CN117899265A - Gelatin-glycerol injectable hydrogel and preparation method and application thereof - Google Patents
Gelatin-glycerol injectable hydrogel and preparation method and application thereof Download PDFInfo
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Landscapes
- Materials For Medical Uses (AREA)
Abstract
The application provides a preparation method of gelatin-glycerin injectable hydrogel, which combines specific gelatin swelling, compound glycerin and gelation methods, and the prepared gelatin-glycerin injectable hydrogel has strong stability and can be used for irradiation sterilization in clinical use. In addition, the gelatin-glycerol injectable hydrogel has good molding effect, no cytotoxicity and cell proliferation promotion effect. In addition, the gelatin-glycerol injectable hydrogel prepared by the preparation method can load functional components to develop functional design, and provides a platform for designing multifunctional bone and soft tissue repair materials.
Description
Technical Field
The application relates to the technical field of biomedical materials, in particular to a gelatin-glycerol injectable hydrogel, a preparation method and application thereof.
Background
Injectable hydrogel materials are considered to be the most proximal biofunctional material to vital tissues due to their high water content, good water retention, excellent biocompatibility and microstructure similar to that of extracellular matrix. The injectable hydrogel can be used as a tissue repair and biological carrier material, can be operated by a minimally invasive technology, has the capability of reducing the wound area and rapidly filling irregular space, and can be widely applied to the fields of tissue repair, drug delivery, implantable medical devices and the like.
The injectable hydrogels can be classified into synthetic polymer hydrogels and natural polymer hydrogels according to material sources, and natural polymer hydrogels have superior biocompatibility and bioactivity compared to synthetic polymer hydrogels, which also make natural polymer hydrogel materials such as collagen, hyaluronic acid, chitosan, cellulose, etc. widely studied and paid attention. However, most natural polymer hydrogels such as hyaluronic acid, chitosan, cellulose and the like have poor stability, and particularly have the problems that the viscosity is difficult to maintain after irradiation sterilization, and although some high-viscosity injectable excipient materials are reported in the prior art, the preparation method is complex (a step of crosslinking and the like is needed, and the like are needed), and a series of problems such as biological safety and the like possibly exist in the materials because of too high proportion of additives such as glycerol and the like, so that the clinical application of the materials is greatly limited. Therefore, there is a need to develop a natural polymer hydrogel which is simple in preparation method, has no cytotoxicity and can be used for irradiation sterilization so as to meet urgent clinical demands.
Disclosure of Invention
The application aims to provide a gelatin-glycerol injectable hydrogel and a preparation method thereof, which are used for improving the stability of the gelatin-glycerol injectable hydrogel and can be used for irradiation sterilization in clinical use.
In a first aspect, the present application provides a method for preparing a gelatin-glycerol injectable hydrogel, comprising the steps of:
gelatin swelling: placing gelatin in water, swelling at 37-45deg.C for 4-12 hr to obtain swelled gelatin;
composite glycerol: adding the swollen gelatin into glycerol at 55-65 ℃ and uniformly mixing to obtain gelatin-glycerol composite sol;
gelation: placing the gelatin-glycerol composite sol at 2-10 ℃ for 1-4 hours to obtain the gelatin-glycerol injectable hydrogel;
wherein, in the gelatin-glycerin injectable hydrogel, the mass percentage of gelatin is 5-10%, the mass percentage of glycerin is 5-20%, and the mass percentage of water is 70-90%.
In one embodiment of the application, the gelatin is 5-10% by mass, the glycerol is 5-10% by mass, and the water is 85-90% by mass.
In one embodiment of the application, the glycerol has a temperature of 58-62 ℃; the gelation temperature is 2-6deg.C, and gelation time is 1-3h.
In one embodiment of the present application, further comprising:
Inorganic metal ion loading: adding inorganic metal ions into the gelatin-glycerol composite sol according to the final concentration of the inorganic metal ions in the gelatin-glycerol composite sol of 0.01-10mmol/L, uniformly mixing, and then gelling.
In one embodiment of the present application, the inorganic metal ion is selected from at least one of magnesium ion, copper ion, zinc ion, and calcium ion.
