Zinc sulfide modified hydrogel with near-infrared light responsiveness-photothermal effect as well as preparation method and application of zinc sulfide modified hydrogel

Abstract

The invention relates to the technical field of medicines, and provides zinc sulfide modified hydrogel with near-infrared light responsiveness-photothermal effect as well as a preparation method and application of the zinc sulfide modified hydrogel. The zinc sulfide modified hydrogel provided by the invention comprises a methacrylated gelatin hydrogel solution and zinc sulfide nanoparticles dispersed in the methacrylated gelatin (GelMA) hydrogel solution. According to the invention, the zinc sulfide nanoparticles are introduced into the GelMA hydrogel by utilizing the biological principle that low frequency-low thermal stimulation can promote the osteogenic differentiation capability of the BMSCs, and the obtained modified hydrogel has good biocompatibility, near-infrared responsiveness and photothermal effect; the prepared bone marrow stem cell biological gel has the advantages that the bone marrow stem cell proliferation capacity, ATP (adenosine triphosphate) energy generation capacity, cell movement activity and osteogenic differentiation capacity can be promoted, the bone marrow stem cell biological gel is expected to be applied to bone tissue injury sites under clinical conditions, a potential available biological gel preparation is provided for promoting bone tissue injury, and the application of the photothermal effect in bone tissue engineering is expanded.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical 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 defect is a common problem in clinical practice, which is caused by various reasons such as trauma, tumor resection, and congenital diseases. At present, bone defect repair remains a major challenge in the field of orthopedics.

[0003] As is well known, appropriate warm physiotherapy during the healing period of patients with bone injuries such as fractures can promote bone injury healing. However, bone tissue injuries are generally located deep in the tissue, and it is difficult for general external warm physiotherapy to effectively act on deep tissue injuries. In recent years, photothermal / photodynamic therapy (PTT / PDT) has received increasing attention and is becoming an effective and non-invasive strategy for tissue repair and regeneration.

[0004] As a three-dimensional network-structured polymer material, hydrogel has good biocompatibility, adjustable physicochemical properties, and a structure similar to the extracellular matrix, and has received extensive attention in the field of bone tissue engineering. Among them, methacrylated gelatin (GelMA) hydrogel is a synthetic hydrogel based on the natural biopolymer gelatin. It inherits the biological activity of gelatin and endows it with photocrosslinkable characteristics through methacrylation, and can rapidly form a three-dimensional network structure with 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 view of this, the present invention 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 has both 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 invention purposes, the present invention provides the following technical solutions:

[0007] A zinc sulfide modified hydrogel with near-infrared light responsiveness-photothermal effect, comprising 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-photothermal effect is 50-5000 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 5-30% (w / v).

[0010] The present invention also provides a preparation method of the zinc sulfide-modified hydrogel with near-infrared light responsiveness-photothermal effect described in the above solution, including the following steps:

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

[0012] Preferably, the preparation method of the zinc sulfide nanoparticles includes: mixing a thiourea solution and a soluble zinc salt solution to carry out 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 1:1-1.5.

[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 time of the precipitation reaction is 20-30 min.

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

[0017] Preferably, the light source used for photocuring is ultraviolet light, and the irradiation time is 10-30 s.

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

[0019] The present invention provides a zinc sulfide modified hydrogel with near-infrared light responsiveness-photothermal effect, which comprises a methacrylated gelatin hydrogel solution and zinc sulfide nanoparticles dispersed in the methacrylated gelatin hydrogel solution. Zinc sulfide (ZnS) is a semiconductor nanomaterial with unique optoelectronic properties, having good chemical stability, biocompatibility and low toxicity; and zinc sulfide nanoparticles have a certain light absorption ability in the near-infrared region and can generate photothermal effect. The inventors of the present invention found that the osteogenic differentiation ability of BMSCs can be promoted by using low-frequency-low heat stimulation. Based on this biological principle, the present invention introduces ZnS nanoparticles into GelMA hydrogel to provide a zinc sulfide modified hydrogel with near-infrared light responsiveness-photothermal effect (abbreviated as ZnS@GelMA). This zinc sulfide modified hydrogel has both good biocompatibility, near-infrared responsiveness and photothermal effect. Its design idea and principle are as follows: by utilizing the good tissue penetration of near-infrared light and based on the potential light responsiveness of the unique molecular structure of zinc sulfide, under the condition of near-infrared light illumination, the light energy absorbed by zinc sulfide nanoparticles makes the molecules or atoms inside it move more violently, resulting in an appropriate increase in the local temperature of the hydrogel and the surrounding tissues. The thermal effect generated under this condition can promote the proliferation ability, ATP energy generation 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 the bone tissue injury site under clinical conditions, providing a potentially available biological gel preparation for promoting bone tissue injury and expanding the application of photothermal effect in bone tissue engineering. Description of the Drawings

