CN110947034B - Bioactive calcium phosphate/fibrin compounded injectable bone repair hydrogel - Google Patents
Bioactive calcium phosphate/fibrin compounded injectable bone repair hydrogel Download PDFInfo
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
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- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
Abstract
The invention relates to a bioactive calcium phosphate/fibrin compounded injectable bone repair hydrogel, which comprises a mixed gel system consisting of bioactive calcium phosphate, fibrin and thrombin; the bioactive calcium phosphate comprises: amorphous calcium phosphate, and a phospho-base-containing biomolecule and/or a hydrolysate of the phospho-base-containing biomolecule uniformly complexed with the amorphous calcium phosphate through interaction.
Description
Technical Field
The invention relates to a bioactive calcium phosphate/fibrin compounded injectable bone repair hydrogel and a preparation method thereof, in particular to a bioactive calcium phosphate and fibrin solution mixed and then synchronously injected with thrombin solution to form the composite hydrogel which can rapidly fill bone defects and accelerate bone defect repair, belonging to the field of preparation of biological materials for orthopedics department.
Background
Since bone defects caused by trauma, bone disease or bone tumors are a common clinical problem, bone grafting is often the first method of repairing bone defects. Sources of bone graft include autogenous bone, allogeneic bone, xenogeneic bone, and various artificial bones. Autologous bone grafting is often limited by the size and number of available sites for grafting, while adding new trauma; allograft or xenogeneic bone transplantation has the possibility of graft rejection, infection and disease transmission, and influences the bone grafting effect. Therefore, the artificial bone repair material provides a good alternative for the repair of bone defects.
The main solid inorganic component in human bone is in the form of calcium-based phosphate biominerals. According to the characteristics, the development of a bionic strategy for preparing the calcium phosphate-based composite material with the bionic structure, components and functions is one of ideal choices for constructing bone tissue repair materials. The calcium phosphate component in the bone tissue is formed by a controllable natural biological mineralization process, and has good biocompatibility, degradability and bioactivity.
In order to prepare an ideal bone repair material, students at home and abroad develop a variety of calcium phosphate-based bone repair materials by regulating the types and the shapes of calcium phosphate, but the traditional bone repair material has a single structure and function, is poor in biological activity (namely, the time for promoting tissue defect regeneration is too long, generally 2 months or more), and is not ideal in degradability and other properties. Therefore, the preparation of the high-activity injectable bone repair material with bionic structure, components and functions is an ideal choice for constructing the bone tissue repair material which can guide the rapid regeneration of bone tissues and can be implanted in a minimally invasive surgery.
Disclosure of Invention
In view of the above problems, the present invention provides a bioactive calcium phosphate/fibrin composite injectable bone repair hydrogel and a preparation method thereof.
In one aspect, the present invention provides a bioactive calcium phosphate/fibrin composite injectable bone repair hydrogel, comprising a mixed gel system of bioactive calcium phosphate, fibrin and thrombin; the bioactive calcium phosphate comprises: amorphous calcium phosphate, and a phospho-base-containing biomolecule and/or a hydrolysate of the phospho-base-containing biomolecule uniformly complexed with the amorphous calcium phosphate through interaction.
The injectable hydrogel bone repair material prepared by compounding calcium phosphate with high biological activity and fibrin and prepared by a bionic strategy for the first time is mainly characterized by being injectable, and having high biocompatibility and the effects of quickly filling bone defects and promoting regeneration of defective tissues. The composite hydrogel has excellent biocompatibility, can be injected into bone defect positions in a minimally invasive mode to repair bone defects of different shapes, and guides the rapid regeneration of bone tissues.
Preferably, the bioactive calcium phosphate is a spherical-like particle with a particle size of 20-100 nm.
Preferably, the nucleobase-containing biomolecule is selected from at least one of mono-, di-or tri-phosphorylated adenosine, guanosine, uridine, cytidine, thymine adenosine.
Preferably, the product of hydrolysis of the nucleobase-containing biomolecule is a product of hydrolysis of the nucleobase-containing biomolecule to lose part or all of the phosphate.
Preferably, the mass ratio of the bioactive calcium phosphate to the fibrin is (0.001-0.8): 1, preferably (0.1 to 0.7): 1.
preferably, the content ratio of fibrin to thrombin is 1mg: (5-50) IU, preferably 1mg: (10-15) IU.
