CN116474173A

CN116474173A - Preparation method and application of composite hydrogel for promoting bone defect repair

Info

Publication number: CN116474173A
Application number: CN202310569085.4A
Authority: CN
Inventors: 袁宝明; 窦茗瀚; 宋万通; 司星辉; 孙守野; 崔宇韬; 王雁冰; 彭传刚; 吴丹凯
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2023-05-19
Filing date: 2023-05-19
Publication date: 2023-07-25

Abstract

The invention discloses a preparation method and application of composite hydrogel for promoting bone defect repair, comprising the following steps of dissolving IGF-1 in PBS; dissolving 4-arm-PEG-NH2 and ODEX in IGF-1 solution, respectively; mixing 4-arm-PEG-NH2 solution and ODEX solution, and rotating; standing to obtain the composite hydrogel. According to the technical scheme, the composite hydrogel carrying the cytokines is used for being placed into bone defects, so that the repairing and treating effects of the bone defects are improved; the preparation method is mainly prepared by crosslinking 4-arm amino polyethylene glycol and oxidized dextran through Schiff base reaction, and insulin-like growth factor-1 is loaded in the preparation process of the material. IGF-1 has the function of strongly promoting proliferation and differentiation of bone marrow mesenchymal stem cells into bone, and the PEG-ODEX hydrogel provides a good and stable carrier environment for release of IGF-1 and has the function of repairing bone defects.

Description

Preparation method and application of composite hydrogel for promoting bone defect repair

Technical Field

The invention relates to the field of medicines, in particular to a preparation method and application of composite hydrogel for promoting bone defect repair.

Background

The large bone defect of long bone occurring after severe trauma, bone tumor resection and osteomyelitis debridement has been a difficult point of treatment in the orthopedics field. Bone nonunion and other complications caused by the defect of the long bone segment often cause serious consequences such as limb disability and the like, and cause great pain and heavy economic burden to patients. In the current means for clinically treating large bone defects of long bones, autologous bone grafting is still the gold standard. However, in the case of a large bone defect, it is often difficult to obtain sufficient bone graft due to limited sources of autologous bone, and a large number of autologous bone harvesting often suffer from a number of complications, including nerve and vascular injury, infection, chronic pain in the bone supply area, and the like.

In addition to autologous bone grafting, several other approaches have been applied to repair and treatment of long bone large bone defects, including the methods of Ilizarov technology, masqueet technology, and bone tissue engineering. However, the above methods have certain disadvantages, and the Ilizarov technology needs a relatively complex external fixation instrument, and the local pressure of soft tissues often causes continuous pain of limbs of patients in the process of stretching and osteogenesis, and the risks of deviation of force lines, needle tract infection and nerve injury are accompanied. The Masquelet technique forms an induction membrane by implantation of PMMA during the first phase of its treatment, and still relies on autologous bone grafting during the second phase of the treatment. In addition, the Masquelet technique has a long treatment period and also increases the uncertainty in the treatment process.

Bone tissue engineering is one of the hot spots studied in recent years, and the three most important biological elements required for bone tissue engineering include seed cells, extracellular matrix scaffolds, and cytokines that promote growth, differentiation, and angiogenesis. Among them, hydrogel (hydrogel) is one of the hot targets of bone tissue engineering, and as a typical "soft and wet material" containing a three-dimensional network structure, it has a wide application prospect in biomedical fields such as tissue engineering, drug release, and biosensors due to its characteristic characteristics such as viscoelasticity, high water content, and environmental responsiveness.

In summary, how to design a novel composite implant material for improving the repair and treatment effects of bone defects is a problem to be solved by those skilled in the art.

Disclosure of Invention

The embodiment of the invention mainly aims at providing a preparation method and application of composite hydrogel for promoting bone defect repair, and aims at designing a novel composite imbedding material for improving the repair and treatment effects of bone defects.

