CN106730021B - Bioactive glass-modified gelatin composite hydrogel and preparation method thereof - Google Patents
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Abstract
The invention discloses a bioactive glass-modified gelatin composite hydrogel and a preparation method thereof, belonging to the field of biomedical materials. The bioactive glass-modified gelatin composite hydrogel is prepared by the following method: adding the bioactive glass powder into a modified gelatin solution containing a photoinitiator, performing ultrasonic treatment, stirring and dispersing uniformly, pouring into a mold, and irradiating for a certain time by ultraviolet light to obtain the bioactive glass-modified gelatin composite hydrogel. The bioactive glass-modified gelatin composite hydrogel has good bioactivity, osteoinductivity and high water content (more than 80%), and can be used in the fields of nonbearing bone defect repair, drug sustained release, bone tissue engineering and the like.
Description
Technical Field
The invention belongs to the field of preparation of biomedical materials, and particularly relates to bioactive glass-modified gelatin composite hydrogel and a preparation method thereof.
background
Bone defects are easily caused after bone loss due to trauma, tumor resection, deformity correction, infection and the like. Currently, the predominant method of treating bone defects is bone grafting, including both autogenous and allogenic bone. Autologous bone transplantation does not need to consider histocompatibility and immunological rejection, but has limited bone supply sources; the source of the allogeneic bone is wide, but the risk of immunological rejection and infection of blood-borne virus exists. Therefore, research and development of novel artificial bone repair materials are required to meet clinical needs.
The main components of bone tissue are collagen and hydroxyapatite. Based on the bionic angle, the preparation of bone repair materials by using organic and inorganic substances similar to natural bone components has become one of the current research hotspots. The bioactive glass serving as a typical silicate material has good biocompatibility, bone guiding property and bone inducing property, and has wide application in bone defect repair. The gelatin is a product obtained by partial hydrolysis of collagen, has good biocompatibility, and simultaneously, carboxyl groups with negative electricity on a chain can be used as nucleation sites to control and induce the formation of hydroxyapatite. The bioactive glass/gelatin composite material is similar to human bone tissue in composition, has good biocompatibility, has biological characteristics such as osteogenesis inducing effect and the like, and meets the requirements of bone repair materials to a certain extent.
At present, the common method for preparing the bioactive glass/gelatin composite material is to add a certain amount of bioactive glass powder into a gelatin solution, stir the mixture evenly, pour the mixture into a mold, freeze-dry the mixture for molding, then use a cross-linking agent for chemical treatment, and freeze-dry the mixture to finally obtain the composite stent material. However, the composite bone repair material prepared by the above method has the following problems: (1) the preparation process is complex, and a plurality of steps such as molding, freeze drying, chemical crosslinking and the like are required; (2) after the scaffold is formed, the stability needs to be improved through chemical crosslinking, but a crosslinking agent (such as glutaraldehyde) is easy to remain in the scaffold, so that cytotoxicity is caused; (3) the composite bracket has low water content and is not beneficial to the transportation of nutrient substances and metabolites.
The modified gelatin is obtained by reacting the active amino group of the side chain of the gelatin with methacrylic anhydride, and free radical polymerization reaction is carried out under the initiation of ultraviolet light to form hydrogel, so that the defects of poor mechanical property, high degradation rate and the like of the gelatin are overcome. The gelatin hydrogel has good biocompatibility and excellent processability, has a structure similar to that of extracellular matrix, and can provide a good microenvironment for cell adhesion, proliferation and differentiation. Researchers add inorganic nanoparticles (such as gold nanoparticles, silicon dioxide and hydroxyapatite) into gelatin hydrogel to improve the bioactivity and osteogenesis of the composite hydrogel.
