Self-reinforced injectable double-network hydrogel for promoting bone regeneration and preparation method thereof

Abstract

This invention, applicable to the field of biomedical engineering materials technology, provides an injectable, self-reinforcing, double-network hydrogel that promotes bone regeneration and its preparation method. Polyvinyl alcohol (PVA) and bioactive glass (BAG) particles are combined and interpenetrated into a sodium alginate (SA) network to form an interpenetrating network, resulting in an injectable, double-network composite hydrogel. This invention makes the hydrogel easily injectable and suitable for repairing irregularly shaped bone defects. The optimal composition of the hydrogel components by mass is: 6% sodium alginate, 10% polyvinyl alcohol, and 6% bioactive glass. The optimal ratio of sodium alginate sol to bioactive glass / PVA hydrosol is 2:1. With this optimal composition and ratio, the hydrogel gels in less than 3.5 minutes after injection and exhibits a 2300% increase in compressive strength, meeting the mechanical properties required for the osseointegration phase of irregularly shaped bone defects.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical engineering materials, and in particular relates to an injectable double-network hydrogel capable of self-reinforcing and promoting bone regeneration, and a preparation method thereof. Background Art

[0002] Bone defects, commonly caused by trauma, infection, and tumor resection, lead to localized dysfunction and even compromise patient quality of life, often necessitating bone reconstruction. Injectable bone fillers are essential in orthopedic surgery, particularly for the reconstruction of irregular bone loss. To date, the only clinically available device, injectable bone cement, is suboptimal for treating various orthopedic injuries. For example, polymethyl methacrylate (PMMA) reinforcement is not recommended in many cases due to its lack of osteoinductivity and bioresorbability. On the other hand, artificial bone particles, primarily made of hydroxyapatite (HCA), tricalcium phosphate, or bioactive glass (BAG), exhibit high bioactivity and biodegradability. However, these materials face significant challenges in surgical applications, as powdered fillers are mechanically unstable and hard cements are too brittle. Polyethylene glycol (PEG) is biocompatible and possesses the ability to form in situ; however, PEG hydrogels generally lack the mechanical properties to replicate load-bearing systems under in vivo conditions, such as those encountered in bone regeneration applications. These shortcomings limit their application in bone repair. Therefore, great expectations are placed on osteogenic biomaterials with flexible interfaces, tough structures, and potentially high affinity with metallic fixators.

[0003] Injectable hydrogels, formed through an in situ gelation process via physical or chemical reactions, have shown promise as drug carriers and implants for treating skin lesions and other tissue defects. However, hydrogels are too soft to serve as scaffolds for load-bearing tissues, and their weaknesses are more pronounced than those of bulk hydrogels. Although several excellent double-network hydrogels have been reported in recent years, their injectability within physiological conditions has been unsatisfactory. Polyvinyl alcohol (PVA) hydrogels are widely used as scaffolds for tissue engineering applications due to their excellent water solubility, inherent nontoxicity, and chemical stability. However, PVA lacks strong binding strength to surrounding tissues, making it difficult to integrate with surrounding tissues. Therefore, to enhance the bioactivity and biocompatibility of hydrogels, sodium alginate (SA) has been adopted as a composite material for tissue engineering applications due to its bioactivity, nontoxicity, water solubility, biocompatibility, nonimmunogenicity, and biodegradability. Despite their many advantages, these hydrogels lack sufficient mechanical properties to withstand the loads that may occur in certain body parts, such as joints.

[0004] Recent studies have shown that bioactive glass (BAG) is widely used as a scaffold for bone tissue engineering due to its excellent bioactivity. Its bone regenerative capacity is well known, as it can mineralize into surface hydroxyapatite (HCA) and form strong chemical bonds with living bone. Therefore, to improve these properties, BAG inorganic particles are added. Furthermore, studies have shown that ionic extracts of 58S nano-bioactive glass can activate the ERK1 / 2 and p38 pathways in the mitogen-activated protein kinase (MAPK) pathway, upregulating the expression of osteogenic genes and proteins such as Runt-related transcription factor 2 (Runx2), alkaline phosphatase, osteocalcin, and type I collagen, demonstrating superior osteoinduction compared to traditional 45S5 bioactive glass. The typical bone structure is a bicontinuous inorganic and organic network, with water (20-30% v / v) and cells dispersed within the matrix, resulting in extremely strong compressive resistance. Inspired by this, it was hypothesized in dislocation theory that if inorganic particles, even micron-sized ones, could bind to polymer chains, they might form a continuous phase that resists the compressive forces in the matrix. Summary of the Invention