On the other hand, the invention also provides a preparation method of the bioactive calcium phosphate/fibrin compounded injectable bone repair hydrogel, which is characterized in that bioactive calcium phosphate and fibrin solution are mixed and then synchronously injected with thrombin solution to be coagulated into gel, so as to obtain the injectable bone repair hydrogel.
In the invention, bioactive calcium phosphate and fibrin solution are mixed to prepare uniform mixed solution; then, the mixed solution and the thrombin solution are synchronously injected to a bone defect position, and the fibrin is activated by the thrombin, so that the mixed solution and the thrombin solution are mixed and quickly gelatinized, and the injectable composite hydrogel material capable of quickly filling the bone defect and promoting the repair of the bone defect is formed. In the gelling process, certain coordination exists between calcium ions in the bioactive calcium phosphate and carboxyl groups among fibrin, and the mechanical property of the injectable hydrogel is enhanced to a certain extent. The injectable bioactive calcium phosphate/fibrin composite hydrogel material can be used for quickly repairing bone tissues, is biodegradable, and is a good method for preparing a high-activity injectable composite hydrogel-based bone repairing material. The method has important scientific significance and application value for expanding the preparation method and application range of the calcium phosphate-based injectable composite hydrogel material.
Preferably, the mass ratio of the bioactive calcium phosphate to the fibrin is (0.001-0.8): 1; the content of fibrin in the fibrin solution is 10-100 mg/mL, preferably 20-60 mg/mL; the content of thrombin in the thrombin solution is 100-1000 IU/mL; more preferably, the volume ratio of the fibrin solution to the thrombin solution is (0.5-1.5): 1, preferably 1: 1.
preferably, the fibrin solution also comprises NaCl with the content of 1-20 mg/mL.
Preferably, the thrombin solution further comprises CaCl2The content ratio of the thrombin to the calcium chloride can be (500-450) IU:1 mg.
Preferably, the time for the coagulation to form gel is 5 to 60 seconds, preferably 5 to 30 seconds.
The bioactive calcium phosphate/fibrin compounded injectable bone repair hydrogel provided by the invention has higher bioactivity, bone induction force and biodegradability, and is suitable for the biomedical field including bone defect repair.
The preparation method of the injectable bioactive calcium phosphate/fibrin composite hydrogel provided by the invention has the advantages of simple process, convenient operation, no need of complex and expensive equipment and easy realization of industrial production. The injectable bioactive/fibrin composite hydrogel material provided by the invention can realize rapid repair of bone defects for 2 weeks, and has rapid and obvious effect. The injectable bioactive calcium phosphate/fibrin composite hydrogel material has good application prospect as a biomedical material.
Drawings
FIG. 1 is a digital photograph of the bioactive calcium phosphate/fibrin composite hydrogel of example 1;
FIG. 2 is a scanning electron microscope image of a sample of bioactive calcium phosphate/fibrin composite hydrogel in example 1 after lyophilization;
FIG. 3 is a scanning electron micrograph of a fibrin hydrogel sample without added bioactive calcium phosphate after lyophilization;
FIG. 4 is a graph showing cell proliferation of a sample of bioactive calcium phosphate prepared in example 1 co-cultured with human mesenchymal stem cells for 7 d;
FIG. 5 is a graph showing the staining of alkaline phosphatase after co-culturing samples of the bioactive calcium phosphate/fibrin composite hydrogel prepared in example 1 with human mesenchymal stem cells for 7d and 14 d;
FIG. 6 is a schematic diagram of an animal experiment in which injectable hydrogel prepared in example 4 was injected into a bone defect site;
FIG. 7 is a digital photograph and a micro-CT image of the injectable hydrogel prepared in example 4 after repairing rat bone defect for 2 weeks;
fig. 8 is a light-microscopic image of HE staining and Masson staining after repair and sectioning of an injectable hydrogel prepared in example 4 at the site of the defect.
Detailed Description
The present invention is further illustrated by the following examples, which are to be understood as merely illustrative and not restrictive.
In the present disclosure, the bioactive calcium phosphate/fibrin compounded injectable bone repair hydrogel is a mixed gel system of bioactive calcium phosphate, fibrin and thrombin, which has the property of inducing bone formation of bone marrow mesenchymal stem cells, can be injected into a bone defect part in a minimally invasive manner by injection, and can rapidly gel to fill the bone defect and promote the repair of the bone defect. Wherein, the bioactive calcium phosphate can efficiently promote osteogenic differentiation performance of the mesenchymal stem cells.