The invention adopts the principle of photopolymerization reaction to prepare the bioactive glass-modified gelatin composite hydrogel scaffold material. The composite hydrogel has good biocompatibility and osteoinduction capability; the bioactive glass is uniformly dispersed in a three-dimensional network structure and is tightly combined with the modified gelatin, and plasma dissolved products of silicon, calcium, phosphorus and the like can be slowly released; the high water content can ensure the normal transportation of nutrient substances and metabolites; the preparation process is simple, no chemical cross-linking agent is needed, and the preparation method has wide application prospect in the field of bone tissue engineering.
disclosure of Invention
The invention aims to provide a bioactive glass-modified gelatin composite hydrogel and a preparation method thereof, aiming at overcoming the defects of the prior art and materials, so that the composite hydrogel with good bioactivity and bone induction capability can be obtained, can be used for treating bone defects of non-bearing parts, and can realize the repair and regeneration of bone tissues.
The invention is realized by the following technical scheme.
A preparation method of bioactive glass-modified gelatin composite hydrogel comprises the following steps:
1) Fully dissolving modified gelatin and photoinitiator 2-hydroxy-4' - (2-hydroxyethoxy) -2-methyl propiophenone (IRGACURE 2959) in a phosphate buffer solution to obtain a modified gelatin prepolymer solution;
2) Adding bioactive glass powder into the modified gelatin prepolymer solution, and performing ultrasonic dispersion and magnetic stirring to obtain uniformly dispersed mixed slurry;
3) And pouring the mixed slurry into a polytetrafluoroethylene mold, initiating a free radical polymerization reaction by ultraviolet irradiation, and swelling a product after the reaction in a phosphate buffer solution to remove unreacted monomers and a photoinitiator to obtain the bioactive glass-modified gelatin composite hydrogel.
Further, the modified gelatin in the step 1) has a methacrylamide substitution degree of 40-80%.
Further, the concentration of the modified gelatin prepolymer in the modified gelatin prepolymer solution in step 1) is 7 to 15 w/v%, preferably 10 w/v%.
Further, the concentration of the photoinitiator in the phosphate buffer solution in the step 1) is 0.1-1 w/v%.
Further, the bioactive glass powder in the step 2) is micro/nano spherical powder prepared by combining an organic template with a sol-gel method.
further, the ultrasonic dispersion of the step 2) is carried out for 30min and magnetic stirring is carried out for 4 h.
Further, the concentration of the bioactive glass powder in the mixed slurry in the step 2) is 1-10 w/v%.
further, the wavelength of the ultraviolet light in the step 3) is 254-400 nm, the intensity is 5-20 mW/cm 2, the irradiation time is 1-20 min, and the preferred wavelength is 365nm, and the intensity is 5m W/cm 2.
the bioactive glass-modified gelatin composite hydrogel obtained by the preparation method is provided. The bioactive glass-modified gelatin composite hydrogel can be applied to treatment of non-bearing bone defect parts, and can promote repair and regeneration of bone tissues.
Compared with the prior art, the invention has the beneficial effects that:
(1) The invention selects two materials of bioactive glass and modified gelatin to be compounded, and the materials are subjected to free radical polymerization reaction under the irradiation of ultraviolet light to form the composite hydrogel. The composite hydrogel has good biocompatibility and osteoinduction capability; the synthetic materials have wide sources and low price; the preparation method is simple and convenient, and does not need to use a chemical cross-linking agent.
(2) The bioactive glass in the composite hydrogel is uniformly embedded into a three-dimensional network structure formed by the modified gelatin hydrogel and is tightly combined with the modified gelatin in a physical mode (hydrogen bonds), so that silicon, calcium, phosphorus and other ions are slowly released. And the composite hydrogel has higher water content (more than 80 percent), and can ensure the transportation of nutrient substances and metabolic wastes.
(3) The modified gelatin can be used as a good cell carrier, can be gelatinized in a short time at room temperature, and can be combined with bone marrow mesenchymal stem cells, growth factors and the like to construct a functionalized bone tissue engineering.
(4) the bioactive glass-modified gelatin composite hydrogel can be applied to the fields of bone defect repair, drug sustained release carriers, bone tissue engineering and the like.