The technical scheme for solving the technical problems is that the preparation method of the composite hydrogel for promoting bone defect repair comprises the following steps:

dissolving IGF-1 in PBS;

dissolving 4-arm-PEG-NH2 and ODEX in IGF-1 solution, respectively;

mixing 4-arm-PEG-NH2 solution and ODEX solution, and rotating;

standing to obtain the composite hydrogel.

In one embodiment of the invention, the step of dissolving IGF-1 in PBS, the IGF-1 has a solubility of 100 μg/L to 1600 μg/L.

In one embodiment of the present invention, in the step of mixing the 4-arm-PEG-NH2 solution and the ODEX solution and rotating, the weight ratio of the 4-arm-PEG-NH2 solution to the ODEX solution is 2:1.

In one embodiment of the present invention, the step of dissolving 4-arm-PEG-NH2 and ODEX in IGF-1 solution comprises:

dissolving 4-arm-PEG-OH in dichloromethane;

adding methylsulfonyl chloride and triethylamine into the 4-arm-PEG-OH solution, and continuing stirring for reaction;

precipitating the solution of methylsulfonyl chloride and triethylamine with ice anhydrous diethyl ether, and vacuum drying at 45 ℃ to obtain dry powder;

and (3) reacting the dry powder with ammonia water for 7 days, cooling to room temperature, extracting the water phase with dichloromethane, concentrating under reduced pressure, and dripping into cold diethyl ether to obtain 4-arm-PEG-NH2.

In one embodiment of the present invention, the step of dissolving 4-arm-PEG-NH2 and ODEX in IGF-1 solution, respectively, further comprises:

dissolving dextran in distilled water;

adding sodium periodate into the dextran solution, and continuing stirring for reaction for 24 hours;

dialyzing the reacted solution by deionized water;

freeze-drying the dialyzed solution to obtain ODEX.

In order to solve the technical problems, the invention also provides application of the composite hydrogel prepared by the preparation method of the composite hydrogel for promoting bone defect repair in preparation of bone defect repair materials.

According to the technical scheme, the composite hydrogel carrying the cytokines is used for being placed into the bone defect, so that the repairing and treating effects of the bone defect are improved; it is mainly prepared by crosslinking 4-arm aminopolyethylene glycol (4-arm-PEG-NH 2) and Oxidized Dextran (ODEX) through Schiff base reaction, and loading insulin-like growth factor-1 (insulin-like growth factors-1, IGF-1) in the material preparation process. In the application, IGF-1 has the function of strongly promoting proliferation and differentiation of bone marrow mesenchymal stem cells (bone marrow stromal cells, BMSCs) into bone, and the PEG-ODEX hydrogel provides a good and stable carrier environment for the release of IGF-1. Experiments prove that the hydrogel has the effect of repairing bone defects and can be used as a novel scaffold of bone tissue based on bone tissue cells.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to the structures shown in these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic illustration of a composite hydrogel prepared by the method for preparing a composite hydrogel for promoting bone defect repair according to the present invention;

FIG. 2 is a schematic gel-forming diagram of a method of preparing a composite hydrogel for promoting bone defect repair in accordance with the present invention;

FIG. 3 is a schematic view of the electron microscope scan results of the composite hydrogel of the present invention;

FIG. 4 is a schematic view of the results of a rheology experiment of the composite hydrogel of the present invention;

FIG. 5 is a schematic diagram showing the CCK8 detection results after co-culturing the composite hydrogel and BMSCs;

FIG. 6 is a schematic representation of the staining results of live and dead cells after co-culturing the composite hydrogel of the present invention with BMSCs;

FIG. 7 is a graph of in vivo degradation rate of an unloaded hydrogel of the invention;

FIG. 8 is a schematic representation of the in vitro drug release kinetics of the composite hydrogels of the present invention;

FIG. 9 is a schematic diagram of semi-quantitative analysis of alizarin red staining of the composite hydrogel of the invention;

FIG. 10 is a schematic illustration of a composite hydrogel of the present invention for promoting repair of bone defects;

FIG. 11 is a graph showing the effect of IGF-1 at various concentrations on BMSCs proliferation in accordance with the invention.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

It should be noted that all directional indicators (such as up, down, left, right, front, and rear … …) in the embodiments of the present invention are merely used to explain the relative positional relationship, movement, etc. between the components in a particular posture (as shown in the drawings), and if the particular posture is changed, the directional indicator is changed accordingly.