In an alternative embodiment, the mass ratio of bioactive calcium phosphate to fibrin may be (0.001-0.8): 1, preferably (0.1 to 0.7): 1. the addition of the bioactive calcium phosphate can obviously promote the regeneration of bones and improve the bone defect repair effect. If the content is too high (more than 0.8:1), the composite injectable hydrogel contains too much bioactive calcium phosphate, and is not easy to gel. If the content is less (less than 0.001:1), the content of the bioactive calcium phosphate in the composite injectable hydrogel is too low, and the effect of promoting bone defect repair is not obvious.
In an alternative embodiment, the fibrin and thrombin may be present in an amount sufficient for both gels. For example, the content ratio of fibrin to thrombin may be 1mg (5-50) IU.
In alternative embodiments, the bioactive calcium phosphate comprises: amorphous calcium phosphate, and a phospho-base-containing biomolecule and/or a hydrolysate of the phospho-base-containing biomolecule uniformly complexed with the amorphous calcium phosphate through interaction. Wherein the bioactive calcium phosphate is spherical-like particles with the particle size of 20-100 nm. Wherein the nucleobase-containing biomolecule is at least one member selected from the group consisting of mono-, di-and tri-phosphorylated adenosine, guanosine, uridine, cytidine and thymine. The hydrolysis product of a nucleobase-containing biomolecule is a product of hydrolysis of a nucleobase-containing biomolecule to lose part or all of the phosphate.
In one embodiment of the invention, the bioactive calcium phosphate and the fibrin solution are uniformly mixed and then polymerized under the action of the thrombin solution to form the bioactive calcium phosphate/fibrin composite injectable bone repair hydrogel, which has the effects of rapidly filling bone defects and promoting bone defect repair. Wherein, the compounding of the bioactive calcium phosphate obviously improves the bioactivity and the osteoinductivity of the fibrin hydrogel material. The following is an exemplary description of the method of preparing the bioactive calcium phosphate/fibrin composite injectable bone repair hydrogel of the present invention.
And (3) preparing bioactive calcium phosphate. Specifically, adenosine triphosphate and water-soluble calcium salt are used for preparing the bioactive calcium phosphate material under the catalysis of alkaline phosphatase. Wherein, the adenosine triphosphate comprises adenosine triphosphate sodium salt and/or hydrate thereof, and adenosine triphosphate potassium salt and/or hydrate thereof. The water-soluble calcium salt can be calcium chloride and/or its hydrate, and calcium nitrate and/or its hydrate. The obtained bioactive calcium phosphate is an aggregate of 10-100 nanometers, and is compounded with a fibrin solution to obtain the injectable bioactive calcium phosphate/fibrin composite hydrogel under the activation action of thrombin.
As a detailed example of preparing bioactive calcium phosphate, the following steps are included: in a 60mL water system, alkaline phosphatase is used as a catalyst in a weakly alkaline (pH of 8.0-8.5) environment at 36-38 ℃, and a water-soluble calcium salt (such as CaCl)2、Ca(NO3)2) And adenosine disodium triphosphate or adenosine dipotassium triphosphate as reactants to prepare the bioactive calcium phosphate. Wherein the molar ratio of water-soluble calcium salt to phosphorus-containing biomoleculeThe molar ratio is 3: 1, the concentration of water-soluble calcium salt is 0.01 mol per liter, the concentration of adenosine triphosphate salt is 0.003 mol per liter, and the biological active calcium phosphate is obtained by stirring and reacting for 1-3 hours under the action of 0.6mg of alkaline phosphatase.
The fibrin solution is obtained by dissolving fibrin in an aqueous solution, preferably a sodium chloride solution (e.g., 1-20 mg/mL) of a certain concentration, such as physiological saline. Wherein, the content of the fibrin is 10-100 mg/mL, preferably 20-60 mg/mL.
Thrombin was dissolved in a calcium chloride solution (solute water) to obtain a thrombin solution. Wherein the content of the thrombin can be 100-1000 IU/mL. The content ratio of thrombin to calcium chloride can be (500-450) IU:1 mg.