In one embodiment of the present application, further comprising:
Decalcified bone particle loading: according to the mass volume ratio of 1g: adding the decalcified bone particles into the gelatin-glycerol composite sol in 2-10mL, uniformly mixing, and then gelling.
In one embodiment of the application, the decalcified bone particles have a particle size of 250-500 μm.
In one embodiment of the present application, further comprising:
Adding inorganic metal ions into the gelatin-glycerol composite sol according to the final concentration of the inorganic metal ions in the gelatin-glycerol composite sol of 0.01-10mmol/L, and according to the volume ratio of 1g: adding the decalcified bone particles into the gelatin-glycerol composite sol in 2-10mL, uniformly mixing, and then gelling.
In a second aspect, the application provides a gelatin-glycerol injectable hydrogel prepared by the method of preparation of the first aspect of the application.
In a third aspect, the present application provides the use of a gelatin-glycerol injectable hydrogel according to the second aspect of the application for the preparation of a bone repair material and/or a soft tissue repair material.
The application has the beneficial effects that:
The preparation method of the gelatin-glycerol injectable hydrogel provided by the application combines specific gelatin swelling, compound glycerol and gelation methods, and the prepared gelatin-glycerol injectable hydrogel has strong stability, can be used for radiation sterilization in clinical use, and reduces the influence of radiation sterilization on gel viscosity. In addition, the gelatin-glycerol injectable hydrogel has good molding effect, no cytotoxicity and cell proliferation promotion effect. Furthermore, the preparation method of the application is simple, the quality is controllable, and the cost is low.
In addition, the gelatin-glycerol injectable hydrogel prepared by the preparation method can load functional components to develop functional design, and provides a platform for designing multifunctional bone and soft tissue repair materials.
Of course, it is not necessary for any one product or method of practicing the application to achieve all of the advantages set forth above at the same time.
Drawings
In order to more clearly illustrate the embodiments of the application or the technical solutions in the prior art, the drawings used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the application, and other embodiments may be obtained according to these drawings to those skilled in the art.
FIG. 1 is an injectable photograph of a gelatin-glycerol injectable hydrogel prepared in example 1;
FIG. 2 is an injectability picture of the magnesium ion-loaded gelatin-glycerol injectable hydrogel prepared in example 6;
FIG. 3 is an injectable photograph of glycerogelatin prepared in comparative example 1;
FIG. 4 is an injectable photograph of the gelatin-glycerol injectable hydrogel loaded with decalcified bone particles prepared in example 10 before irradiation;
FIG. 5 is an injectable photograph of the gelatin-glycerol injectable hydrogel loaded with decalcified bone particles prepared in example 10 after irradiation;
FIG. 6 is an injectable photograph of glycerogelatin loaded with decalcified bone particles prepared in comparative example 2;
FIG. 7 is an injectability picture of the gelatin-glycerol injectable hydrogel loaded with decalcified bone particles prepared in example 11;
FIG. 8 is a graph showing the result of analysis of cytotoxicity CCK-8 of the gelatin-glycerol injectable hydrogel prepared in example 1;
FIG. 9 is a graph showing the results of cytotoxicity CCK-8 analysis of gelatin-glycerol injectable hydrogels with different glycerol ratios;
FIG. 10 is a graph showing the results of analysis of the expression of Vascular Endothelial Growth Factor (VEGF) by using the magnesium ion-loaded gelatin-glycerol injectable hydrogel prepared in example 6.
Detailed Description
The following description of the embodiments of the present application will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present application, but not all embodiments. All other embodiments obtained by the person skilled in the art based on the present application fall within the scope of protection of the present application.
In a first aspect, the present application provides a method for preparing a gelatin-glycerol injectable hydrogel, comprising the steps of:
gelatin swelling: placing gelatin in water, swelling at 37-45deg.C for 4-12 hr to obtain swelled gelatin;
composite glycerol: adding the swollen gelatin into glycerol at 55-65 ℃ and uniformly mixing to obtain gelatin-glycerol composite sol;
gelation: placing the gelatin-glycerol composite sol at 2-10 ℃ for 1-4 hours to obtain the gelatin-glycerol injectable hydrogel;
wherein, in the gelatin-glycerin injectable hydrogel, the mass percentage of gelatin is 5-10%, the mass percentage of glycerin is 5-20%, and the mass percentage of water is 70-90%.
