liquid state, while the GelMA hydrogel and ZnS@GelMA hydrogel after UV irradiation (LAP+) are both solidified, and the ZnS@GelMA hydrogel solidifies faster than the GelMA hydrogel, indicating that the addition of zinc sulfide can promote the cross-linking and curing of the GelMA hydrogel.

[0056] Example 4 Proliferation ability detection

[0057] 1. Detect cell proliferation ability by CCK-8 method. Based on the principle that CCK-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt) can be reduced by dehydrogenase in living cells to a highly water-soluble orange-yellow formazan product, and the amount of the generated formazan product is proportional to the number of living cells, the cell proliferation ability or cell number can be quantitatively analyzed by measuring its absorbance at a wavelength of about 450nm. The specific method is as follows: (1) The experimental groups are blank control group (Con), GelMA group and ZnS@GelMA group. (2) GelMA or ZnS@GelMA hydrogel (zinc sulfide content of 100μg / mL) is added to a 96-well culture plate at a volume of 100μL per well and irradiated with ultraviolet light for 1min for curing and cross-linking. (3) 100 μL of culture medium containing 1000 bone marrow stem cells (BMSCs) was added to each well and cultured in a 37°C incubator. During this period, an 808 nm near-infrared light source (NIR) with a power of 1.5 W / cm 2) Irradiate for 10 minutes. (4) After 72 hours, aspirate the culture medium in the culture wells, add 100 μL of a reaction solution containing 10 wt% CCK-8 to each well, and continue incubating in a 37°C incubator for 1 hour. (5) Use a spectrophotometer to measure the absorbance of the reaction wells at 450 nm and record the values.

[0058] 2. Detect cell proliferation ability by fluorescence method. The specific method is as follows: (1) The experimental groups were blank control group (Con), GelMA group and ZnS@GelMA group. (2) GelMA or ZnS@GelMA hydrogel (zinc sulfide content of 100 μg / mL) was added to a 96-well culture plate at a volume of 100 μL per well, and irradiated with 405 nm ultraviolet light for 1 min for curing and crosslinking. (3) 100 μL of culture medium containing 1000 green fluorescent GFP-labeled bone marrow stem cells (BMSCs) was added to each well and cultured in a 37 ° C incubator for 72 hours. During this period, an 808 nm near-infrared light source NIR (power of 1.5 W / cm 2 (4) Using an inverted fluorescence microscope, observe and photograph the cell proliferation. A control group without near-infrared light was also set up.

[0059] The results of the CCK-8 assay to detect cell proliferation are as follows Figure 3 As shown in the figure, the test results of cell proliferation ability detected by fluorescence method are as follows Figure 4 As shown. Figure 3 It can be seen that the cell proliferation ability of the ZnS@GelMA hydrogel group was significantly higher than that of the blank group and the GelMA hydrogel group. Figure 4 It can be seen that under NIR- (no near-infrared light irradiation) conditions, the cell proliferation ability of the GelMA hydrogel group and the ZnS@GelMA hydrogel group was slightly higher than that of the blank control CON group. Under NIR+ (near-infrared light irradiation) conditions, the cell proliferation ability of the ZnS@GelMA hydrogel group with 100 μg / mL ZnS added was significantly higher than that of the other two groups.

[0060] Example 5 Cell Viability Detection

[0061] The intracellular ATP content was determined by chemiluminescence to analyze the cell viability. The specific method is as follows: (1) The experimental groups were blank control group (Con), GelMA group and ZnS@GelMA group. (2) GelMA or ZnS@GelMA hydrogel was added to a 6-well culture plate at a volume of 100 μL per well and irradiated with 405 nm ultraviolet light for 1 min for curing and crosslinking. (3) GelMA or ZnS@GelMA hydrogel (100 μg / mL) was added to a 96-well culture plate at a volume of 100 μL per well and irradiated with ultraviolet light for 1 min for curing and crosslinking. (4) 100 μL of culture medium containing 1000 bone marrow stem cells (BMSCs) was added to each well and cultured in a 37°C incubator for 72 hours. During this period, an 808 nm near-infrared light source NIR (power of 1.5 W / cm 2 ) irradiate for 10 min. (5) Take out the cell culture plate and equilibrate it at room temperature for 10 min. Add 100 μL CellTiter-Lumi TM (6) Oscillate at room temperature for 2 minutes to promote cell lysis, then incubate at room temperature (approximately 25°C) for 10 minutes to allow the luminescence signal to stabilize. (7) Perform chemiluminescence detection using a multifunctional microplate reader with chemiluminescence detection function and record the results.

[0062] The test results are as follows Figure 5 As shown, Figure 5 The results showed that under NIR+ conditions, the intracellular ATP content of the ZnS@GelMA hydrogel group with 100 μg / mL ZnS was significantly higher than that of the other two groups, and its cell viability was significantly enhanced.

