Preparation method and application of a high-strength, high inorganic ratio and suitably degradable organic-inorganic composite hydrogel

Abstract

The present invention discloses a preparation method and application of a high-strength, high inorganic ratio and suitable degradability organic-inorganic composite hydrogel, belonging to the technical field of biomedical tissue engineering. The hydrogel contains 30 wt% to 70 wt% of calcium phosphate nanoclusters and 30 wt% to 70 wt% of sodium alginate. This organic-inorganic composite hydrogel is beneficial to the proliferation and mineralization of bone cells, achieving more ideal bone guidance. The experimental results prove that it has good cell compatibility and can rapidly promote the repair of rat cranial bone defects (the repair time of the 8 mm limit bone injury is shortened to 4 weeks). Compared with the autologous bone graft material with the best clinical repair effect in the control group, the healing effect is more significant. The high-strength organic-inorganic composite hydrogel prepared by the present invention is a single network structure, showing good mechanical properties, while taking into account excellent and tissue-matched degradability, which is beneficial to hard tissue repair.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical tissue engineering, and specifically relates to a preparation method and application of a high-strength, high-inorganic ratio and suitable degradability organic-inorganic composite hydrogel. Background Art

[0002] Jawbone destruction caused by tumors, trauma, inflammation, etc. will affect the appearance and function of patients. Approximately 2 million patients worldwide need bone graft repair every year. Autologous bone grafting is considered the gold standard for clinical bone grafting, but there are problems such as limited donor tissue and the need to create a second surgical site; although allogeneic bone grafting has sufficient sources, there are potential problems such as immune rejection and virus transmission. Therefore, how to obtain a bionic artificial bone repair material to quickly and effectively promote the repair of large-area bone defects is the key to solving this problem.

[0003] Bone tissue is composed of hydroxyapatite crystals and collagen fibers arranged in parallel, and is a typical organic-inorganic composite material. Therefore, organic-inorganic composite bone repair materials have become one of the research hotspots in the field of tissue engineering in recent years. The organic matrices involved include synthetic ones (such as polyvinyl alcohol, PCL, etc.) and natural ones (such as sodium alginate, collagen, etc.). Among them, sodium alginate in natural organic matrices, a natural polysaccharide extracted from brown algae such as kelp or sargassum, has broad application prospects in fields such as wound dressings and biomedical tissue engineering due to its excellent biosafety and biocompatibility. It has been approved by the US Food and Drug Administration for biomedical applications and has advantages in tissue engineering and drug delivery applications.

[0004] Inorganic matrices include calcium phosphate, calcium carbonate, silicon minerals, iron minerals, etc. Calcium phosphate widely exists in the bones of animals. Therefore, calcium phosphate-based organic-inorganic composites are a type of advanced functional materials currently under research. Research shows that the mechanical strength of such composites is closely related to the size of calcium phosphate nanoparticles. Small-sized calcium phosphate (1 - 5 nm) is beneficial to improving the mechanical properties of the materials. However, there are still challenges in the preparation technology of small-sized calcium phosphate in existing research, resulting in poor mechanical properties of sodium alginate-based organic-inorganic composites. Especially in the bone regeneration and repair treatment in the oral wet environment, the material is prone to ulceration and cannot play an early supporting role, leading to a long osteogenesis cycle and poor osteogenic mechanical properties. In addition, research shows that biomaterials with mechanical properties matching those of bone are also beneficial for the proliferation and mineralization of bone cells, and play a good role in bone conduction and bone induction. Therefore, solving the phase separation problem between the organic and inorganic matrices to improve the mechanical properties of the hydrogel is the key to improving the bone defect repair effect. During tissue regeneration, the scaffold material gradually degrades, providing space for the new tissue, while maintaining sufficient structural integrity to support the growth and function of cells. However, if the material occupies space for a long time, it will limit the migration and proliferation of cells, affect the natural healing process of the tissue, and may even trigger an inflammatory response, increasing the risk of infection. Therefore, having good mechanical properties and a degradation rate matching that of the tissue is of great significance for tissue regeneration. The patent application with the publication number CN115414527A discloses an organic-inorganic integrated structure formed by ultra-small amorphous calcium phosphate nanoclusters (with a diameter of about 1 nm), sodium alginate, and polyvinyl alcohol, which is similar to the structure of biomineralized collagen. There are potential problems in the degradation of this double-network hydrogel in vivo, which may cause adverse reactions such as high antigenicity. At the same time, traditional nanocomposite hydrogels will agglomerate with the increase in the filler content, resulting in poor uniformity. Summary of the Invention