In the application, gelatin and glycerol are used as raw materials, the specific methods of gelatin swelling, compound glycerol and gelation are combined, and the hydroxyl in the glycerol and the carboxyl in the gelatin are used for forming hydrogen bond acting force, so that the stability of a gel network is improved, the preparation process is simple, the method can be used for radiation sterilization in clinical use, and the influence of radiation sterilization on the gel viscosity is reduced. In addition, the gelatin-glycerol injectable hydrogel has good molding effect, no cytotoxicity and cell proliferation promotion effect.
In the present application, the choice of water is not particularly limited as long as the object of the present application can be achieved, and for example, purified water, ultrapure water, and water for injection can be selected.
In one embodiment of the application, the gelatin is 5-10% by mass, the glycerol is 5-10% by mass, and the water is 85-90% by mass.
The inventors found in the study that the mass percentage of gelatin, glycerol and water in the gelatin-glycerol injectable hydrogel is controlled within the above range, and the gelatin-glycerol injectable hydrogel has better molding effect and loading capacity, has no cytotoxicity and shows the effect of promoting cell proliferation.
In one embodiment of the application, the glycerol has a temperature of 58-62 ℃; the gelation temperature is 2-6deg.C, and gelation time is 1-3h.
The inventor finds in the research that the temperature of the glycerol is controlled within the range, so that the viscosity of the glycerol can be reduced, the fluidity of the glycerol can be increased, and the glycerol can be conveniently and uniformly mixed with the gelatin solution; the gelation temperature and time are controlled within the above ranges, so that the gelatin-glycerin composite sol can be rapidly gelled to prepare gelatin-glycerin injectable gel.
In one embodiment of the present application, further comprising:
Inorganic metal ion loading: adding inorganic metal ions into the gelatin-glycerol composite sol according to the final concentration of the inorganic metal ions in the gelatin-glycerol composite sol of 0.01-10mmol/L, uniformly mixing, and then gelling.
In one embodiment of the present application, the inorganic metal ion is selected from at least one of magnesium ion, copper ion, zinc ion, and calcium ion.
The inventors found in the research that the loading of magnesium ions, copper ions, zinc ions and calcium ions by adopting the preparation method of the application can endow gelatin-glycerin injectable hydrogel with different functions, for example, magnesium ions have vascularization promoting capability, copper ions have antibacterial effect, zinc ions can promote angiogenesis, calcium ions can promote osteogenic differentiation and the like. The gelatin-glycerol injectable hydrogel loaded with the metal ion can be applied according to the functional requirements of different tissue repair, and can meet various repair requirements.
In one embodiment of the application, the inorganic metal ion is selected from magnesium ions.
The inventor finds that the magnesium ion loading can endow gelatin-glycerol injectable hydrogel with vascularization promoting capability by adopting the preparation method provided by the application, and has better tissue repair promoting effect.
In one embodiment of the present application, further comprising:
Decalcified bone particle loading: according to the mass volume ratio of 1g: adding the decalcified bone particles into the gelatin-glycerol composite sol in 2-10mL, uniformly mixing, and then gelling.
In the present application, the source of the decalcified bone particles is not particularly limited as long as the object of the present application can be achieved, and for example, bovine-derived decalcified bone particles may be selected.
In one embodiment of the application, the decalcified bone particles have a particle size of 250-500 μm.
The inventor finds that the preparation method is adopted to load the decalcified bone particles, and the prepared gelatin-glycerin injectable hydrogel loaded with the decalcified bone particles can effectively solve the problems that the decalcified bone particles are poor in formability, cannot form good defect repair forms and the like in operation, can better play the bone conduction and bone induction performances of the decalcified bone, and plays a role in better promoting bone repair and bone regeneration.
In one embodiment of the present application, further comprising:
Adding inorganic metal ions into the gelatin-glycerol composite sol according to the final concentration of the inorganic metal ions in the gelatin-glycerol composite sol of 0.01-10mmol/L, and according to the volume ratio of 1g: adding the decalcified bone particles into the gelatin-glycerol composite sol in 2-10mL, uniformly mixing, and then gelling.