[0005] The purpose of the present invention is to provide an injectable double-network hydrogel that self-reinforces and promotes bone regeneration and a preparation method thereof, in order to solve the problems raised in the above background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] An injectable double-network hydrogel for self-reinforcement and bone regeneration promotion, the hydrogel comprising the following components by mass fraction: 4-6% sodium alginate, 10-15% polyvinyl alcohol, and 4-6% bioactive glass; the optimal ratio of the sodium alginate sol to the bioactive glass / polyvinyl alcohol hydrosol is 2:1; the bioactive glass is 58S nano-bioactive glass prepared by a sol-gel method, and the chemical composition of the 58S nano-bioactive glass is 58% SiO2, 33% CaO, and 9% P2O5 in molar percentage;

[0008] The sodium alginate forms a first hydrogel network with a coagulation effect under physical action, and the bioactive glass interpenetrates the polyvinyl alcohol to form a second bioactive gel network. The first hydrogel network and the second bioactive gel network are connected by Ca in the bioactive glass. 2+ As a cross-linking agent, chemical cross-linking is formed.

[0009] Furthermore, the optimal composition of the components in the hydrogel by mass fraction is: 6% sodium alginate, 10% polyvinyl alcohol and 6% bioactive glass.

[0010] Furthermore, the preparation method of the sodium alginate hydrosol is as follows: dissolving sodium alginate in deionized water, stirring with a magnetic stirrer at room temperature until dissolved, and allowing to stand until air bubbles are exhausted to obtain the sodium alginate hydrosol.

[0011] Furthermore, in the preparation method of the sodium alginate hydrosol, the stirring rate is 200-400 rpm / min, the stirring time is 30-60 min, and the standing time is 6-12 h.

[0012] Furthermore, the preparation method of the polyvinyl alcohol hydrosol is: dissolving polyvinyl alcohol in deionized water, heating until dissolved, and stirring until air bubbles are exhausted to obtain the polyvinyl alcohol hydrosol.

[0013] Furthermore, in the preparation method of the polyvinyl alcohol hydrosol, the heating temperature is greater than 90° C., the stirring rate is 200-400 rpm / min, and the stirring time is not less than 6 hours.

[0014] Furthermore, the preparation method of the bioactive glass hydrosol is as follows: adding the bioactive glass into a polyvinyl alcohol solution, stirring at room temperature until dissolved, and obtaining the polyvinyl alcohol hydrosol containing the bioactive glass.

[0015] Furthermore, in the preparation method of the bioactive glass hydrosol, the stirring rate is 200-400 rpm / min and the stirring time is 30-60 min.

[0016] The preparation method of an injectable double-network hydrogel that self-reinforces and promotes bone regeneration includes the following steps: respectively loading a polyvinyl alcohol hydrosol containing bioactive glass and a sodium alginate hydrosol into double syringes, injecting them into a mold at the same time, stirring and mixing them evenly at room temperature, and standing for reaction to obtain the injectable double-network hydrogel that self-reinforces and promotes bone regeneration.

[0017] Furthermore, the ratio of the sodium alginate hydrosol to the polyvinyl alcohol hydrosol containing bioactive glass is 1:1-3:1, the stirring rate is 200-400 rpm / min, the stirring time is 0-1 min, and the standing time is 3.5-14.5 min.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] (1) The injectable double-network hydrogel of the present invention has a simple manufacturing process, and the proportions of the components are optimized to make it suitable for injection applications. At the same time, its good rheological properties enable it to adapt to bone defects of different shapes.

[0020] (2) In the injectable double-network hydrogel system of the present invention, polyvinyl alcohol forms a continuous colloidal network rather than being dispersed only in the continuous phase alginate network. Therefore, the interpenetrating double networks in the gel network structure improve the mechanical strength of the hydrogel.