Drawings
FIGS. 1a and 1b are scanning electron micrographs of the bioactive glass-modified gelatin composite hydrogel obtained in example 1 of the present invention at different magnifications;
FIGS. 1c and 1d are scanning electron micrographs of the bioactive glass-modified gelatin composite hydrogel obtained in example 2 of the present invention at different magnifications;
FIGS. 1e and 1f are scanning electron micrographs of the bioactive glass-modified gelatin composite hydrogel obtained in example 3 of the present invention at different magnifications;
FIG. 2 is an infrared spectrum of the bioactive glass-modified gelatin composite hydrogel obtained in example 2 of the present invention;
FIG. 3 is a graph showing the water content of the bioactive glass-modified gelatin composite hydrogel obtained in examples 1, 2 and 3 of the present invention;
FIG. 4 is a live-dead staining pattern of the bioactive glass-modified gelatin composite hydrogel obtained in example 2 of the present invention after 24 h coculture with mouse bone marrow mesenchymal stem cells.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Example 1
(1) The preparation method of the bioactive glass powder comprises the following steps:
The bioactive glass powder is prepared by combining an organic template with a sol-gel method, and the specific synthetic process is as follows: firstly, 40 g of dodecylamine is added into a mixed solution of 250 ml of deionized water and 800 ml of absolute ethyl alcohol, and the mixture is stirred in a water bath kettle at the temperature of 40 ℃ until the dodecylamine is completely dissolved; then 202 ml of tetraethoxysilane is added and stirred for 30 min; adding 24 ml triethyl phosphate, stirring for 30 min; 137 g of calcium nitrate tetrahydrate is added; continuously stirring the obtained milky white solution for 3 hours; and finally, centrifuging the glass sol to obtain white precipitate, drying the white precipitate in a drying box at the temperature of 60 ℃ for 24 hours, and sintering the dried white precipitate in a muffle furnace at the temperature of 650 ℃ for 3 hours to obtain the bioactive glass powder.
(2) preparation steps of the modified gelatin (degree of substitution 40%):
The modified gelatin is obtained by the reaction of active amino of a gelatin side chain and methacrylic anhydride, and the specific synthetic process is as follows: adding 2 g of gelatin into 20 ml of phosphate buffer solution, and stirring in a constant-temperature water bath kettle at 60 ℃ to prepare 10% gelatin solution; after the gelatin is completely dissolved, 0.25 ml of methacrylic anhydride is added, and the reaction is continued for 4 hours; transferring to a constant temperature water bath kettle at 40 ℃ after the reaction is finished, adding 40 ml of phosphate buffer solution for dilution to terminate the reaction, and continuing stirring for 30 min; subsequently transferred to a 12k dialysis bag for dialysis for 7 d to remove unreacted methacrylic anhydride and inorganic salts from the solution; finally, the solution is pre-frozen in a refrigerator at the temperature of-80 ℃, and the modified gelatin (with the degree of substitution of 40%) is obtained after freeze drying.
(3) the synthesis process of the bioactive glass-modified gelatin composite hydrogel comprises the steps of adding 1 g of modified gelatin (with the substitution degree of 40%) and 0.01 g of photoinitiator IRGACURE 2959 into 10 ml of phosphate buffer solution, stirring the mixture in a constant-temperature water bath kettle at the temperature of 60 ℃ until the mixture is completely dissolved, then adding 0.1 g of bioactive glass powder, carrying out ultrasonic dispersion for 30min, carrying out magnetic stirring for 4h to uniformly disperse the bioactive glass in the solution, finally adding the mixed slurry into a polytetrafluoroethylene mold, irradiating the mixed slurry for 20 min with ultraviolet light of 365nm and 5 mW/cm 2 to obtain the bioactive glass-modified gelatin composite hydrogel, and swelling the obtained composite hydrogel in the phosphate buffer solution for 24 h to remove unreacted monomers and the photoinitiator.