In the present application, inorganic metal ion loading and/or decalcified bone particle loading is performed after gelatin swelling and glycerin complexing and before gelation. The steps of loading the inorganic metal ions and loading the decalcified bone particles are not indicative of the order of the inorganic metal ions and the decalcified bone particles, and can be performed according to different orders as required. In addition, the gelatin-glycerol injectable hydrogel of the present application does not represent simultaneous loading of inorganic metal ions and decalcified bone particles, and may be added according to different needs, for example, (1) loading only inorganic metal ions; (2) loading only decalcified bone particles; (3) supporting inorganic metal ions and decalcified bone particles simultaneously.
In a second aspect, the application provides a gelatin-glycerol injectable hydrogel prepared by the method of preparation of the first aspect of the application.
In one embodiment of the application, the gelatin-glycerol injectable hydrogel comprises 5-10% gelatin, 5-20% glycerol, 70-90% water by weight.
In a third aspect, the present application provides the use of a gelatin-glycerol injectable hydrogel according to the second aspect of the application for the preparation of a bone repair material and/or a soft tissue repair material.
The inventor finds that the gelatin-glycerin injectable hydrogel has good formability and injectability, can be used for irradiation sterilization, can be used as a carrier material to load functional inorganic metal ions and decalcified bone particles, and has the effect of better promoting bone and soft tissue repair.
Hereinafter, embodiments of the present application will be described in more detail with reference to examples and comparative examples. The various tests and evaluations were carried out according to the following methods. Unless otherwise specified, "parts" and "%" are mass references.
The experimental materials and methods used in the examples below are conventional materials and methods unless otherwise specified.
The method for preparing the bovine-derived decalcified bone powder is self-made by Hangzhou Huamai medical science and technology Co-Ltd, and comprises the following specific steps:
(1) Pretreatment: taking cortical bone of cattle of 20-30 months old, removing surface soft tissues and periosteum by a physical scraping mode, cutting into bone blocks with the thickness of 3-20mm, crushing into bone particles with the particle size of 250-500 mu m, mixing the bone particles with purified water according to the mass-volume ratio of feed liquid of 1g to 5mL, oscillating and cleaning for 6 times, and 10min each time;
(2) And (3) disinfection: mixing bone particles with 0.2wt% of peracetic acid solution according to the mass-volume ratio of 1g to 5mL of the feed liquid, oscillating for 120min, discarding the peracetic acid solution, adding purified water, stirring and cleaning for 6 times, and 5min each time;
(3) Degreasing: mixing the sterilized bone particles with isopropanol according to the mass-volume ratio of 1g to 5ml of the feed liquid, oscillating for 5 hours, discarding the isopropanol, adding 0.05mol/L MES buffer solution (pH=5.5), stirring and cleaning for 6 times, and 5 minutes each time;
(4) Decellularization: mixing defatted bone particles with 0.8wt% of sodium dodecyl ether sulfate solution according to the mass volume ratio of 1g to 5ml of feed liquid, placing into a low-temperature water tank (4 ℃), stirring for 24 hours, discarding the sodium dodecyl ether sulfate solution, mixing with 0.5wt% of triton X-100 solution according to the mass volume ratio of 1g to 5ml of feed liquid, placing into a low-temperature water tank (4 ℃) for 24 hours, stirring, discarding the triton X-100 solution, adding 0.05mol/L MES buffer solution (pH=5.5), stirring and cleaning for 6 times, and 10 minutes each time;
(5) Decalcification: 8g of sucrose is added into 100mL of hydrochloric acid solution (0.5 mol/L) to obtain decalcification solution; according to the mass volume ratio of 1g:8mL of the bone particles after cell removal are mixed with decalcification liquid, and the mixture is placed into a low-temperature water tank (4 ℃), stirred for 4 hours, and the liquid is changed every hour; discarding decalcification solution, adding 0.1mol/L phosphate buffer solution (pH=7.8), stirring and cleaning for 3 times each for 10min, cleaning until the solution is neutral, stirring and cleaning for 7 times each for 10min with purified water;
(6) And (3) freeze-drying: spreading the decalcified bone matrix in a steel plate mold, freeze-drying and packaging;
(7) And (3) sterilization: the decalcified bone matrix after packaging is preserved at normal temperature after irradiation sterilization of 20 kGy.