[0021] (3) The injectable double-network hydrogel system of the present invention is rich in bioactive glass, which can undergo ion exchange with body fluids to generate hydroxyapatite, further achieving significant self-enhancement of compressive strength. The optimal composition and optimal ratio of the hydrogel have a gelation time of less than 3.5 minutes after injection. After soaking in simulated body fluid for 0-14 days, the compressive strength can gradually achieve a significant self-enhancement effect, and the compressive strength can be "self-enhanced" by 2300%, which can meet the mechanical properties required for irregular-shaped bone defects during the bone integration stage.

[0022] (4) The injectable double-network hydrogel of the present invention is composed of an organic-inorganic composite repair material. It not only has a high-water content gel network similar to the extracellular hydrogel matrix, but also contains calcium, phosphorus and other ions. The acidity and alkalinity in the gel network are regulated by the weak alkaline ions dissolved from the bioactive glass, which meets the microenvironment for the proliferation and differentiation of osteoblasts.

[0023] (5) The injectable double-network hydrogel of the present invention has good biocompatibility, can enrich calcium, phosphorus ions and trace elements released by mineralization, and can promote osteoblast differentiation and alkaline phosphatase activation.

[0024] (6) The injectable double-network hydrogel of the present invention overcomes some of the shortcomings of existing tissue engineering scaffolds for bone defects. It is a composite of multiple substances, maintaining the advantages of the original substances and solving the shortcomings of the performance of a single original material.

[0025] (7) The injectable double-network hydrogel of the present invention can enrich calcium, phosphorus ions and trace elements released by mineralization, and has the effect of rapidly inducing bone regeneration and promoting bone repair in micro-injuries, fractures and bone defects. At the same time, it has good coagulation ability, which improves the bone repair effect and provides a new idea for the treatment of bone defects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the rheological property optimization result of the PVA / SA / BAG hydrogel prepared in the present invention, wherein the mass fraction of PVA is 10% and the mass fraction of SA is 6%.

[0027] Figure 2 Schematic diagram of the formation of gel by injection of SA hydrosol and PVA / BAG hydrosol prepared in Example 1 of the present invention.

[0028] Figure 3 This is the FTIR graph of the mineralization of PVA / SA / BAG hydrogel prepared in Example 2 of the present invention.

[0029] Figure 4 This is the XRD pattern of the mineralization of the PVA / SA / BAG hydrogel prepared in Example 2 of the present invention.

[0030] Figure 5 Schematic diagram of the mechanical strength of the mineralized PVA / SA / BAG hydrogel prepared in Example 2 of the present invention.

[0031] Figure 6 This is a statistical graph of the in vitro coagulation results of the PVA / SA / BAG hydrogel prepared in Example 3 of the present invention.

[0032] Figure 7 Schematic diagram of the degradation of PVA / SA / BAG hydrogel prepared in Example 4 of the present invention.

[0033] Figure 8 Schematic diagram of the cytotoxicity results of the PVA / SA / BAG hydrogel prepared in Example 1 of the present invention on mouse embryonic osteoblast precursor cells (MC3T3-E1).

[0034] Figure 9 This is a statistical graph showing the effect of the PVA / SA / BAG hydrogel prepared in Example 1 of the present invention on the ALP activity of mouse embryonic osteoblast precursor cells (MC3T3-E1). DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0036] The specific implementation of the present invention is described in detail below with reference to specific embodiments.

[0037] An embodiment of the present invention provides an injectable double-network hydrogel for self-reinforcement and bone regeneration promotion. The hydrogel comprises the following components by mass fraction: 4-6% sodium alginate, 10-15% polyvinyl alcohol, and 4-6% bioactive glass. The optimal ratio of the sodium alginate sol to the bioactive glass / polyvinyl alcohol hydrosol is 2:1. The bioactive glass is 58S nano-bioactive glass prepared by a sol-gel method. The chemical composition of the 58S nano-bioactive glass is 58% SiO2, 33% CaO, and 9% P2O5 in molar percentage.

[0038] The sodium alginate forms a first hydrogel network with a coagulation effect under physical action, and the bioactive glass interpenetrates the polyvinyl alcohol to form a second bioactive gel network. The first hydrogel network and the second bioactive gel network are connected by Ca in the bioactive glass. 2+As a cross-linking agent, chemical cross-linking is formed.