[0020] Figure 1 It is the detection result of the heat generation effect of the zinc sulfide modified hydrogel by light irradiation in Example 2, where the upper figure is the detection result of the thermal imager, and the lower figure is the change of temperature with the light irradiation time;

[0021] Figure 2 It is the detection result of the hydrogel curing ability in Example 3;

[0022] Figure 3 It is the test result of detecting the cell proliferation ability by CCK-8 method in Example 4;

[0023] Figure 4 It is the test result of detecting the cell proliferation ability by fluorescence method in Example 4;

[0024] Figure 5 It is the detection result of cell viability in Example 5;

[0025] Figure 6 It is the detection result of the alkaline phosphatase ALP activity in Example 6;

[0026] Figure 7 It is the detection result of cell mineralization ability in Example 7;

[0027] Figure 8 For the Ca in Example 8 2+ Concentration detection result;

[0028] Figure 9 For the osteogenic specific gene detection result in Example 9. Detailed implementation mode

[0029] The present invention provides a zinc sulfide modified hydrogel with near-infrared light responsiveness-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 50-5000 μg / mL, specifically it can be 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, and preferably 100 μg / mL.

[0031] In the present invention, the average particle size of the zinc sulfide nanoparticles is preferably 20-40 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 further 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 amino substitution degree is preferably 25-95%. In a specific embodiment of the present invention, the methacrylated gelatin hydrogel solution is a commercially available product, with the model number EFL-GM-60 ， Among them, the concentration of methacrylated gelatin is 10% (w / v), the concentration of the photoinitiator (LAP) is 0.25% (w / v), the amino substitution degree is 60±5%, and it is purchased from Suzhou Yongqinquan Intelligent Equipment Co., Ltd.

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

[0034] Mix zinc sulfide nanoparticles and a methacrylated gelatin hydrogel solution to obtain the zinc sulfide-modified hydrogel with near-infrared light-responsive photothermal effect.

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

[0036] In the present invention, the preparation method of the zinc sulfide nanoparticles preferably includes: mixing a thiourea solution and a soluble zinc salt solution to carry out 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 - 20 mg / mL, specifically can be 15 mg / mL. The pH value of the thiourea solution is preferably 7.35 - 7.45, preferably 7.3 - 7.4; the preparation method of the thiourea solution is preferably: dissolving thiourea in water, and then adjusting the pH value to 7.35 - 7.45 with ammonia water to obtain the thiourea solution; in the specific embodiments 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; 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 time of the precipitation reaction is preferably 20 - 30 min, specifically can be 20 min, 25 min or 30 min; the temperature of the precipitation reaction is preferably room temperature; in the specific embodiments of the present invention, the soluble zinc salt solution is preferably dropped into the thiourea solution for the precipitation reaction, and the time of the precipitation reaction starts to count after the dropping of the soluble zinc salt solution is completed.

[0040] After the precipitation reaction is completed, in the present invention, the obtained product liquid is preferably centrifuged, and the solid product is collected and then washed and dried to obtain the zinc sulfide nanoparticles; the rotation speed of the centrifugation is preferably 12000 rpm, and the time is preferably 10 min; the washing is preferably carried out by alternately washing with deionized water and ethanol; the temperature of the drying is preferably 60 °C, and the drying is preferably vacuum drying.

[0041] The present invention also provides a zinc sulfide modified hydrogel solidified product, which is characterized in that it is obtained by photocuring the zinc sulfide modified hydrogel with near-infrared light responsiveness-photothermal effect described in the above solution or the zinc sulfide modified hydrogel with near-infrared light responsiveness-photothermal effect prepared by the preparation method described in the above solution; 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 10-30 s.

[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 irradiation.