Example 1
(1) Gelatin swelling: placing 2g of gelatin in 34g of purified water, and swelling for 8 hours at 37 ℃ to obtain swollen gelatin;
(2) Composite glycerol: adding the swelled gelatin into 4g of glycerin heated to 60 ℃, and uniformly stirring and mixing to obtain gelatin-glycerin composite sol;
(3) Gelation: placing the gelatin-glycerol composite sol at 4 ℃ for 2 hours to obtain the gelatin-glycerol injectable hydrogel.
Examples 2 to 5
Example 1 was repeated except that the parameters were adjusted according to table 1.
Example 6
(1) Gelatin swelling: placing 2g of gelatin in 34g of purified water, and swelling for 8 hours at 37 ℃ to obtain swollen gelatin;
(2) Composite glycerol: adding the swelled gelatin into 4g of glycerin heated to 60 ℃, and uniformly stirring and mixing to obtain gelatin-glycerin composite sol;
(3) Inorganic metal ion loading: adding magnesium chloride into the gelatin-glycerol composite sol according to the final concentration of magnesium ions in the gelatin-glycerol composite sol of 10mmol/L, and stirring and mixing uniformly;
(4) Gelation: and placing the gelatin-glycerol composite sol loaded with magnesium ions at the temperature of 4 ℃ for 2 hours to obtain the gelatin-glycerol injectable hydrogel loaded with magnesium ions.
Examples 7 to 9
Example 6 is the same except that the relevant parameters are adjusted according to table 2.
Example 10
(1) Gelatin swelling: placing 2g of gelatin in 34g of purified water, and swelling for 8 hours at 37 ℃ to obtain swollen gelatin;
(2) Composite glycerol: adding the swelled gelatin into 4g of glycerin heated to 60 ℃, and uniformly stirring and mixing to obtain gelatin-glycerin composite sol;
(3) Decalcified bone particle loading: according to the mass volume ratio of 1g:3mL of decalcified bone particles (bovine decalcified bone powder, self-made by Hangzhou Huamai medical science and technology Co., ltd., see the description above) with the particle size range of 250-500 μm are added into the gelatin-glycerin composite sol, and stirred and mixed uniformly;
(4) Gelation: placing the gelatin-glycerin composite sol loaded with the decalcified bone particles at the temperature of 4 ℃ for 2 hours to obtain the gelatin-glycerin injectable hydrogel loaded with the decalcified bone particles.
Example 11
(1) Gelatin swelling: 5g of gelatin is placed in 90g of purified water and swelled for 8 hours at 37 ℃ to obtain swelled gelatin;
(2) Composite glycerol: adding the swelled gelatin into 5g of glycerin heated to 60 ℃, and uniformly stirring and mixing to obtain gelatin-glycerin composite sol;
(3) Decalcified bone particle loading: according to the mass volume ratio of 1g:3mL of decalcified bone particles (bovine decalcified bone powder, self-made by Hangzhou Huamai medical science and technology Co., ltd., see the description above) with the particle size range of 250-500 μm are added into the gelatin-glycerin composite sol, and stirred and mixed uniformly;
(4) Gelation: placing the gelatin-glycerin composite sol loaded with the decalcified bone particles at the temperature of 4 ℃ for 2 hours to obtain the gelatin-glycerin injectable hydrogel loaded with the decalcified bone particles.
Comparative example 1
Weighing 2g of gelatin, placing in a weighed evaporating dish, adding 80g of water for soaking for 1h, draining excessive water, adding 18g of glycerol, heating on a water bath until the gelatin is dissolved, filtering in a container, and cooling until the gelatin is coagulated to obtain the glycerogelatin.