[0039] In the embodiment of the present invention, preferably, soaking in simulated body fluid for 0-14 days can gradually achieve a significant self-enhancement effect of compressive strength, with the compressive strength "self-enhancing" by 2300%; and has the best coagulation effect.

[0040] As a preferred embodiment of the present invention, the optimal composition of the components in the hydrogel in terms of mass fraction is: 6% sodium alginate, 10% polyvinyl alcohol and 6% bioactive glass.

[0041] In an embodiment of the present invention, preferably, the hydrogel with the optimal composition and optimal ratio has a gelation time of less than 3.5 minutes after injection, and can gradually achieve a significant self-enhancement effect of compressive strength by soaking in simulated body fluid for 0-14 days, with the compressive strength "self-enhanced" by 2300%, which can meet the mechanical properties required for irregular-shaped bone defects during the bone integration stage.

[0042] As a preferred embodiment of the present invention, the preparation method of the sodium alginate hydrosol is: dissolving sodium alginate in deionized water, stirring with a magnetic stirrer at room temperature until dissolved, and allowing to stand until air bubbles are exhausted to obtain the sodium alginate hydrosol.

[0043] As a preferred embodiment of the present invention, in the preparation method of the sodium alginate hydrosol, the stirring rate is 200-400 rpm / min, the stirring time is 30-60 min, and the standing time is 6-12 h.

[0044] In the embodiment of the present invention, preferably, the stirring rate is 200 rpm / min, the stirring time is 30 min, and the standing time is 6 h.

[0045] As a preferred embodiment of the present invention, the preparation method of the polyvinyl alcohol hydrosol is: dissolving polyvinyl alcohol in deionized water, heating until dissolved, and stirring until air bubbles are exhausted to obtain the polyvinyl alcohol hydrosol.

[0046] As a preferred embodiment of the present invention, in the method for preparing the polyvinyl alcohol hydrosol, the heating temperature is greater than 90° C., the stirring rate is 200-400 rpm / min, and the stirring time is not less than 6 hours.

[0047] In the embodiment of the present invention, preferably, the heating temperature is 95° C., the stirring rate is 200 rpm / min, and the stirring time is 6 h.

[0048] As a preferred embodiment of the present invention, the preparation method of the bioactive glass hydrosol is as follows: adding bioactive glass to a polyvinyl alcohol solution, stirring at room temperature until dissolved, to obtain the polyvinyl alcohol hydrosol containing bioactive glass.

[0049] As a preferred embodiment of the present invention, in the method for preparing the bioactive glass hydrosol, the stirring rate is 200-400 rpm / min, and the stirring time is 30-60 min.

[0050] The preparation method of an injectable double-network hydrogel that self-reinforces and promotes bone regeneration includes the following steps: respectively loading a polyvinyl alcohol hydrosol containing bioactive glass and a sodium alginate hydrosol into double syringes, injecting them into a mold at the same time, stirring and mixing them evenly at room temperature, and standing for reaction to obtain the injectable double-network hydrogel that self-reinforces and promotes bone regeneration.

[0051] As a preferred embodiment of the present invention, the ratio of the sodium alginate hydrosol to the polyvinyl alcohol hydrosol containing bioactive glass is 1:1-3:1, the stirring rate is 200-400 rpm / min, the stirring time is 0-1 min, and the standing time is 3.5-14.5 min.

[0052] In the embodiment of the present invention, preferably, the ratio of sodium alginate hydrosol to polyvinyl alcohol hydrosol containing bioactive glass is 2: 1. The stirring rate is 200 rpm / min, the stirring time is 1 min, and the standing time is 3.5 min.

[0053] After the ratio of each component is optimized (as shown in Table 1), it is suitable for injection application. At the same time, after the rheological properties are optimized (as shown in Table 1), Figure 1 It can adapt to bone defects of different shapes.

[0054] Table 1 Ratio of raw materials for the preparation of PVA / SA / BAG hydrogel

[0055]

[0056]

[0057] Example 1

[0058] This embodiment provides a double-network structure composite hydrogel based on sodium alginate, polyvinyl alcohol and bioactive glass.