Furthermore, descriptions such as those referred to as "first," "second," and the like, are provided for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying an order of magnitude of the indicated technical features in the present disclosure. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature. In the description of the present invention, the meaning of "several", "a plurality" or "a plurality" is at least two, such as two, three, etc., unless specifically defined otherwise.

In the present invention, unless specifically stated and limited otherwise, the terms "connected," "affixed," and the like are to be construed broadly, and for example, "affixed" may be a fixed connection, a removable connection, or an integral body; can be mechanically or electrically connected; either directly or indirectly, through intermediaries, or both, may be in communication with each other or in interaction with each other, unless expressly defined otherwise. The specific meaning of the above terms in the present invention can be understood by those of ordinary skill in the art according to the specific circumstances.

In addition, the technical solutions of the embodiments of the present invention may be combined with each other, but it is necessary to be based on the fact that those skilled in the art can implement the technical solutions, and when the technical solutions are contradictory or cannot be implemented, the combination of the technical solutions should be considered as not existing, and not falling within the scope of protection claimed by the present invention.

The invention provides a preparation method of composite hydrogel for promoting bone defect repair, and aims to design a novel composite implantation material for improving the repair and treatment effects of bone defects.

The following are examples, wherein the experimental reagents and equipment in all examples are as follows:

experimental reagent: 4-arm-PEG-OH, methylene chloride, methylsulfonyl chloride, triethylamine, anhydrous diethyl ether, ammonia water, dextran and sodium periodate are all purchased from Aladin, DMEM/F12 complete medium, fetal Bovine Serum (FBS), rabbit bone marrow mesenchymal stem cells, PBS and IGF-1 lyophilized powder are all purchased from Gibco, CCK8 kit, live and dead staining kit and alizarin red staining kit are all purchased from Beijing Biyun, and IGF-1Elisa kit is purchased from river-derived organisms.

Experimental equipment: OHAUS-Adventure electronic balance was obtained from Orhaus corporation; millipore Direct-Q8 UV ultra-pure water machine was obtained from Merck Mibo, germany; MS7-H550-S constant temperature heating magnetic stirrer and dialysis bag were obtained from the laboratory instruments stock company of Xinghuang, beijing; the ultra-low temperature refrigerator is taken from Thermo Scientific; the LABCONCO freeze dryer was obtained from LABCONCO company in the united states.

The specific structure of the method for preparing a composite hydrogel for promoting bone defect repair according to the present invention will be described in the following examples:

example 1

A preparation method of composite hydrogel for promoting bone defect repair, which comprises the following steps:

1. preparation of 4-arm-PEG-NH2

Dissolving raw material 4-arm-PEG-OH in dichloromethane;

adding methylsulfonyl chloride and triethylamine into the solution, and continuing stirring for reaction;

precipitating the above solution with ice anhydrous diethyl ether, and vacuum drying at 45deg.C to obtain dry powder;

the dry powder and ammonia water react for 7 days, after the reaction is cooled to room temperature, dichloromethane is used for extracting water phase, decompression concentration is carried out, and the water phase is dripped into cold diethyl ether, thus obtaining the 4-arm-PEG-NH2.

2. Preparation of ODEX

Weighing raw material dextran, and dissolving in distilled water;

adding the sodium periodate solution, and continuously stirring and reacting for 24 hours;

dialyzing the solution with deionized water;

freeze-drying the above solution to obtain ODEX.

3. Preparation of IGF-1 solution

IGF-1 was dissolved in PBS.

It will be appreciated that the IGF-1 has a solubility of 100 μg/L to 1600 μg/L

Preferably, the IGF-1 has a solubility of 400 μg/L.