Comparative example 2
(1) Gelatin swelling: placing 2g of gelatin in 80g of purified water, and swelling for 8 hours at 37 ℃ to obtain swollen gelatin;
(2) Composite glycerol: adding the swelled gelatin into 18g of glycerin heated to 60 ℃, and uniformly stirring and mixing to obtain gelatin-glycerin composite sol;
(3) Gelation: placing the gelatin-glycerol composite sol at 4 ℃ for 2 hours to obtain glycerol gelatin;
(2) According to the mass volume ratio of 1g:3mL of decalcified bone particles (bovine decalcified bone powder, self-made by Hangzhou Huamai medical science and technology Co., ltd., see the description above) with the particle size range of 250-500 μm are added into glycerogelatin, and stirred and mixed uniformly;
(3) And (3) placing the glycerogelatin loaded with the decalcified bone particles at the temperature of 4 ℃ for 2 hours to obtain the glycerogelatin loaded with the decalcified bone particles.
Comparative examples 3 to 4
Example 1 was repeated except that the parameters were adjusted according to table 1.
TABLE 1
TABLE 2
Injectability evaluation
The gelatin-glycerin injectable hydrogels prepared in examples 1 to 5, the magnesium ion-loaded gelatin-glycerin injectable hydrogel prepared in example 6, the copper ion-loaded gelatin-glycerin injectable hydrogel prepared in example 7, the zinc ion-loaded gelatin-glycerin injectable hydrogel prepared in example 8, and the calcium ion-loaded gelatin-glycerin injectable hydrogel prepared in example 9 were injected using 1mL syringe (no needle), respectively, and the time required for pushing out a certain length of gelatin-glycerin injectable hydrogel using the same force was recorded, and the results showed that the gelatin-glycerin injectable hydrogels prepared in examples 1 to 9 were all smoothly pushed out (see table 3) and had a good molding effect. The injectable pictures of the gelatin-glycerin injectable hydrogel prepared in example 1 and the magnesium ion-loaded gelatin-glycerin injectable hydrogel prepared in example 6 are shown in fig. 1 and 2, respectively.
The glycerogelatin prepared in comparative example 1 was injected using a 1mL syringe (no needle), and the results showed that the glycerogelatin was very poor in molding effect (see fig. 3) although it could be injected by pushing, and could not be used as an injection gel.
The decalcified bone particles are widely applied to bone repair and bone regeneration due to their bone conduction and bone induction properties, but the decalcified bone particles have the problems of poor formability in operation, inability to form good defect repair morphology and the like, so that the decalcified bone particles are limited in clinical use. According to the application, the gelatin-glycerin injectable hydrogel loaded with decalcified bone particles prepared in example 10 is subjected to irradiation sterilization by using a 20KGy irradiation dose, 1mL syringes (without needles) are respectively used before and after irradiation sterilization, and the time required for pushing out a gel with a certain length by using the same force is recorded, and the results are shown in fig. 4, fig. 5 and Table 3, wherein fig. 4 is an injectable picture before irradiation of the gelatin-glycerin injectable hydrogel loaded with decalcified bone particles, and fig. 5 is an injectable picture after irradiation of the gelatin-glycerin injectable hydrogel loaded with decalcified bone particles, and as can be seen from fig. 4, fig. 5 and Table 3, the gelatin-glycerin injectable hydrogel loaded with decalcified bone particles before and after irradiation can be smoothly pushed in by using the syringes (18G needles), and the gelatin-glycerin injectable hydrogel prepared by the method has good stability and can be used for sterilization and decalcification bone particle shaping. The decalcified bone particles are loaded by the gelatin-glycerin injectable hydrogel, have good formability and injectability before and after irradiation, can effectively solve the problems that the decalcified bone particles are easy to fall off, have poor formability and the like when being singly used, are simple to operate, and can realize better bone repair and regeneration effects.
The gelatin-glycerin injectable hydrogel loaded with decalcified bone particles prepared in example 11 and the glycerin gelatin loaded with decalcified bone particles prepared in comparative example 2 were injected using a 1mL syringe (no needle), respectively, and the time required to push out a gel of a certain length using the same force was recorded, and as a result, it was revealed that the glycerin gelatin loaded with decalcified bone particles prepared in comparative example 2 was easy to break and poor in molding effect although it could be pushed out (as shown in fig. 6); the gelatin-glycerin injectable hydrogel loaded with decalcified bone particles prepared in example 11 was smoothly injected (see table 3) and had a good molding effect (as shown in fig. 7). The results show that the gelatin-glycerol injectable hydrogel can be smoothly injected after the decalcified bone particles are loaded, has a better molding effect, and can realize better bone repair and regeneration effects; in contrast, the glycerogelatin of comparative example 2 was easily broken after loading the decalcified bone particles, and was poor in moldability, and did not achieve the bone repair and regeneration effects well.