[0059] The hydrogel comprises the following components by weight: 6% sodium alginate, 10% polyvinyl alcohol, and 6% bioactive glass, with the ratio of sodium alginate to polyvinyl alcohol hydrosol containing bioactive glass being 2:1. The preparation of the PVA / BAG hydrogel specifically includes the following steps:

[0060] S1: Preparation of sodium alginate hydrosol (SA)

[0061] 6 g of sodium alginate was weighed and dissolved in 100 mL of deionized water. The mixture was stirred at room temperature for 30 min using a magnetic stirrer at a stirring rate of 200 rpm / min. The mixture was allowed to stand for 6 h and the air bubbles were removed to obtain a sodium alginate hydrosol with a content of 6%.

[0062] S2: Preparation of polyvinyl alcohol hydrosol (PVA)

[0063] 5 g of polyvinyl alcohol was weighed and dissolved in 50 mL of deionized water, and heated and stirred at 95° C. for 6 h until dissolved and air bubbles were expelled to obtain a polyvinyl alcohol hydrosol with a content of 10%.

[0064] S3: Preparation of PVA / BAG hydrogel

[0065] 3 g of bioactive glass (BAG) was weighed and dissolved in the above polyvinyl alcohol hydrosol, and stirred at room temperature for 30 min at a stirring rate of 200 rpm / min to obtain a polyvinyl alcohol hydrosol containing bioactive glass, wherein the bioactive glass content was 6% and the polyvinyl alcohol content was 10%.

[0066] S4: Preparation of PVA / SA / BAG hydrogel

[0067] The sodium alginate sol and the polyvinyl alcohol hydrosol containing bioactive glass were loaded into double syringes at a ratio of 2:1, injected into a mold and evenly mixed for 1 minute, and allowed to react for 3.5 minutes to obtain an injectable double-network hydrogel that self-reinforces and promotes bone regeneration.

[0068] Figure 2 Schematic diagram of the prepared PVA / SA / BAG hydrogel injected through a syringe. Figure 2 (a) SA hydrosol and PVA / BAG hydrosol have excellent injectability. Figure 2 (b) PVA / SA / BAG hydrogel can form a gel in 3.5 min.

[0069] Example 2

[0070] The preparation method of PVA / SA / BAG hydrogel is the same as that of Example 1, except that 1 mL of PVA / SA / BAG hydrogel is immersed in 4 mL of simulated body fluid (SBF) and subjected to in vitro mineralization reaction in a shaker at 37°C. The hydrogel is taken out after mineralization for 0-28 days, washed with deionized water and anhydrous ethanol three times in sequence, and then vacuum freeze-dried for further characterization. The infrared spectrum (FTIR), X-ray diffraction pattern (XRD) and mechanical strength of PVA / SA / BAG hydrogel are shown in Figure 2. Figure 3 、 Figure 4 and Figure 5 shown.

[0071] like Figure 3 The following are the FTIR spectra of PVA-SA-BAG hydrogel after being immersed in SBF for 0d, 4d, 7d, 14d and 28d. -1 The broad bands shown in the range of 2926cm are the stretching vibrations of OH and the stretching vibrations of hydrogen between polysaccharide and alginate molecules. -1 and 2876cm -1 The two absorption bands at 1411cm are CH symmetric and asymmetric stretching vibrations. -1 and 1591cm -1 The two absorption bands at 0d are the symmetric stretching and asymmetric stretching of COO-. Compared with the hydrogels at 0d of mineralization, the peaks related to hydroxyapatite appeared in the FTIR spectrum of the hydrogels immersed in simulated body fluid (SBF). After 28d of mineralization, 3 and PO4 were observed in the FTIR spectrum. 3- The characteristic vibration absorption peak related to the functional group is 563 cm -1 、620cm -1 and 1035cm -1 With the increase of mineralization time, PO4 3- The peak intensity gradually increased, and apatite continued to form on the surface of PVA / SA / BAG hydrogel.