[0063] Example 6 Alkaline phosphatase ALP activity detection

[0064] The early osteogenic differentiation ability of cells was evaluated by alkaline phosphatase (ALP) activity assay. The specific methods are as follows: (1) The experimental groups were blank control group (Con), GelMA group and ZnS@GelMA group. (2) GelMA or ZnS@GelMA hydrogel (100 μg / mL) was added to a 6-well culture plate at a volume of 1000 μL per well and irradiated with 405 nm UV light for 1 min for curing and crosslinking. (3) 1500 μL of 2×10 5 The culture medium of bone marrow stem cells (BMSCs) was placed in a 37℃ incubator for 24 hours. (4) The osteogenic differentiation induction culture medium was replaced and induced in a 37℃ incubator for 72 hours. During this period, an 808nm near-infrared light source (NIR) with a power of 1.5W / cm 2) irradiate for 10 minutes. (5) Take a 6-well plate that has been induced for 3 days of osteogenic differentiation, discard the osteogenic differentiation induction medium, wash the cell growth surface with PBS twice, and discard the residual PBS liquid; add 600μL of ALP extraction lysis solution, and place it in a 37℃ environment for incubation for 15 minutes. (6) Use a clean cell scraper to gently scrape the cells in each well, transfer the lysis suspension into a 1.5mL EP tube, centrifuge at 4℃, 14000rpm for 10 minutes, and transfer the supernatant to a new EP tube, which is the extracted ALP protein sample solution. (7) Take a new 96-well plate, add 50μL of ALP reaction substrate solution to each well, continue to add 50μL ALP buffer solution to each well, tap gently to mix, add 10μL ALP protein sample solution to the detection well, place it in a constant temperature environment at 37℃, and incubate it for 15 minutes. (8) Remove the 96-well plate and observe with the naked eye that a clear light yellow color change has appeared in the test wells. Then, add 110 μL of 0.5 M NaOH solution to each reaction well to terminate the reaction. (9) Use a microplate reader with a wavelength of 405 nm to measure the corresponding absorbance value (OD value).

[0065] The test results are as follows Figure 6 As shown, Figure 6 The results showed that under NIR- conditions, the intracellular alkaline phosphatase activity of the GelMA hydrogel group and the ZnS@GelMA hydrogel group was slightly higher than that of the blank control CON group. Under NIR+ conditions, the cell proliferation ability of the ZnS@GelMA hydrogel group with 100 μg / mL ZnS was significantly higher than that of the other two groups.

[0066] Example 7 Mineralization Capacity Detection

[0067] The mineralization capacity of cells was assessed by Alizarin red staining of mineralized nodules. The specific test method was as follows: (1) The experimental groups were blank control group (Con), GelMA group and ZnS@GelMA group. (2) GelMA or ZnS@GelMA hydrogel (100 μg / mL) was added to a 6-well culture plate at a volume of 1000 μL per well and irradiated with 405 nm UV light for 1 min for curing and crosslinking. (3) 1500 μL of 2×10 5 The culture medium of bone marrow stem cells (BMSCs) was placed in a 37℃ incubator for 24 hours. (4) The osteogenic differentiation induction culture medium was replaced and induced in a 37℃ incubator for 14 days. During this period, an 808nm near-infrared light source (NIR) (power of 1.5W / cm2) was used every 24 hours. 2) Irradiate for 10 minutes. (5) Take the 6-well plate that has been induced for 14 days of osteogenic differentiation, discard the osteogenic differentiation induction medium, wash the cell growth surface twice with PBS, and discard the residual PBS liquid; add 2 mL of 70% ethanol to each well, place it in a constant temperature environment at 4°C, and fix it for 1 hour. (6) Take out the 6-well plate and equilibrate it to room temperature, discard the 70% ethanol fixative in the well, and wash it twice with ultrapure water. (7) Add 2 mL of 40 mM Alizarin Red Solution (ARS, pH = 4.2) to each well and stain it at room temperature. After observing obvious bright red coloration, immediately discard the ARS staining solution. (8) Wash it with ultrapure water 5 times, place it on a horizontal shaker at 100 rpm, and continue to shake it gently for 15 minutes to try to remove non-specific staining. (9) Collect images in the transmission mode of the scanner.

[0068] The test results are as follows Figure 7 shown. Figure 7 The results showed that under NIR- conditions, the cell mineralization ability of the GelMA hydrogel group and the ZnS@GelMA hydrogel group was higher than that of the blank control CON group. Under NIR+ conditions, the cell mineralization ability of the GelMA hydrogel group and the ZnS@GelMA hydrogel group was further enhanced, and the ZnS@GelMA hydrogel group was significantly higher than the GelMA hydrogel group.

[0069] Example 8 Calcium ion (Ca 2+ ) concentration determination

[0070] (1) Take the 6-well culture plate after ARS staining in Example 6, add 10% w / v CPC, and incubate at 37°C for 30 minutes to completely dissolve the ARS stain. (2) Collect the CPC solution into a clean EP tube, dilute it 10 times with ultrapure water, take out 200 μL and add it to a 96-well plate. (3) Measure the OD value with a microplate reader at a wavelength of 562 nm. (4) Collect the cell membrane sheets in the secondary wells of each culture plate using RIPA lysis buffer, extract the total protein, and determine the protein concentration of each protein using the BCA method. (5) Calculate Ca using the ARS standard curve 2+ The relative concentration of Ca 2+ Calculate the Ca of the sample using a concentration curve 2+ concentration.