TABLE 3 Table 3
Cytotoxicity of cells
The gelatin-glycerol injectable hydrogels prepared in examples 1 to 5 were diluted to 0.2g/mL using a complete culture medium (89 vol% high sugar medium/10 vol% fetal bovine serum/1 vol% double antibody), added to a plate-incubated L-929 fibroblast culture plate (96 wells, purchased from Kunming cell bank of China academy of sciences) at 100. Mu.L/well, 5 duplicate wells were made, and a complete culture medium without gelatin-glycerol injectable hydrogel was set as a blank. The plates were placed in a 37℃5% CO 2 incubator for 24h, the growth of the cells was observed under an inverted microscope, and cytotoxicity CCK-8 assay was performed according to the kit protocol (CCK-8 kit, manufacturer: shanghai Biyun biotechnology Co., ltd.). The results showed that none of the gelatin-glycerol injectable hydrogels prepared in examples 1 to 5 was cytotoxic and showed an effect of promoting cell proliferation. The cytotoxicity CCK-8 assay results of the gelatin-glycerol injectable hydrogel prepared in example 1 are shown in fig. 8, and compared with the blank control group, the cell viability of the gelatin-glycerol injectable hydrogel prepared in example 1 is significantly increased (< 0.001) and the effect of promoting cell proliferation is shown. Therefore, the gelatin-glycerol injectable hydrogel prepared by the application has no cytotoxicity and has the effect of promoting cell proliferation.
The gelatin-glycerol injectable hydrogels prepared in example 4 (5% glycerol excipient), example 1 (10% glycerol excipient), example 5 (20% glycerol excipient), comparative example 3 (30% glycerol excipient) and comparative example 4 (40% glycerol excipient) were diluted to two concentrations of 0.2g/mL and 0.1g/mL using the complete culture solution (89 vol% high sugar medium/10 vol% fetal bovine serum/1 vol% diab), respectively, and added to the plate-incubated L-929 fibroblast culture plates (96 wells, purchased from the academy of sciences of china, kunming cell bank) at 100 μl/well for 5 multiple wells, while setting the complete culture solution without gelatin-glycerol injectable hydrogels as a blank control. The plates were placed in a 37℃5% CO 2 incubator for 24 hours, the growth of the cells was observed under an inverted microscope, and cytotoxicity CCK-8 assay (CCK-8 kit, manufacturer: shanghai Biyun Biotechnology Co., ltd.) was performed according to the kit instructions, and the results are shown in FIG. 9, wherein 100 represents a gelatin-glycerol injectable hydrogel dilution group of 0.2g/mL and 50 represents a gelatin-glycerol injectable hydrogel dilution group of 0.1 g/mL. The results of fig. 9 show that the gelatin-glycerol injectable hydrogels prepared in example 4, example 1 and example 5 were non-cytotoxic, but the gelatin-glycerol injectable hydrogels prepared in comparative example 3 and comparative example 4 were cytotoxic.
Vascular Endothelial Growth Factor (VEGF) expression
The magnesium ion-loaded gelatin-glycerol injectable hydrogel prepared in example 6 was diluted to 0.2g/mL using a complete culture medium (89 vol% high sugar medium/10 vol% fetal bovine serum/1 vol% double antibody), added to a plate (96 wells, commercially available from Qingqi (Shanghai) Biotechnology development Co., ltd.) of Human Umbilical Vein Endothelial Cells (HUVEC) after plating incubation, 100. Mu.L/well was prepared, 3 duplicate wells were made, and a complete culture medium without gelatin-glycerol injectable hydrogel was set as a blank control, and the gelatin-glycerol injectable hydrogel set was subjected to synchronous treatment using the gelatin-glycerol injectable hydrogel of example 1. The plates were incubated at 37℃in a 5% CO 2 incubator for 24h, cell culture supernatants were collected, and VEGF concentration in the supernatants was measured according to the ELISA kit (manufacturer: whan enzyme Biotechnology Co., ltd.) for kit instructions, as shown in FIG. 10, and the VEGF expression of the magnesium-loaded gelatin-glycerol injectable hydrogel group was significantly increased (P < 0.05) compared to the blank control group and gelatin-glycerol injectable hydrogel, indicating that the introduction of magnesium ions could improve the angiogenesis promoting ability of the material.