[0072] like Figure 4 The following are XRD patterns of PVA / SA / BAG hydrogel after immersion in SBF for 0d, 4d, 7d, 14d and 28d. When the PVA / SA / BAG hydrogel was mineralized in simulated body fluid (SBF) for 0-28d, the hydrogel showed several new characteristic diffraction peaks. Through crystal phase analysis and comparison with STANDARD HCA cards, it was found that the positions of these new diffraction peaks were consistent with the characteristic peak positions of hydroxyapatite (HCA) crystals, corresponding to the (211) and (222) crystal planes corresponding to ~32° and ~46° in HCA crystals, respectively. When the PVA / SA / BAG hydrogel was mineralized in simulated body fluid, a very broad peak (2θ=20°) was shown, which was a new peak formed by the cross-linking of polyvinyl alcohol (PVA) and sodium alginate (SA). With the increase of mineralization time, the intensity of the characteristic diffraction peak of the PVA / SA / BAG hydrogel gradually increased, indicating that the hydroxyapatite content increased and the crystallinity further improved, which is basically consistent with the results of SEM. XRD experiments confirmed the gradual formation of hydroxyapatite, consistent with results reported for other bioactive glass-containing hydrogels. The diffraction peaks corresponding to the PVA / SA / BAG hydrogel were sharp, similar to the crystalline structure of bone, indicating the ability to induce bone defect repair.

[0073] like Figure 5The figure shows the compressive properties of PVA / SA / BAG hydrogel after immersion in SBF for 0, 4, 7, and 14 days. With the increase in mineralization time, the compressive strength of PVA / SA / BAG hydrogel shows a significant self-enhancement effect. When the PVA / SA / BAG hydrogel is mineralized in simulated body fluid (SBF) for 0 days, the hydrogel cannot withstand a weight of 500g, and the gel height is negligible (recorded as 0.1cm for ease of calculation). After 4 days of mineralization, the hydrogel can withstand a weight of 1000g, and the gel height is 2.1cm. After 14 days of mineralization, the hydrogel can withstand a weight greater than 1000g, and the gel height is 2.4cm. Therefore, the PVA / SA / BAG hydrogel is "active" and shows a significant "self-enhancement" phenomenon during the in vitro mineralization process in simulated body fluid (SBF), with the compressive strength increasing by 2300%.

[0074] Example 3

[0075] The preparation method of PVA / SA / BAG hydrogel is the same as that of Example 1, except that PVA / SA / BAG hydrogels with different BAG contents are prepared, wherein the PVA content is 10%, the SA content is 6%, and the BAG content is 4-6%. Commercial inorganic bioactive bone graft substitutes (IBBGS) and PVA / SA / BAG hydrogels of various proportions are cut into equal volumes and placed in beakers, with blank beakers as the control group. First, 0.1 mL of whole blood is dripped onto the surface of each sample. The control group directly drips an equal amount of blood into a beaker, and then adds 20 μL of 0.2 mol / L CaCl2 solution for coagulation determination. All beakers are incubated at 37° C. for 5 min. Subsequently, 25 mL of distilled water is added to each beaker, and the free red blood cells are hemolyzed in the water. The solution in each beaker is measured by ultraviolet method, and the absorbance at 540 nm is recorded. The absorbance of the control group solution is used as the reference value. The coagulation index (BCI) calculation formula is:

[0076]

[0077] like Figure 6The following is a statistical chart of the in vitro coagulation results of PVA / SA / BAG hydrogels. When the PVA / SA / BAG hydrogels contained 10% PVA, 6% SA, and 4-6% BAG, the BCI values ​​of the different BAG content groups did not differ significantly and were all lower than those of the control group. When the PVA / SA / BAG hydrogels contained 10% PVA, 6% SA, and 6% BAG, the BCI value of the hydrogels was 63.41±1.45, significantly lower than that of commercial IBBGS (88.88±1.56), further demonstrating that the PVA / SA / BAG hydrogels have excellent hemostatic properties. The adsorption capacity and porous structure of the PVA / SA / BAG hydrogels provide more active sites for blood cell adhesion and aggregation, which is conducive to thrombus formation. The red blood cells encapsulated in the blood clots are not easily destroyed in water.