Mixing the bioactive calcium phosphate with the fibrin solution to obtain a mixed solution A. Wherein the mass ratio of the bioactive calcium phosphate to the fibrin is 0.001: 1-0.8: 1. In addition, the stirring and mixing time of the bioactive calcium phosphate and the fibrin solution can be 5min to 30 min.
The mixed solution A and thrombin solution are injected synchronously (preferably at the same injection rate) to form gel rapidly, and the injectable bone repair hydrogel compounded by bioactive calcium phosphate/fibrin is obtained. Wherein, the content ratio of the fibrin to the thrombin can be 1mg (5-50) IU, preferably 1mg (10-20) IU. The time for quick gelling of the collagen fiber solution and the thrombin can be 5 s-30 s.
As an example of a method for preparing a bioactive calcium phosphate/fibrin composite injectable bone repair hydrogel, the method comprises the following steps: dissolving fibrin in sodium chloride solution with certain concentration, mixing the obtained bioactive calcium phosphate with certain mass into the fibrin solution, stirring and mixing for 5-30 min under aseptic condition, and recording the mixed solution as solution A. A certain amount of thrombin was dissolved in a calcium chloride solution of a certain concentration, and the solution was designated as solution B. And (3) uniformly mixing the solution A and the solution B by using a double-channel needle cylinder to prepare the bioactive calcium phosphate/fibrin composite hydrogel, wherein the gelling time generally needs 5-60 s.
The invention has the following advantages:
the bioactive calcium phosphate/fibrin compounded injectable bone repair hydrogel provided by the invention has higher bioactivity, bone induction force and biodegradability, and is suitable for the biomedical field including bone defect repair. The preparation method has the advantages of simple preparation process, convenient operation, no need of expensive equipment and easy realization of industrial production.
The present invention will be described in further detail with reference to examples. It is also to be understood that the following examples are illustrative of the present invention and are not to be construed as limiting the scope of the invention, and that certain insubstantial modifications and adaptations of the invention by those skilled in the art may be made in light of the above teachings. For example, the following examples use CaCl2And adenosine disodium triphosphate, but as mentioned above, other suitable water soluble calcium salts, water soluble adenosine triphosphates may be used instead. The following examples are also illustrative of suitable ranges of reaction temperatures, times, amounts of charge, and the like, and those skilled in the art can select the reaction temperature, time, amount of charge, and the like within the suitable ranges by the description herein, and are not limited to the specific values exemplified below.
Example 1
In a water bath at 37 ℃, 110mg of adenosine disodium triphosphate and 66mg of CaCl are sequentially added2Dissolving in 60mL of deionized water, adding 0.6mg of alkaline phosphatase, keeping the pH value of a reaction system between 8.0 and 8.5 in the whole reaction process, and reacting for 2 hours under magnetic stirring. And washing the product with water and ethanol for 3 times respectively, and drying to obtain the bioactive calcium phosphate, wherein the bioactive calcium phosphate is spherical-like particles with the particle size of 20-100 nm. At room temperature, 100mg of fibrinogen was dissolved in 2.5 ml of deionized water containing 22.5 mg of sodium chloride, 10mg of bioactive calcium phosphate was dispersed in the above fibrinogen solution with stirring to form solution A, and 1125IU of thrombin was dissolved in 2.5 ml of deionized water containing 11.1 mg of calcium chloride to form solution B (thrombin solution). Respectively placing the solution A and the solution B into two syringe barrels, connecting the two syringe barrels through a double-channel needle head, injecting at the same injection rate and pushing out,an injectable bioactive calcium phosphate/fibrin composite hydrogel was obtained as shown in figure 1. And a control group without adding bioactive calcium phosphate was set to obtain a fibrin hydrogel sample. The bioactive calcium phosphate/fibrin composite hydrogel after freeze drying is of a porous structure, the surface of the bioactive calcium phosphate/fibrin composite hydrogel is rough compared with the penetrated fibrin hydrogel, the SEM characteristic diagram of a freeze-dried bioactive calcium phosphate/fibrin composite hydrogel sample is shown in figure 2, the SEM characteristic diagram of the freeze-dried fibrin hydrogel sample is shown in figure 3, and as can be seen from figures 2 and 3, the internal microscopic pore size of the composite hydrogel is not changed after the bioactive calcium phosphate