Different inorganic metal ions have different functions in tissue repair, for example copper ions have antibacterial effect, zinc ions promote angiogenesis, and calcium ions promote osteogenic differentiation. In addition to the magnesium ion-loaded gelatin-glycerin injectable hydrogel, the application also prepares the copper ion-loaded gelatin-glycerin injectable hydrogel, the zinc ion-loaded gelatin-glycerin injectable hydrogel and the calcium ion-loaded gelatin-glycerin injectable hydrogel in examples 7 to 9 respectively, and can be applied according to the functional requirements of different tissue repair to meet various repair requirements.
The preparation method of the gelatin-glycerol injectable hydrogel provided by the application combines specific gelatin swelling, compound glycerol and gelation methods, and the prepared gelatin-glycerol injectable hydrogel can be used for irradiation sterilization in clinical use. In addition, the gelatin-glycerol injectable hydrogel has good molding effect, no cytotoxicity and cell proliferation promotion effect. Furthermore, the preparation method of the application is simple, the quality is controllable, and the cost is low. In addition, the gelatin-glycerol injectable hydrogel prepared by the preparation method can load functional components to develop functional design, and provides a platform for designing multifunctional bone and soft tissue repair materials.
The foregoing description of the preferred embodiments of the application is not intended to limit the application to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the application are intended to be included within the scope of the application.
Claims (10)
1. A method for preparing a gelatin-glycerol injectable hydrogel comprising the steps of:
gelatin swelling: placing gelatin in water, swelling at 37-45deg.C for 4-12 hr to obtain swelled gelatin;
composite glycerol: adding the swollen gelatin into glycerol at 55-65 ℃ and uniformly mixing to obtain gelatin-glycerol composite sol;
gelation: placing the gelatin-glycerol composite sol at 2-10 ℃ for 1-4 hours to obtain the gelatin-glycerol injectable hydrogel;
wherein, in the gelatin-glycerin injectable hydrogel, the mass percentage of gelatin is 5-10%, the mass percentage of glycerin is 5-20%, and the mass percentage of water is 70-90%.
2. The preparation method according to claim 1, wherein the mass percentage of gelatin is 5-10%, the mass percentage of glycerin is 5-10%, and the mass percentage of water is 85-90%.
3. The method of claim 1, wherein the glycerol is at a temperature of 58-62 ℃; the gelation temperature is 2-6deg.C, and gelation time is 1-3h.
4. The method of manufacturing according to claim 1, further comprising:
Inorganic metal ion loading: adding inorganic metal ions into the gelatin-glycerol composite sol according to the final concentration of the inorganic metal ions in the gelatin-glycerol composite sol of 0.01-10mmol/L, uniformly mixing, and then gelling.
5. The method according to claim 4, wherein the inorganic metal ion is at least one selected from the group consisting of magnesium ion, copper ion, zinc ion, and calcium ion.
6. The method of manufacturing according to claim 1, further comprising:
Decalcified bone particle loading: according to the mass volume ratio of 1g: adding the decalcified bone particles into the gelatin-glycerol composite sol in 2-10mL, uniformly mixing, and then gelling.
7. The method according to claim 6, wherein the decalcified bone particles have a particle diameter of 250 to 500. Mu.m.
8. The method of manufacturing according to claim 1, further comprising:
Adding inorganic metal ions into the gelatin-glycerol composite sol according to the final concentration of the inorganic metal ions in the gelatin-glycerol composite sol of 0.01-10mmol/L, and according to the volume ratio of 1g: adding the decalcified bone particles into the gelatin-glycerol composite sol in 2-10mL, uniformly mixing, and then gelling.
9. A gelatin-glycerol injectable hydrogel prepared by the preparation method according to any one of claims 1 to 8.
10. Use of the gelatin-glycerol injectable hydrogel according to claim 9 for the preparation of bone repair material and/or soft tissue repair material.
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