Example 2
(1) the preparation method of the bioactive glass powder comprises the following steps:
The bioactive glass powder is prepared by combining an organic template with a sol-gel method, and the specific synthetic process is as follows: firstly, adding 4 g of dodecylamine into a mixed solution of 25 ml of deionized water and 80 ml of absolute ethyl alcohol, and stirring in a water bath kettle at 40 ℃ until the dodecylamine is completely dissolved; then 20.2 ml of ethyl orthosilicate is added and stirred for 30 min; adding 2.4 ml triethyl phosphate, stirring for 30 min; adding 13.7 g of calcium nitrate tetrahydrate; continuously stirring the obtained milky white solution for 3 hours; and finally, centrifuging the glass sol to obtain white precipitate, drying the white precipitate in a drying box at the temperature of 60 ℃ for 24 hours, and sintering the dried white precipitate in a muffle furnace at the temperature of 650 ℃ for 5 hours to obtain the bioactive glass powder.
(2) preparation steps of the modified gelatin (degree of substitution 60%):
the modified gelatin is obtained by the reaction of active amino of a gelatin side chain and methacrylic anhydride, and the specific synthetic process is as follows: adding 20 g of gelatin into 200 ml of phosphate buffer solution, and stirring in a constant-temperature water bath kettle at 60 ℃ to prepare 10% gelatin solution; after the gelatin is completely dissolved, 10 ml of methacrylic anhydride is added, and the reaction is continued for 4 hours; after the reaction is finished, transferring the mixture into a water bath kettle with the constant temperature of 40 ℃, adding 500 ml of phosphate buffer solution for dilution to terminate the reaction, and continuing stirring for 30 min; subsequently transferred to a 12k dialysis bag for dialysis for 7 d to remove unreacted methacrylic anhydride and inorganic salts from the solution; finally, the solution is pre-frozen in a refrigerator at the temperature of 80 ℃ below zero, and the modified gelatin (with the substitution degree of 60%) is obtained after freeze drying.
(3) The synthesis process of the bioactive glass-modified gelatin composite hydrogel comprises the steps of adding 1 g of modified gelatin (with the substitution degree of 60%) and 0.05 g of photoinitiator IRGACURE 2959 into 10 ml of phosphate buffer solution, stirring the mixture in a constant-temperature water bath kettle at the temperature of 60 ℃ until the mixture is completely dissolved, then adding 0.5 g of bioactive glass powder, carrying out ultrasonic dispersion for 30min, carrying out magnetic stirring for 4h to uniformly disperse the bioactive glass in the solution, finally adding the mixed slurry into a polytetrafluoroethylene mold, irradiating the mixed slurry for 10 min with ultraviolet light of 365nm and 5 mW/cm 2 to obtain the bioactive glass-modified gelatin composite hydrogel, and swelling the obtained composite hydrogel in the phosphate buffer solution for 24 h to remove unreacted monomers and the photoinitiator.
Example 3
(1) The preparation method of the bioactive glass powder comprises the following steps:
The bioactive glass powder is prepared by combining an organic template with a sol-gel method, and the specific synthetic process is as follows: firstly, adding 8 g of dodecylamine into a mixed solution of 50 ml of deionized water and 160 ml of absolute ethyl alcohol, and stirring in a water bath kettle at 40 ℃ until the dodecylamine is completely dissolved; then adding 40.4 ml of ethyl orthosilicate and stirring for 30 min; adding 4.8 ml triethyl phosphate, stirring for 30 min; adding 27.4 g of calcium nitrate tetrahydrate; continuously stirring the obtained milky white solution for 3 hours; and finally, centrifuging the glass sol to obtain white precipitate, drying the white precipitate in a drying box at the temperature of 60 ℃ for 24 hours, and sintering the dried white precipitate in a muffle furnace at the temperature of 650 ℃ for 3 hours to obtain the bioactive glass powder.