[0078] Example 4

[0079] The preparation method for PVA / SA / BAG hydrogel was the same as in Example 1, except that the hydrogel after vacuum freeze-drying was first weighed and the initial mass was recorded. The hydrogel was then immersed in SBF solution and placed in a shaker at 37°C and 50 rpm. After mineralization for 0-28 days, the hydrogel was removed, washed three times with deionized water, and vacuum freeze-dried before being weighed and the residual mass recorded. The residual mass percentage was calculated using the following formula:

[0080]

[0081] like Figure 7 The figure shows the degradation performance of the PVA / SA / BAG hydrogel after immersion in SBF for 0, 4, 7, 14, and 28 days. When the PVA / SA / BAG hydrogel was immersed in simulated body fluid (SBF) for 0-7 days, the residual mass ratio of the hydrogel gradually increased. This is due to the formation of hydroxyapatite (HCA) on the surface of the hydrogel, which not only increased the mass of the hydrogel but also slowed down the degradation of the hydrogel to a certain extent. When the hydrogel was immersed in simulated body fluid (SBF) for 7-28 days, the residual mass ratio of the hydrogel gradually decreased. This is because as the immersion time increases, the degradation rate of the hydrogel is greater than the formation rate of hydroxyapatite. When the hydrogel was immersed in simulated body fluid (SBF) for 28 days, the degradation rate of the hydrogel was 28.3%.

[0082] Example 5

[0083] The method of detecting cell activity using CCK-8 was used to evaluate the cytotoxic effect of the PVA-SA-BAG hydrogel extract prepared in Example 1 on mouse embryonic osteoblast precursor cells (MC3T3-E1). The specific operation steps are as follows: First, mouse embryonic osteoblast precursor cells (MC3T3-E1) were seeded in a 96-well plate at a density of 5000 cells / well, and then placed in a CO2 incubator to culture and adhere to the wall overnight. Subsequently, the original culture medium was aspirated and replaced with complete culture medium containing PVA / SA / BAG hydrogel extract, with 5 parallel replicates set for each concentration. The cells were cultured in an incubator for 1d, 4d, and 7d, respectively. After culture, the cells were washed once with PBS and 100 μL of fresh culture medium (containing 10% CCK-8) was added to each well. Place in an incubator and incubate for 1h. Finally, a microplate reader was used to detect and record the absorbance at a wavelength of 450nm.

[0084] like Figure 8 The figure shows the effect of PVA / SA / BAG hydrogel on the proliferation of MC3T3-E1 cells. When the hydrogel was co-cultured with MC3T3-E1 cells for 1 day, 4 days, and 7 days, the proliferation rate of MC3T3-E1 cells gradually increased with the increase in culture time, and the difference was not significant compared with the control group (P<0.05), indicating that PVA / SA / BAG hydrogel had no cytotoxicity to MC3T3-E1 cells and had good cell compatibility. Therefore, PVA / SA / BAG hydrogel material has excellent biocompatibility and can be used as a safe gel carrier for the treatment of bone defects.

[0085] Example 6

[0086] Alkaline phosphatase (ALP) secreted by osteoblasts is one of the important detection indicators of osteoblasts. Detection of ALP activity in osteoblasts can reflect the differentiation of osteoblasts. MC3T3-E1 cells were cultured at 5×10 4 cells / cm 2 The cells were seeded at a density of 100 μg / mL in a 6-well plate and cultured in a differentiation medium containing or not containing PVA / SA / BAG extract for 7 and 14 days, and then digested with 0.25% trypsin and collected. ALP activity in cell lysates was measured using an ALP kit according to the manufacturer's instructions. The collected cells were washed 1-2 times with PBS, and 200 μL of TritonX-100 reagent was added to each well to lyse the cells for 30 minutes (cell breakage was observed under a microscope). The protein concentration in the cell lysate was measured using a BCA protein assay kit. The specific operation of the ALP activity detection experiment is shown in Table 2:

[0087] Table 2 ALP activity detection procedure

[0088]

[0089] ALP activity was normalized based on protein concentration according to the manufacturer's instructions. Three replicates were performed for each group.