4. Preparation of composite hydrogels

Dissolving 4-arm-PEG-NH2 in IGF-1 solution;

dissolving ODEX in IGF-1 solution;

mixing 4-arm-PEG-NH2 solution and ODEX solution, and standing to obtain composite hydrogel as shown in figure 1.

It will be appreciated that the weight ratio of 4-arm-PEG-NH2 solution to ODEX solution is 2:1. Since the reaction of 4-arm-PEG-NH2 and ODEX is rapid, it is necessary to dissolve 4-arm-PEG-NH2 and ODEX in IGF-1 solution containing PBS, respectively, and the material formed during the reaction of 4-arm-PEG-NH2 and ODEX can directly physically encapsulate IGF-1 to form a composite hydrogel, as shown in FIG. 2.

The composite hydrogel carrying the cytokines is used for being placed into bone defects, so that the repairing and treating effects of the bone defects are improved; it is mainly prepared by crosslinking 4-arm aminopolyethylene glycol (4-arm-PEG-NH 2) and Oxidized Dextran (ODEX) through Schiff base reaction, and loading insulin-like growth factor-1 (insulin-like growth factors-1, IGF-1) in the material preparation process. In the application, IGF-1 has the function of strongly promoting proliferation and differentiation of bone marrow mesenchymal stem cells (bone marrow stromal cells, BMSCs) into bone, and the PEG-ODEX hydrogel provides a good and stable carrier environment for the release of IGF-1. Experiments prove that the hydrogel has the effect of repairing bone defects and can be used as a novel scaffold of bone tissue based on bone tissue cells

Example 2

A preparation method of a composite hydrogel scaffold for promoting bone defect repair, which comprises the following steps:

dissolving IGF-1 in PBS;

dissolving 4-arm-PEG-NH2 and ODEX in IGF-1 solution, respectively;

mixing 4-arm-PEG-NH2 solution and ODEX solution, and rotating;

placing the mixed solution into a grinding tool, and standing; the composite hydrogel for promoting bone defect repair is obtained, and the composite hydrogel is in the form of a bracket.

Example 3

Step 1, preparing 4-arm-PEG-NH2:

(1) 20.0g of 4-arm-PEG-OH was weighed out and dissolved in 100mL of methylene chloride.

(2) Then respectively adding 2.3g of methylsulfonyl chloride and 1.1g of triethylamine into the solution in the step (1), and continuing stirring and reacting for 24 hours;

(3) After the reaction was completed, the solution in (2) was precipitated with ice anhydrous diethyl ether, and then dried under vacuum at 45 ℃ until the weight was constant;

(4) And (3) reacting the dry powder obtained in the step (3) with 100mL of ammonia water for 7 days, cooling to room temperature, extracting the water phase with dichloromethane, concentrating to 50mL under reduced pressure, and dripping into cold diethyl ether to obtain 4-arm-PEG-NH2, and preserving in a refrigerator at 20 ℃.

Step 2, preparing ODEX:

(1) 2.0g of dextran is weighed and put into a 100mL dry flask, distilled water is added under the condition of room temperature, and the mixture is slowly stirred until the dextran is dissolved;

(2) 792mg of sodium periodate is added to the solution in (1), and stirring is continued for 24 hours at room temperature;

(3) Dialyzing the solution of (2) with deionized water for 3 days (molecular weight 3500 Da) (for removing crude product);

(4) Freezing the solution in (3) at-80 ℃ overnight, transferring to a freeze dryer for drying for 24 hours, and finally freeze-drying the solution to obtain the ODEX which is preserved at-20 ℃.

Step 3, preparing unloaded hydrogel:

(1) The final products obtained in step 1, step 2 were dissolved in PBS at 10wt% (weight fraction 10%, meaning 10% by weight, i.e. 10 parts of the substance to be dissolved and 90 parts of water), respectively;

(2) The 4-arm-PEG-NH2 solution of (1) was mixed with the ODEX solution in a weight ratio of 2:1, and after 10s rotation, 400. Mu.L of the mixture was added to a 24-well plate and left for 15min, to empty the hydrogel.