is compounded in the fibrin hydrogel. FIG. 4 is a cell proliferation graph of the bioactive calcium phosphate sample prepared in example 1 co-cultured with human mesenchymal stem cells for 7 days, and it can be seen that the efficiency of formation of clones of human mesenchymal stem cells is 0.98%, 0.99%, 1.32% and 1.23% at the material concentrations of 0. mu.g/ml, 100. mu.g/ml, 300. mu.g/ml and 500. mu.g/ml, respectively. Fig. 5 is a graph showing the staining of alkaline phosphatase of the sample of the bioactive calcium phosphate/fibrin composite hydrogel prepared in example 1 after co-culturing with human mesenchymal stem cells for 7d and 14d, and it can be seen from the graph that the human mesenchymal stem cells after co-culturing with the material for 7d and 14d have significant expression of alkaline phosphatase in the cells, and the expression of 14d is significantly greater than 7d, which indicates that the material can promote osteogenic differentiation of human mesenchymal stem cells. The bioactive calcium phosphate material contained in the composite hydrogel has good biocompatibility and the performance of promoting osteogenic differentiation of bone marrow mesenchymal stem cells (see figures 4 and 5). Compared with the common hydroxyapatite serving as an orthopedic repair material, the bioactive calcium phosphate material of the composite hydrogel has a higher expression effect of promoting the alkaline phosphatase in the mesenchymal stem cells, and shows that the material can obviously promote the osteogenic differentiation of the mesenchymal stem cells.
Example 2
At room temperature, 100mg of fibrinogen was dissolved in 3.0 ml of deionized water containing 22.5 mg of sodium chloride, 10mg of the bioactive calcium phosphate prepared in example 1 was dispersed in the above fibrinogen solution with stirring to form solution A, and 1125IU of thrombin was dissolved in 3.0 ml of deionized water containing 11.1 mg of calcium chloride to form solution B. And respectively placing the solution A and the solution B into two syringe barrels, connecting the two syringe barrels through a double-channel needle head, and injecting and pushing out at the same injection rate to obtain the injectable bioactive calcium phosphate/fibrin composite hydrogel.
Example 3
At room temperature, 100mg of fibrinogen was dissolved in 2.0 ml of deionized water containing 22.5 mg of sodium chloride, 50 mg of the bioactive calcium phosphate prepared in example 1 was dispersed in the above fibrinogen solution with stirring to form solution A, and 1125IU of thrombin was dissolved in 2.0 ml of deionized water containing 11.1 mg of calcium chloride to form solution B. And respectively placing the solution A and the solution B into two syringe barrels, connecting the two syringe barrels through a double-channel needle head, and injecting and pushing out at the same injection rate to obtain the injectable bioactive calcium phosphate/fibrin composite hydrogel.
Example 4
At room temperature, 100mg of fibrinogen was dissolved in 2.5 ml of deionized water containing 22.5 mg of sodium chloride, 30 mg of the bioactive calcium phosphate described in example 1 was dispersed in the above fibrinogen solution under mechanical agitation to form solution A, and 1125IU of thrombin was dissolved in 2.5 ml of deionized water containing 11.1 mg of calcium chloride to form solution B. Respectively placing the solution A and the solution B into two syringe barrels, connecting the two syringe barrels through a double-channel needle, injecting and pushing out at the same injection rate, and injecting into the bone defect position, referring to the animal experiment schematic diagram of fig. 6. Fig. 7 is a digital photograph and a Micro-CT image of a blank control group, a control group (fibrin gel), and an injectable hydrogel prepared in this example 4 after repairing rat bone defect for 2 weeks, and it can be seen from the Micro CT reconstruction image and the cross-sectional view of the bone defect site that the bone defect site in the experimental group for repairing bone defect with composite bioactive calcium phosphate hydrogel is basically repaired, while the new bone formation at the bone defect site in the experimental group for fibrin hydrogel of blank control group is less, and the repairing effect is inferior to that of the experimental group for composite bioactive calcium phosphate hydrogel. FIG. 8 is a light microscope picture of HE staining and Masson staining after repairing and slicing the defect part, more bone and ossein appear in the tissues around the bone defect of the composite bioactive calcium phosphate hydrogel experimental group, and the bone defect repairing effect is obvious. From fig. 7 and 8, it was found that the injectable bioactive calcium phosphate/fibrin composite hydrogel had the effects of rapidly filling a bone defect and promoting regeneration of defective tissues.