[0090] ALP activity was used as a marker of early osteogenic differentiation to explore the differentiation-inducing ability of PVA / SA / BAG hydrogel. The effect of PVA / SA / BAG hydrogel on ALP activity of MC3T3-E1 cells was measured on days 7 and 14. Figure 9 As shown, at 7 days of culture, the ALP staining area in the PVA / SA / BAG hydrogel group was significantly larger than that in the control group. At 14 days of culture, the ALP staining area increased significantly, indicating further differentiation of MC3T3-E1 cells. ALP activity in the PVA / SA / BAG hydrogel group was significantly higher than that in the control group at both 7 and 14 days of culture (P < 0.0001), reaching its maximum at 14 days. These results indicate that PVA / SA / BAG hydrogels can promote the differentiation of MC3T3-E1 cells, with ALP activity reaching its peak at 14 days.

[0091] The above are only preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, several variations and improvements can be made without departing from the concept of the present invention. These should also be regarded as the scope of protection of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. Self-reinforced injectable double-network hydrogel for promoting bone regeneration, characterized in that: The hydrogel comprises the following components by mass fraction: 6% sodium alginate, 10% polyvinyl alcohol, and 6% bioactive glass; the ratio of the sodium alginate hydrosol to the polyvinyl alcohol hydrosol containing the bioactive glass is 2:1; the bioactive glass is 58S nano-bioactive glass prepared by a sol-gel method, and the molar percentages of the chemical components of the 58S nano-bioactive glass are 58% SiO2, 33% CaO, and 9% P2O5; The sodium alginate forms a first hydrogel network with a coagulation effect under physical action, and the bioactive glass interpenetrates the polyvinyl alcohol to form a second bioactive gel network. The first hydrogel network and the second bioactive gel network are connected by Ca in the bioactive glass. 2+ As a cross-linking agent, chemical cross-linking is formed.

2. The self-reinforced injectable double-network hydrogel for promoting bone regeneration according to claim 1, characterized in that: The preparation method of the sodium alginate hydrosol is as follows: dissolving sodium alginate in deionized water, stirring with a magnetic stirrer at room temperature until dissolved, and allowing the solution to stand until air bubbles are exhausted, thereby obtaining the sodium alginate hydrosol.

3. The self-reinforced injectable double-network hydrogel for promoting bone regeneration according to claim 2, characterized in that: In the preparation method of the sodium alginate hydrosol, the stirring rate is 200-400 rpm, the stirring time is 30-60 min, and the standing time is 6-12 h.

4. The self-reinforced injectable double-network hydrogel for promoting bone regeneration according to claim 1, characterized in that: The preparation method of the polyvinyl alcohol hydrosol is as follows: dissolving polyvinyl alcohol in deionized water, heating until dissolved, and stirring until air bubbles are exhausted to obtain the polyvinyl alcohol hydrosol.

5. The self-reinforced injectable double-network hydrogel for promoting bone regeneration according to claim 4, characterized in that: In the preparation method of the polyvinyl alcohol hydrosol, the heating temperature is greater than 90° C., the stirring rate is 200-400 rpm, and the stirring time is not less than 6 hours.

6. The self-reinforced injectable double-network hydrogel for promoting bone regeneration according to claim 1, characterized in that: The preparation method of the polyvinyl alcohol hydrosol containing bioactive glass is as follows: adding bioactive glass into polyvinyl alcohol solution, stirring at room temperature until dissolved, and obtaining the polyvinyl alcohol hydrosol containing bioactive glass.

7. The self-reinforced injectable double-network hydrogel for promoting bone regeneration according to claim 6, characterized in that: In the preparation method of the polyvinyl alcohol hydrosol containing bioactive glass, the stirring rate is 200-400 rpm and the stirring time is 30-60 min.

8. The method for preparing the self-reinforced injectable double-network hydrogel for promoting bone regeneration according to any one of claims 1 to 7, characterized in that: The method comprises the following steps: respectively loading polyvinyl alcohol hydrosol containing bioactive glass and sodium alginate hydrosol into double syringes, injecting them into a mold at the same time, stirring and mixing them evenly at room temperature, standing for reaction, and obtaining an injectable double-network hydrogel that self-reinforces and promotes bone regeneration.

9. The method for preparing the self-reinforced injectable double-network hydrogel for promoting bone regeneration according to claim 8, characterized in that: The ratio of the sodium alginate hydrosol to the polyvinyl alcohol hydrosol containing bioactive glass is 2:1, the stirring rate is 200-400 rpm, the stirring time is 0-1 min, and the standing time is 3.5-14.5 min.

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