Step 4, preparing composite hydrogel:

(1) Dissolving IGF-1 freeze-dried powder in PBS, and adjusting the concentration to 400 mug/L;

(2) The final products prepared in step 1 and step 2 were dissolved in the solutions of (1) at 10wt%, respectively:

(3) Mixing the 4-arm-PEG-NH2 solution of (2) and the ODEX solution in a weight ratio of 2:1, rotating for 10s, adding 400 mu L of the mixture into a 24-pore plate, and standing for 15min to obtain the composite hydrogel.

Characterization of the composite hydrogels:

(1) The resulting composite hydrogel was frozen at-80 ℃ for 12 hours, and the frozen samples were lyophilized for 24 hours. Scanning electron microscopy and rheological experiments were performed on the freeze-dried composite hydrogels. As shown in FIG. 3 and FIG. 4, the hydrogel has a micro cross-linked network porous structure, and the pore diameter is between 50 and 100 μm. The pore size is also the optimal pore size of the bone tissue engineering biological implant, and the compact porous structure ensures the loading and controlled release characteristics of small molecular drugs and protein drugs. The hydrogels have a storage modulus G 'greater than the corresponding loss modulus G' and exhibit gel properties.

Biocompatibility of empty hydrogel with composite hydrogel and analysis of BMSCs proliferation:

(1) BMSC was placed in DMEM/F12 medium containing 10% fetal bovine serum at 37℃with 5% CO ₂ Is cultured in a cell culture vessel. BMSCs cultured to the third generation were used for evaluation of all in vitro cell experiments.

(2) The experimental process is that 1×10 ⁴ Density of individuals/wells BMSCs cell suspensions were inoculated into blank wells (condral, ctrl group), culture wells containing empty hydrogel (hydrogel, H group) and composite hydrogel (pure hydrogel incorporated IGF-1, H/IGF-1 group), respectively.

(3) After 1 and 3 days incubation in the incubator, a working solution of calcein-AM/PI stain was prepared for soaking the samples according to the instructions of the live dead cell staining kit. After soaking each group of samples for 15 minutes at 4 ℃ in the dark, the working solution is removed, PBS is used for washing twice, redundant dye is washed away, and the living and dead conditions of cells are observed under a fluorescence microscope. As shown in fig. 5, the stained number of BMSCs viable cells per group showed a significant trend of increasing from day 1 to day 3. As shown in FIG. 6, there was no significant difference in the number of dead cells between the three groups at each time point. This indicates that both the unloaded hydrogel and the composite hydrogel have good biocompatibility.

(4) A cell counting kit (Cell counting kit-8, CCK-8) was used to examine the specific effect of the complex system on BMSCs cell proliferation. 1X 104 BMSCs were inoculated into the H group and H/IGF-1 group, respectively.

(5) Experiments were performed at each time point after co-culturing with each set of hydrogels for 1, 3 and 7 days, respectively. The complete medium in each set of wells was changed, 10% by volume of CCK-8 working fluid was added to the wells and incubated at 37℃and 5% CO2 for 2 hours. The absorbance of each set of reaction solutions was measured at 450nm using a microplate reader. The results are shown in fig. 5, and BMSCs cells of each group exhibited a significant tendency to proliferate and have good cell activity over the 7 day experimental period. The proliferation rates of the blank hole group and the empty hydrogel group are not different in days 3 and 7, so that the cell proliferation of the composite hydrogel group is more obvious, and the other groups are far beyond each other; it was revealed that the empty hydrogel had no effect on the proliferation activity of BMSCs, whereas the composite hydrogel carrying IGF-1 had an effect of promoting the proliferation of BMSCs. It is further demonstrated that the hydrogels have good biocompatibility.