[0043] The present invention also provides the application of the zinc sulfide modified hydrogel with near-infrared light responsiveness-photothermal effect described in the above solution, the zinc sulfide modified hydrogel with near-infrared light responsiveness-photothermal effect prepared by the preparation method described in the above solution, or the zinc sulfide modified hydrogel solidified product described in the above solution in the preparation of products 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-photothermal effect provided by the present invention and its solidified product can generate a photothermal effect under near-infrared light irradiation, can promote the proliferation ability, ATP energy generation ability, cell motility and osteogenic differentiation ability of bone marrow stem cells, and at the same time has good biocompatibility, and has broad application prospects in the field of bone defect repair. In a specific embodiment of the present invention, the wavelength of the light source for the near-infrared light irradiation is preferably 808 nm, and the power is preferably 1.5 W / cm 2 。

[0045] Next, the technical solutions in the present invention will be clearly and completely described in conjunction with the embodiments in the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0046] Example 1

[0047] Add 11 drops of 7 wt% ammonia water to 10 mL of 15 mg / mL thiourea solution to obtain a weakly basic thiourea solution with a pH value of 7.4; prepare a 0.3 mol / L zinc sulfate solution, and slowly add the zinc sulfate solution to the above-mentioned weakly basic thiourea solution with a pipette gun, controlling the molar ratio of thiourea to zinc ions to be 1:1. After the addition is complete, stir magnetically at room temperature for 30 min. After the reaction at room temperature ends, collect the complex product zinc sulfide by centrifugation (12,000 rpm, 10 min), and wash the zinc sulfide product alternately with deionized water and ethanol until the supernatant is colorless, and then dry it under vacuum at 60 °C to obtain zinc sulfide nanoparticles.

[0048] Add the collected zinc sulfide nanoparticles to the finished methacrylated gel (GelMA) hydrogel solution (EFL-GM-60) in a sterile operating table, place it in a magnetic stirrer, and stir at a speed of 300 rpm / min for 30 min at room temperature to thoroughly mix to obtain zinc sulfide modified hydrogel (ZnS@GelMA). By controlling the addition amount of zinc sulfide nanoparticles, ZnS@GelMA hydrogels with the contents of zinc sulfide nanoparticles being 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 are obtained respectively.

[0049] Usage conditions of ZnS@GelMA hydrogel: This hydrogel is liquid at room temperature, has good fluidity, and is easy to perform necessary operations such as spreading on plates and coating during general cell culture. After completing the above operations, if solidification and shaping are required, use a 405 nm light source to irradiate for 20 s to gel it.

[0050] Example 2 Detection of light-induced heat generation effect of zinc sulfide modified hydrogel

[0051] Analyze cell viability by using cell ATP energy production. The specific method is as follows: Use the different concentrations of ZnS@GelMA hydrogels prepared in Example 1 as raw materials. Add GelMA hydrogel and different concentrations of ZnS@GelMA hydrogels to 1.5 mL EP tubes, and the addition amount is 1000 μL for each. Cure and crosslink them by irradiating with ultraviolet light of 405 nm for 1 min. Use a near-infrared light source NIR of 808 nm (power is 1.5 W / cm 2 ) to irradiate for 10 min. During this period, use an infrared thermal imager to detect and photograph and record the near-infrared light-induced heat generation effect of each group of hydrogels every 1 min.

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

[0053] Example 3 Detection of Hydrogel Curing Ability

[0054] Using the principle of ultraviolet light cross-linking and curing the hydrogel, the curing ability of the hydrogel was detected. The specific method was as follows: 200 μL of GelMA hydrogel or ZnS@GelMA hydrogel (zinc sulfide content was 100 μg / mL) was added to a 1.5 mL EP tube. Ultraviolet light at 405 nm was used to irradiate for 0, 5, 10, 30, and 60 s respectively for curing cross-linking. Observe and photograph the gelation situation of the hydrogel. At the same time, a GelMA hydrogel without ultraviolet light irradiation was set as the control group.

[0055] The test results are as Figure 2 shown, Figure 2 The results in show that the GelMA hydrogel without ultraviolet light irradiation (LAP-) was in a liquid state, and both the GelMA hydrogel and ZnS@GelMA hydrogel after ultraviolet light irradiation (LAP+) were cured. Compared with the GelMA hydrogel, the ZnS@GelMA hydrogel had a faster curing speed, indicating that the addition of zinc sulfide could promote the cross-linking and curing of the GelMA hydrogel.