Comparative example 1
At room temperature, 100mg of fibrinogen was dissolved in 2.5 ml of deionized water containing 22.5 mg of sodium chloride, 100mg of the bioactive calcium phosphate described in example 1 was dispersed in the above fibrinogen solution under mechanical agitation to form solution A, and 1125IU of thrombin was dissolved in 2.5 ml of deionized water containing 11.1 mg of calcium chloride to form solution B. The solution A and the solution B are respectively placed into two syringe barrels, the two syringe barrels are connected through a double-channel needle head, and then the injection is pushed out at the same injection speed, and the injectable hydrogel is not easy to gel due to excessive bioactive calcium phosphate.
Table 1 shows the composition and performance parameters of the injectable hydrogels prepared in examples 1-4 of the present invention:
| fibrinogen/mg | Bioactive calcium phosphate/mg | Thrombin/IU | |
| Example 1 | 100mg | 10mg | 1125IU |
| Example 2 | 100mg | 10 | 1125IU |
| Example 3 | 100mg | 50mg | 1125IU |
| Example 4 | 100mg | 30mg | 1125IU |
| Control group | 100mg | 0mg | 1125IU |
| Comparative example 1 | 100mg | 100mg | 1125IU |
。
Industrial applicability: the preparation method has the advantages of simple preparation process, convenient operation, no need of complex and expensive equipment and easy realization of industrial production. The injectable bioactive calcium phosphate/fibrin composite hydrogel prepared by the preparation method has good application prospect when being used as a biomedical material in the fields of hard tissue repair and the like.
Claims (10)
1. A bioactive calcium phosphate/fibrin composite injectable bone repair hydrogel is characterized in that the injectable bone repair hydrogel is a mixed gel system consisting of bioactive calcium phosphate, fibrin and thrombin; the mass ratio of the bioactive calcium phosphate to the fibrin is (0.1-0.7): 1; the content ratio of the fibrin to the thrombin is 1mg: (5-50) IU;
the bioactive calcium phosphate comprises: amorphous calcium phosphate, and a phosphoryl group-containing biomolecule and/or a hydrolysate of the phosphoryl group-containing biomolecule that is uniformly compounded with the amorphous calcium phosphate through interaction; the bioactive calcium phosphate is spherical-like particles with the particle size of 20-100 nm;
mixing bioactive calcium phosphate and a fibrin solution, synchronously injecting the mixture with a thrombin solution, and coagulating into gel to obtain the injectable bone repair hydrogel, wherein the time for coagulating into gel is 5-60 seconds; the injectable bone repair hydrogel can realize the rapid repair of bone defects for 2 weeks.
2. The injectable bone repair hydrogel of claim 1, wherein said nucleobase-containing biomolecule is selected from at least one of mono-, di-or tri-phosphorylated adenosine, guanosine, uridine, cytidine, thoracic adenosine.
3. The injectable bone repair hydrogel of claim 1, wherein the product of hydrolysis of the nucleobase-containing biomolecule is a product of hydrolysis of the nucleobase-containing biomolecule with loss of some or all of phosphate.
4. The injectable bone repair hydrogel according to claim 1, wherein the fibrin to thrombin content ratio is 1mg: (10-15) IU.
5. The injectable bone repair hydrogel according to claim 1, wherein the fibrin solution has a fibrin content of 10 to 100 g/L; the content of thrombin in the thrombin solution is 100-1000 IU/mL.
6. The injectable bone repair hydrogel according to claim 5, wherein the fibrin solution has a fibrin content of 20 to 60 mg/mL.
7. The injectable bone repair hydrogel according to claim 5, wherein the volume ratio of the fibrin solution to the thrombin solution is (0.5-1.5): 1.
8. the injectable bone repair hydrogel according to claim 7, wherein the volume ratio of fibrin solution and thrombin solution is 1: 1.
9. the injectable bone repair hydrogel according to claim 1, further comprising NaCl in an amount of 1 to 20 mg/mL; and/or CaCl is also included in the thrombin solution2And the content ratio of the thrombin to the calcium chloride is (500-450) IU:1 mg.
10. The injectable bone repair hydrogel according to claim 1, wherein the time to gel is 5 to 30 seconds.
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