(6) The IGF-1 solution was used in step 4 (1) at the following concentrations: 0 μg/L,20 μg/L,50 μg/L,100 μg/L,200 μg/L,400 μg/L,800 μg/L,1600 μg/L; different composite hydrogels were formed and analysis of BMSCs proliferation was performed separately: the results are shown in FIG. 11, where the proliferation of BMSCs was most pronounced at a concentration of 400. Mu.g/L IGF-1 solution.

In vivo degradation analysis of empty hydrogels:

(1) After isoflurane anesthesia of SD rats, shaving the two sides of the back, injecting hydrogel subcutaneously, and injecting 0.4mL on each side;

(2) Rats were sacrificed on days 0, 3, 7,14,21,28, 35, the skin surrounding the injection was removed, fascia was removed, the residual hydrogel removed was weighed, and degradation was observed. As shown in FIG. 7, the hydrogel swells by absorbing water in the early stage and is degraded smoothly, and the whole process lasts for about 35 days, so that long-time degradation is realized, and the requirement of bone formation is met.

Drug release analysis of composite hydrogels:

(1) The Elisa method is adopted to detect the drug release condition of the composite hydrogel in vitro. The concentration of the drug loaded into the gel when in vitro drug release is detected is improved to 100 mug/L. After the preparation of the composite hydrogel, the composite hydrogel was placed in deionized water at 37 ℃ and the soaking solutions were collected at day 1,2,3,5,7,14,21,28, respectively. After collection, the same amount of deionized water is added for continuous soaking.

(2) Preparing working solution according to the specification of the Elisa kit, mixing the soaking solution collected in the step (1) with the working solution, adding the mixture into a pore plate with the bottom coated with an antibody in the kit, incubating for 30 minutes, measuring absorbance of each sample at a wavelength of 450nm after a color reaction, calculating the concentration of the drug in the soaking solution according to a standard curve, and evaluating the drug release condition. The results are shown in FIG. 8, which shows the release kinetics of the H/IGF-1 complex hydrogel drug over a 35 day period, showing a relatively good sustained drug release profile.

As shown in fig. 8, the release rate of the drug was fast at the initial stage, the total release percentages at the first day were 25.8% ± 3.2%, respectively, and the release rate of the drug was significantly slow in the next 28 days, exhibiting a good slow release effect. By day 28 and 35, the total amount of drug released from the H/IGF-1 complex hydrogel was 86.9% + -1.8% and 89.1% + -2.3%, respectively, indicating that little drug was released from day 28 to day 35, thus stopping the recovery of the drug extract. In a drug delivery architecture, the drug release process is mediated primarily by the degradation of the material and the diffusion of the drug itself. Since the hydrogel has not been degraded in the early stages of release, the drug is released mainly in a freely diffused form. Meanwhile, the rate of the medicine during diffusion is closely related to the pore size of the hydrogel, and the larger the pore size is, the easier the medicine is to diffuse through the network structure of the hydrogel, so that the medicine release is accelerated. The pore size of the hydrogel is about 50-100 mu m, and the water-swelling capacity of the hydrogel is high although the pore size is small, so that IGF-1 can be more easily diffused to the outside. However, the burst release phase is not long, and as the hydrogel swells to the limit and the hydrogel gradually degrades, the release of the drug becomes mainly dominated by the slow degradation of the material, so that the drug release rate is slowed down, and a good drug release profile is exhibited. In general, the composite hydrogel system constructed by the application can realize good drug release results.

Effect of empty hydrogel and composite hydrogel on BMSC osteogenic differentiation:

(1) The effect of the composite hydrogel on the osteogenic differentiation of BMSCs was evaluated and examined under induction of osteogenic induction medium. BMSCs at 1X10 ⁵ Density of individuals/wells was inoculated into Ctrl, H and H/IGF-1 well plates, respectively, and cultured with osteogenic induction medium.

(2) After induction day 21, alizarin red staining was performed separately, cells were washed twice with PBS and fixed with 4% paraformaldehyde for 30 min at 37 ℃. Alizarin red solution was then added to the immobilized cells and incubated for 30 minutes at room temperature.