[0056] Example 4 Detection of Proliferation Ability

[0057] 1. The cell proliferation ability was detected by the CCK-8 method. Using the principle that CCK-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid phenyl)-2H-tetrazolium monosodium salt) can be reduced by dehydrogenases in living cells to form a highly water-soluble orange-yellow formazan product, and the amount of the formed formazan product is proportional to the number of living cells, the cell proliferation ability or the number of cells can be quantitatively analyzed by measuring its absorbance at a wavelength of about 450 nm. The specific method was as follows: (1) The experimental groups were the blank control group (Con), the GelMA group, and the ZnS@GelMA group. (2) GelMA or ZnS@GelMA hydrogel (zinc sulfide content was 100 μg / mL) was added to a 96-well culture plate at a volume of 100 μL per well, and ultraviolet light was irradiated for 1 min for curing cross-linking. (3) 100 μL of the culture solution containing 1000 bone marrow stem cells (BMSCs) was added to each well and cultured in an incubator at 37 °C. During this period, every 24 hours, a near-infrared light source NIR at 808 nm (power was 1.5 W / cm 2) Irradiate for 10 min. (4) After 72 hours, aspirate the culture medium in the culture wells, and add 100 μL of reaction solution containing 10 wt% CCK-8 to each well, and continue to incubate in a 37 °C incubator for 1 hour. (5) Use a spectrophotometer to measure the absorbance value of the reaction wells at 450 nm, and record and count.

[0058] 2. Detect the cell proliferation ability by fluorescence method. The specific method is as follows: (1) The experimental groups are blank control group (Con), GelMA group and ZnS@GelMA group. (2) Add GelMA or ZnS@GelMA hydrogel (zinc sulfide content is 100 μg / mL) to a 96-well culture plate at a volume of 100 μL per well, and irradiate with 405 nm ultraviolet light for 1 min for curing and crosslinking. (3) Add 100 μL of culture medium containing 1000 green fluorescent GFP-labeled bone marrow stem cells (BMSCs) to each well, and culture in a 37 °C incubator for 72 hours. During this period, use a near-infrared light source NIR at 808 nm (power is 1.5 W / cm 2 ) Irradiate for 10 min. (4) Use an inverted fluorescence microscope to observe and photograph and record the cell proliferation situation. At the same time, set up an experimental group without near-infrared light irradiation as a control.

[0059] The test results of detecting the cell proliferation ability by CCK-8 method are as Figure 3 shown, and the test results of detecting the cell proliferation ability by fluorescence method are as Figure 4 shown. According to Figure 3 it can be seen that the cell proliferation ability of the ZnS@GelMA hydrogel group is significantly higher than that of the blank group and the GelMA hydrogel group. According to Figure 4 it can be seen that under the condition of NIR - (without near-infrared light irradiation), the cell proliferation ability of the GelMA hydrogel group and the ZnS@GelMA hydrogel group is slightly higher than that of the blank control CON group. Under the condition of NIR + (with near-infrared light irradiation), the cell proliferation ability of the ZnS@GelMA hydrogel group with 100 μg / mL ZnS added is significantly higher than the other two groups.

[0060] Example 5 Cell Viability Detection

[0061] The intracellular ATP content was measured by chemiluminescence method to analyze cell viability. The specific 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 was added to a 6-well culture plate at a volume of 100 μL per well and cured by ultraviolet light irradiation at 405 nm for 1 min. (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 cured by ultraviolet light irradiation for 1 min. (4) 100 μL of culture medium containing 1000 bone marrow stem cells (BMSCs) was added to each well and cultured in an incubator at 37 °C for 72 hours. During this period, the cells were irradiated with a near-infrared light source NIR at 808 nm (power: 1.5 W / cm 2 ) for 10 min every 24 hours. (5) The cell culture plate was taken out and equilibrated at room temperature for 10 min, and 100 μL of CellTiter-Lumi TM luminescence assay reagent was added to each well. (6) The plate was shaken at room temperature for 2 min to promote cell lysis, and then incubated at room temperature (about 25 °C) for 10 min to stabilize the luminescence signal. (7) Chemiluminescence detection was performed using a multifunctional microplate reader with chemiluminescence detection function, and the results were recorded and statistically analyzed.

[0062] The test results are as Figure 5 shown. Figure 5 The results in

[0063] showed that under the NIR+ condition, the intracellular ATP content in the ZnS@GelMA hydrogel group with 100 μg / mL ZnS added was significantly higher than that in the other two groups, and its cell viability was significantly enhanced.