[0005] Aiming at the deficiencies in the prior art, the purpose of the present invention is to provide a high-strength, high-inorganic ratio, and suitable degradability organic-inorganic composite hydrogel for bone defect repair, its preparation method and application. By copolymerizing and crosslinking nanoparticles (inorganic polyion clusters) with sodium alginate, the mechanical properties of the material are enhanced. The prepared high-strength, high-inorganic ratio, and suitable degradability hydrogel material can be applied to skull repair. For the extreme model of 8 mm bone injury, good bone repair effects can be obtained in 4 weeks.

[0006] To achieve the above purpose, the specific technical solutions adopted by the present invention are as follows:

[0007] The present invention provides a preparation method for a high-strength, high-inorganic ratio, and suitable degradability organic-inorganic composite hydrogel, and the steps are as follows:

[0008] (1) Triethylamine was used as a stabilizer, calcium salt as a calcium source, and phosphoric acid as a phosphorus source were added to an organic solvent to produce a calcium phosphate nanocluster solution;

[0009] (2) The calcium phosphate nanocluster solution was blended with an aqueous sodium alginate solution to form a homogeneous emulsion;

[0010] (3) The emulsion was evaporated to dryness to obtain a composite film;

[0011] (4) The composite film prepared in step (3) was immersed in water until swelling equilibrium was reached to obtain the high-strength, high inorganic ratio and suitable degradability organic-inorganic composite hydrogel with an ordered structure.

[0012] The high-strength hydrogel material prepared by the present invention exhibits good mechanical properties, and at the same time takes into account excellent biocompatibility / degradability, which is beneficial to hard tissue repair.

[0013] Preferably, in step (1), the density of the calcium phosphate nanocluster solution is 12.5 mg / mL, and the average size of the nanoparticles in the calcium phosphate nanocluster solution is 1.7 nm.

[0014] Specifically, the preparation method of the calcium phosphate nanoclusters is as follows:

[0015] 11.76 g of calcium chloride dihydrate was dissolved in 880 mL of absolute ethanol and stirred for 1 hour. Subsequently, an absolute ethanol solution containing phosphoric acid (volume percentage 85%) and triethylamine (i.e., 4.18 mL of 85% phosphoric acid + 220 mL of triethylamine + 800 mL of absolute ethanol) was added dropwise, and the mixture was vigorously stirred for 12 hours. After standing at room temperature for 24 hours, a white precipitate accumulated at the bottom of the solution. The solution above the white precipitate was removed by suction to obtain the calcium phosphate nanocluster solution.

[0016] Preferably, in step (2), the blending volume ratio of the calcium phosphate nanocluster solution to the sodium alginate solution is 0.25-1:1; the mass fraction of the sodium alginate solution is 0.5 wt%, and the viscosity is 200±20 mPa·s.

[0017] The cross-linking method of the hydrogel determines its physical and chemical properties. There are stronger ionic bonds between small-sized calcium phosphate nanoclusters and the molecular chains of sodium alginate, making the gel form a tight network. At the same time, the sodium alginate molecules endow the composite hydrogel with a certain deformation ability, and the maximum stress during the stretching process can reach 200 kPa.

[0018] Specifically, the conditions for the blending are: the stirring speed is 500 rpm, and the stirring time is 3 hours.

[0019] Specifically, in step (3), the emulsion is poured into a cell culture dish with a diameter of 10 cm and naturally evaporated and dried at 25°C for 4 - 7 days, where the volume of the emulsion is 50 - 100 mL.

[0020] Preferably, in step (4), the thickness of the organic - inorganic composite hydrogel is 0.2 - 1.0 mm.

[0021] The present invention also provides an organic - inorganic composite hydrogel with high strength, appropriate inorganic content and suitable degradability prepared by the described preparation method. The organic - inorganic composite hydrogel contains 30 - 70 wt% calcium phosphate and 30 - 70 wt% sodium alginate. The organic - inorganic composite hydrogel contains 30 - 60 wt% calcium phosphate and 40 - 70 wt% sodium alginate.

[0022] More preferably, the composite hydrogel contains 54 wt% calcium phosphate nanoclusters and 46 wt% sodium alginate.