(3) The mineralized nodules of each group were dissolved in 10% cetylpyridinium chloride solution and the reaction solution was assayed for semi-quantitative analysis by measuring absorbance at 562 nm. The results are shown in FIG. 9, with H/IGF-1 groups showing the deepest calcium nodule staining and the largest stained area in all groups on day 21. Semi-quantitative analysis of alizarin red staining also confirmed the experimental results, which indicated that the number of calcified nodules was significantly higher in the H/IGF-1 group than in the other groups. Differentiation of osteoblasts is a continuous process in which differentiated cells secrete mineralized extracellular matrix, thereby promoting deposition of minerals (e.g., calcium). Thus, calcium deposition is a marker of mature osteoblasts. The results of alizarin red staining experiments indicate that IGF-1 released by the composite hydrogel promotes in vitro osteogenesis of BMSCs.

Example 4

Application of composite hydrogel to bone defect repair:

the preparation methods of the empty hydrogel and the composite hydrogel are the same as in example 3;

(1) 30 New Zealand white rabbits were randomly divided into 3 groups of 10. The experimental animals were anesthetized with 3% (w/v) pentobarbital at a dose of 50mg/kg and operated. Anesthesia of the back skin, sterilization of the operation area, hole towel spreading, longitudinal incision of the distal radius side of the left forelimb, separation of fascia and muscle layer by layer, and exposure of radius.

(2) A small electric saw is used for cutting off the radius with the length of 2cm to cause the defect with the length of 2cm, and the periosteum with the length of 0.5cm above and below the defect is cleaned. The Ctrl group directly sews the wound layer by layer, the H group sews the wound layer by layer after injecting blank hydrogel at the defect, and the H/IGF-1 group sews the wound layer by layer after injecting composite hydrogel at the defect. Intramuscular injection of penicillin (1.5 mg/kg) was performed for each experimental animal within 3 days after the operation to prevent infection.

(3) Animals were sacrificed by air injection from the ear margin intravenously 12 weeks after surgery under anesthesia and radius specimens were collected. As shown in FIG. 10, the Ctrl group and H group bone defects were not repaired, bone nonunion was formed, only a small amount of new bone was seen at the defect, and the broken ends were rat tail-shaped, forming atrophic bone nonunion. The H/IGF-1 group defect is filled with a large amount of new bone tissue, the broken ends are connected, and the bone healing condition is excellent.

The present invention is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present invention are intended to be included in the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims

1. The preparation method of the composite hydrogel for promoting bone defect repair is characterized by comprising the following steps of:

dissolving IGF-1 in PBS;

dissolving 4-arm-PEG-NH2 and ODEX in IGF-1 solution, respectively;

mixing 4-arm-PEG-NH2 solution and ODEX solution, and rotating;

standing to obtain the composite hydrogel.

2. The method of claim 1, wherein the step of dissolving IGF-1 in PBS, wherein the IGF-1 is soluble in 100 μg/L to 1600 μg/L.

3. The method of claim 2, wherein in the step of mixing and spinning the 4-arm-PEG-NH2 solution and the ODEX solution, the weight ratio of the 4-arm-PEG-NH2 solution to the ODEX solution is 2:1.

4. The method of preparing a composite hydrogel for promoting bone defect repair according to claim 1, wherein the steps of dissolving 4-arm-PEG-NH2 and ODEX in IGF-1 solution respectively comprise:

dissolving 4-arm-PEG-OH in dichloromethane;

5. The method of claim 1, wherein the step of dissolving 4-arm-PEG-NH2 and ODEX in IGF-1 solution, respectively, further comprises:

dissolving dextran in distilled water;

dialyzing the reacted solution by deionized water;

freeze-drying the dialyzed solution to obtain ODEX.

6. Use of a composite hydrogel prepared by the method for preparing a composite hydrogel for promoting repair of a bone defect according to any one of claims 1 to 5 in preparing a bone defect repair material.