[0064] The early osteogenic differentiation ability of cells was evaluated by detecting the alkaline phosphatase (ALP) activity. The specific 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 cured by ultraviolet light irradiation at 405 nm for 1 min. (3) 1500 μL of culture medium containing 2×10 5 bone marrow stem cells (BMSCs) was added to each well and cultured in an incubator at 37 °C for 24 hours. (4) The culture medium was replaced with osteogenic differentiation induction medium and induced in an incubator at 37 °C for 72 hours. During this period, the cells were irradiated with a near-infrared light source NIR at 808 nm (power: 1.5 W / cm 2)Irradiate for 10 min. (5) Take the 6-well plate after osteogenic induction for 3 days, discard the osteogenic differentiation induction culture medium, wash the cell growth surface with PBS twice, and discard the residual PBS; add 600 μL of ALP extraction and lysis solution, and place it in an environment at 37 °C for static incubation for 15 min. (6) Gently scrape the cells in each well with a clean cell scraper, transfer the lysate suspension into a 1.5 mL EP tube, centrifuge at 14,000 rpm at 4 °C for 10 min, 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 50 μL of ALP reaction substrate solution to each well, then continue to add 50 μL of ALP buffer to each well, gently tap and mix, add 10 μL of ALP protein sample solution to the detection wells, and place it in a constant temperature environment at 37 °C for static incubation for 15 min. (8) Take out the 96-well detection plate. After visually observing an obvious light yellow color change in the detection wells, continue to add 110 μL of 0.5 M NaOH solution to each reaction well to terminate the reaction. (9) Select an enzyme-labeled instrument with a wavelength of 405 nm to measure the corresponding absorbance value (OD value).

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

[0066] Example 7 Mineralization ability detection

[0067] The cell mineralization ability was evaluated by alizarin red staining of mineralized nodules. The specific test method is as follows: (1) The experimental groups were blank control group (Con), GelMA group and ZnS@GelMA group. (2) Add GelMA or ZnS@GelMA hydrogel (100 μg / mL) to the 6-well culture plate in an amount of 1000 μL per well, and irradiate with 405 nm ultraviolet light for 1 min for curing and crosslinking. (3) Add 1500 μL of culture medium containing 2×10 5 bone marrow stem cells (BMSCs) to each well, and place it in an incubator at 37 °C for 24 hours. (4) Replace it with osteogenic differentiation induction culture medium and induce it in an incubator at 37 °C for 14 days. During this period, use a near-infrared light source NIR (power of 1.5 W / cm 2)Irradiate for 10 min. (5) Take the 6-well plate after osteogenic induction for 14 days, discard the osteogenic differentiation induction culture medium, wash the cell growth surface with PBS twice, and discard the residual PBS completely; 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 fixing solution in the wells, and wash it twice with ultrapure water. (7) Add 2 mL of 40 mM alizarin red solution (ARS, pH = 4.2) to each well, stain it at room temperature, and immediately discard the ARS staining solution after observing obvious bright red staining. (8) Wash it 5 times with ultrapure water, place it on a horizontal shaker at 100 rpm, and continue to gently shake and wash for 15 min to wash away non-specific staining as much as possible. (9) Collect images in transmission mode with a scanner.

[0068] The test results are as Figure 7 shown. Figure 7 The results in it show that under the NIR- condition, the cell mineralization ability of the GelMA hydrogel group and the ZnS@GelMA hydrogel group is higher than that of the blank control CON group. Under the NIR+ condition, the cell mineralization ability of the GelMA hydrogel group and the ZnS@GelMA hydrogel group is further enhanced, and the ZnS@GelMA hydrogel group is 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, incubate it at 37 °C for 30 min to completely dissolve the ARS staining. (2) Collect the CPC dissolution solution into a clean EP tube, dilute it 10 times with ultrapure water, and take out 200 μL and add it to a 96-well plate. (3) Measure the OD value at a wavelength of 562 nm with an enzyme-labeled instrument. (4) Collect the cell membrane sheets in the secondary wells of each culture plate with RIPA lysis buffer, extract the total protein, and measure the respective protein concentrations by the BCA method. (5) Calculate the relative concentration of Ca 2+ through the ARS standard curve, and calculate the Ca 2+ concentration of the sample through the standard Ca 2+ concentration curve.