(2) preparation steps of the modified gelatin (the degree of substitution is 80%):
The modified gelatin is obtained by the reaction of active amino of a gelatin side chain and methacrylic anhydride, and the specific synthetic process is as follows: firstly, adding 5 g of gelatin into 50 ml of phosphate buffer solution, and stirring in a constant-temperature water bath kettle at 60 ℃ to prepare 10% gelatin solution; after the gelatin is completely dissolved, adding 5 ml of methacrylic anhydride, and continuing to react for 4 hours; after the reaction is finished, transferring the mixture into a water bath kettle with the constant temperature of 40 ℃, adding 100 ml of phosphate buffer solution for dilution to terminate the reaction, and continuing stirring for 30 min; subsequently transferred to a 12k dialysis bag for dialysis for 7 d to remove unreacted methacrylic anhydride and inorganic salts from the solution; and finally, pre-freezing the solution in a refrigerator at the temperature of-80 ℃, and freeze-drying to obtain the modified gelatin (the substitution degree is 80%).
(3) The synthesis process of the bioactive glass-modified gelatin composite hydrogel comprises the steps of adding 1 g of modified gelatin (with the substitution degree of 80%) and 0.1 g of photoinitiator IRGACURE 2959 into 10 ml of phosphate buffer solution, stirring the mixture in a constant-temperature water bath kettle at the temperature of 60 ℃ until the mixture is completely dissolved, then adding 1 g of bioactive glass powder, carrying out ultrasonic dispersion for 30min, carrying out magnetic stirring for 4h to uniformly disperse the bioactive glass in the solution, finally adding the mixed slurry into a polytetrafluoroethylene mold, irradiating the mixed slurry for 1 min with ultraviolet light of 365nm and 5 mW/cm 2 to obtain the bioactive glass-modified gelatin composite hydrogel, and swelling the obtained composite hydrogel in the phosphate buffer solution for 24 h to remove unreacted monomers and the photoinitiator.
-1 -1 -1The performance of the bioactive glass-modified gelatin composite hydrogels obtained in examples 1, 2 and 3 is characterized, the scanning electron micrographs obtained are shown in fig. 1a, 1b, 1C, 1d, 1e and 1f, all three bioactive glass-modified gelatin composite hydrogels show a honeycomb-shaped connected porous structure with a pore size range of 200-.
Claims (5)
1. A preparation method of bioactive glass-modified gelatin composite hydrogel is characterized by comprising the following steps:
1) fully dissolving modified gelatin and photoinitiator 2-hydroxy-4' - (2-hydroxyethoxy) -2-methyl propiophenone in a phosphate buffer solution to obtain a modified gelatin prepolymer solution;
2) Adding bioactive glass powder into the modified gelatin prepolymer solution, and performing ultrasonic dispersion and magnetic stirring to obtain uniformly dispersed mixed slurry;
3) Pouring the mixed slurry into a polytetrafluoroethylene mold, initiating a free radical polymerization reaction under the irradiation of ultraviolet light, and swelling a product after the reaction in a phosphate buffer solution to remove unreacted monomers and a photoinitiator so as to obtain the bioactive glass-modified gelatin composite hydrogel;
The substitution degree of methacrylamide in the modified gelatin in the step 1) is 40-80%;
The concentration of the photoinitiator in the step 1) in the phosphate buffer solution is 0.1-1 w/v%;
the concentration of the bioactive glass powder in the step 2) in the mixed slurry is 1-10 w/v%;
The wavelength of the ultraviolet light in the step 3) is 254-400 nm, the intensity is 5-20 mW/cm 2, and the irradiation time is 1-20 min.
2. the method for preparing the bioactive glass-modified gelatin composite hydrogel according to claim 1, wherein the method comprises the following steps: the concentration of the modified gelatin prepolymer in the modified gelatin prepolymer solution in the step 1) is 7-15 w/v%.
3. the method for preparing the bioactive glass-modified gelatin composite hydrogel according to claim 1, wherein the method comprises the following steps: the bioactive glass powder in the step 2) is micro/nano spherical powder prepared by combining an organic template with a sol-gel method.
4. the method for preparing the bioactive glass-modified gelatin composite hydrogel as claimed in claim 1, wherein the wavelength of the ultraviolet light is 365nm, and the intensity is 5m W/cm 2.
5. A bioactive glass-modified gelatin composite hydrogel obtained by the production method according to any one of claims 1 to 4.
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