[0071] The test results are as follows Figure 8 As shown, Figure 8 The results showed that under NIR- conditions, the intracellular calcium ion concentrations in the GelMA hydrogel and ZnS@GelMA hydrogel groups were higher than those in the blank control CON group. Under NIR+ conditions, the intracellular calcium ion concentrations in the GelMA hydrogel and ZnS@GelMA hydrogel groups increased further, and the concentration in the ZnS@GelMA hydrogel group was significantly higher than that in the GelMA hydrogel group. This demonstrates that the near-infrared light responsiveness of the ZnS@GelMA hydrogel can significantly enhance the mineralization capacity of cells in vitro.

[0072] Example 9 Detection of osteoblast-specific genes (BSP, OCN)

[0073] The osteogenic differentiation ability of cells was evaluated by detecting osteogenic specific matrix proteins. The specific methods are as follows: (1) The experimental groups were blank control group (Con), GelMA group and ZnS@GelMA group. (2) GelMA or ZnS@GelMA hydrogel (100 μg / mL) was added to 6-well culture plates at a volume of 1000 μL per well and irradiated with 405 nm UV light for 1 min for curing and crosslinking. (3) 1500 μL of 2×10 5 The culture medium of bone marrow stem cells (BMSCs) was placed in a 37℃ incubator for 24 hours. (4) The osteogenic differentiation induction culture medium was replaced and induced in a 37℃ incubator for 14 days. During this period, an 808nm near-infrared light source (NIR) (power of 1.5W / cm2) was used every 24 hours. 2 ) Irradiate for 10 minutes. (5) Take the 6-well plate after 14 days of osteogenic induction and harvest the cells by trypsin digestion. (6) Collect total RNA using an RNA extraction kit. (7) Detect the expression levels of BSP and OCN by reverse transcription and real-time fluorescence quantitative PCR.

[0074] The test results are as follows Figure 9 As shown, Figure 9 The results in the study showed that under NIR+ conditions, the expression levels of BSP and OCN in the GelMA hydrogel and ZnS@GelMA hydrogel groups were significantly higher than those in the CON control group, and the expression levels in the ZnS@GelMA hydrogel group were significantly higher than those in the GelMA hydrogel group. This demonstrates that the near-infrared light responsiveness of the ZnS@GelMA hydrogel can significantly enhance the osteogenic differentiation of cells.

[0075] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Application of zinc sulfide modified hydrogel with near-infrared light responsiveness and photothermal effect or its cured product in the preparation of products for repairing bone defects, characterized in that: The zinc sulfide modified hydrogel with near-infrared light responsiveness and photothermal effect comprises a methacrylated gelatin hydrogel solution and zinc sulfide nanoparticles dispersed in the methacrylated gelatin hydrogel solution; the content of the zinc sulfide nanoparticles in the zinc sulfide modified hydrogel with near-infrared light responsiveness and photothermal effect is 50-5000 μg / mL; the zinc sulfide modified hydrogel with near-infrared light responsiveness and photothermal effect and its cured product generate a photothermal effect under near-infrared light irradiation; the components of the methacrylated gelatin hydrogel solution include a photoinitiator, methacrylated gelatin and water; and the concentration of methacrylated gelatin in the methacrylated gelatin hydrogel solution is 5-30 w / v%.

2. The use according to claim 1, characterized in that The preparation method of the zinc sulfide modified hydrogel with near-infrared light responsiveness and photothermal effect comprises the following steps: The zinc sulfide nanoparticles and the methacrylated gelatin hydrogel solution are mixed to obtain the zinc sulfide modified hydrogel with near-infrared light responsiveness and photothermal effect.

3. The use according to claim 2, characterized in that The preparation method of the zinc sulfide nanoparticles comprises: mixing a thiourea solution and a soluble zinc salt solution to perform a precipitation reaction to obtain the zinc sulfide nanoparticles; the molar ratio of thiourea in the thiourea solution to zinc ions in the soluble zinc salt solution is 1:1-1.

5.

4. The use according to claim 3, characterized in that The concentration of the thiourea solution is 10-20 mg / mL, and the pH value is 7.35-7.45; The soluble zinc salt includes one or more of zinc sulfate, zinc nitrate and zinc chloride; the concentration of the soluble zinc salt solution is 0.3-0.4 mol / L.

5. The use according to claim 3 or 4, characterized in that The precipitation reaction time is 20 to 30 minutes.

6. The use according to claim 3 or 4, characterized in that The solidified material is obtained by photocuring a zinc sulfide modified hydrogel with near-infrared light responsiveness and photothermal effect.

7. The use according to claim 6, characterized in that The light source used for the photocuring is ultraviolet light, and the irradiation time is 10 to 30 seconds.

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