[0071] The test results are as Figure 8 shown, Figure 8 and the results in it show that under the NIR- condition, the intracellular calcium ion concentration of the GelMA hydrogel group and the ZnS@GelMA hydrogel group is higher than that of the blank control CON group. Under the NIR+ condition, the intracellular calcium ion concentration of the GelMA hydrogel group and the ZnS@GelMA hydrogel group further increases, and the ZnS@GelMA hydrogel group is significantly higher than the GelMA hydrogel group. It is proved that the near-infrared light responsiveness of the ZnS@GelMA hydrogel can significantly enhance the in vitro mineralization ability of cells.

[0072] Example 9 Detection of Osteoblast-Specific Genes (BSP, OCN)

[0073] The osteogenic differentiation ability of cells was evaluated by detecting osteoblast-specific matrix proteins. The specific 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 cured and crosslinked by ultraviolet light irradiation at 405 nm for 1 min. (3) 1500 μL of culture medium containing 2×10 5 bone marrow stem cells (BMSCs) was added to each well and cultured in an incubator at 37 °C for 24 hours. (4) The culture medium was replaced with osteogenic differentiation induction medium and induced in an incubator at 37 °C for 14 days. During this period, the cells were irradiated with a near-infrared light source NIR at 808 nm (power: 1.5 W / cm 2 ) for 10 min every 24 hours. (5) The cells in the 6-well plate after 14 days of osteogenic induction were collected by trypsin digestion. (6) Total RNA was collected using an RNA extraction kit. (7) The expression levels of BSP and OCN were detected by reverse transcription and real-time fluorescence quantitative PCR.

[0074] The test results are as Figure 9 shown. Figure 9 The results in

[0075] showed that under the NIR+ condition, the expression levels of BSP and OCN in the GelMA hydrogel group and the ZnS@GelMA hydrogel group were significantly higher than those in the CON control group, and the ZnS@GelMA hydrogel group was significantly higher than the GelMA hydrogel group. It was proved that the near-infrared light responsiveness of the ZnS@GelMA hydrogel could significantly enhance the osteogenic differentiation ability of cells.

[0075] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A zinc sulfide modified hydrogel with near-infrared light responsiveness and photothermal effect, characterized in that: The invention comprises a methacrylated gelatin hydrogel solution and zinc sulfide nanoparticles dispersed in the methacrylated gelatin hydrogel solution.

2. The zinc sulfide modified hydrogel with near-infrared light responsiveness and photothermal effect according to claim 1, characterized in that: The content of zinc sulfide nanoparticles in the zinc sulfide modified hydrogel with near-infrared light responsiveness and photothermal effect is 50-5000 μg / mL.

3. The zinc sulfide modified hydrogel with near-infrared light responsiveness and photothermal effect according to claim 1, characterized in that: The components of the methacrylic gelatin hydrogel solution include a photoinitiator, methacrylic gelatin and water; the concentration of methacrylic gelatin in the methacrylic gelatin hydrogel solution is 5-30% (w / v).

4. The method for preparing the zinc sulfide modified hydrogel having near-infrared light responsiveness and photothermal effect according to any one of claims 1 to 3, characterized in that: The following steps are involved: 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.

5. The preparation method according to claim 4, 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 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.

6. The preparation method according to claim 5, 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.

7. The preparation method according to claim 5 or 6, characterized in that: The precipitation reaction time is 20 to 30 minutes.

8. A zinc sulfide modified hydrogel solidified material, characterized in that: The method is obtained by photocuring the zinc sulfide modified hydrogel with near-infrared light responsiveness and photothermal effect as described in any one of claims 1 to 3 or the zinc sulfide modified hydrogel with near-infrared light responsiveness and photothermal effect prepared by the preparation method as described in any one of claims 4 to 7.

9. The zinc sulfide modified hydrogel solidified material according to claim 8, characterized in that: The light source used for the photocuring is ultraviolet light, and the irradiation time is 10 to 30 seconds.

10. Use of the zinc sulfide modified hydrogel with near-infrared light responsiveness and photothermal effect as described in any one of claims 1 to 3, the zinc sulfide modified hydrogel with near-infrared light responsiveness and photothermal effect prepared by the preparation method as described in any one of claims 4 to 7, or the zinc sulfide modified hydrogel solidified material as described in claim 8 or 9 in the preparation of products for repairing bone defects.

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