[0023] The hydrogel prepared by the present invention using ultra - small - sized calcium phosphate nanoclusters can achieve continuous and stable uniformity. The inorganic content ratio in the hydrogel can be increased to a maximum of 69.4 wt% without material collapse.

[0024] Preferably, the strain range of the organic - inorganic composite hydrogel is 33% - 66%, the maximum tensile strength range is 30 - 200 kPa, and the elastic modulus range is 34 - 154 kPa.

[0025] Preferably, the degradation characteristic of the organic - inorganic composite hydrogel is that it can degrade at least 20% within one week. For the organic - inorganic composite hydrogel prepared by the present invention, due to the poor cross - linking degree between the organic and inorganic components, water molecules in the aqueous solution will act with sodium alginate, thus destroying the interaction between the organic and inorganic components, resulting in the collapse of the organic - inorganic composite hydrogel, that is, degradation occurs.

[0026] The present invention also provides an application of the organic - inorganic composite hydrogel prepared by the described preparation method, or the organic - inorganic composite hydrogel as a bionic periosteum in the preparation of medical materials for repairing hard tissues.

[0027] Preferably, the hard tissue is the skull.

[0028] More preferably, the damage diameter of the skull is 8 mm (the size of the extreme bone defect).

[0029] In the embodiment of the present invention, the organic - inorganic composite hydrogel with high strength, high inorganic ratio and suitable degradability is applied to a rat model of 8 - mm extreme skull defect. The repair effect is significantly better than that of autologous bone transplantation within 4 weeks.

[0030] The present invention also provides a medical material for repairing hard tissues, which comprises an organic-inorganic composite hydrogel prepared by the preparation method described, or the organic-inorganic composite hydrogel.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) In the present invention, sodium alginate is used as the organic structural unit and calcium phosphate oligomer as the inorganic structural unit, and the two are crosslinked by ionic bonds. Due to the small size of the calcium phosphate oligomer, the bond strength of the ionic bond is increased, significantly improving the mechanical strength of the material, and obtaining an organic-inorganic composite single-network hydrogel with mechanical stability in a wet environment. The obtained material also has good biocompatibility. In addition, the material preparation method is simple and controllable. Compared with the disadvantages that most other high-strength single-network hydrogels require complex chemical synthesis, this simple method has practical significance for industrial applications.

[0033] (2) The high-strength organic-inorganic composite hydrogel prepared by the present invention is beneficial to the proliferation and mineralization of bone cells, achieving more ideal bone guidance. The experimental results prove that it has good cell compatibility and can quickly promote the repair of rat skull defects (the repair time of the 8-mm limit bone injury is shortened to 4 weeks). Compared with the autologous bone graft material with the best clinical repair effect in the control group, the healing effect is more significant.

[0034] (3) In the prior art, there are problems with the degradation of double-network hydrogel materials in vivo, which may cause adverse reactions such as high antigenicity. The high-strength organic-inorganic composite hydrogel prepared by the present invention is a single-network structure, showing good mechanical properties, and at the same time taking into account excellent and tissue-matched degradability, which is beneficial to hard tissue repair. Description of the Drawings

[0035] Figure 1 It is the appearance diagram of the high-strength organic-inorganic composite hydrogel obtained in Example 2 of the present invention and the appearance diagram of the control group.

[0036] Figure 2 It is the transmission electron microscope (TEM) diagram of the high-strength organic-inorganic composite hydrogel obtained in Example 2 of the present invention and the TEM diagram of the control group.

[0037] Figure 3 It is the inorganic calcium phosphate proportion diagram of the organic-inorganic composite hydrogels obtained in Examples 1 to 3 of the present invention; the data points respectively represent the inorganic calcium phosphate proportion of a sample; 0.0398, 0.0004, <0.0001 all represent significance.

[0038] Figure 4Comparison charts of tensile stress-strain results and elastic modulus results of the organic-inorganic composite hydrogels obtained in Examples 1 to 3 of the present invention; the data points respectively represent the elastic modulus results of one sample; 0.431 indicates no significance, and 0.0004 and <0.0001 both indicate significance.

[0039] Figure 5 It is the degradation situation of the organic-inorganic composite hydrogel obtained in Examples 1 to 3 of the present invention within 1 week.

[0040] Figure 6 It is the degradation situation of the high-strength organic-inorganic composite hydrogel prepared in Example 2 under the skin of rats within 1 week.

[0041] Figure 7 It is the micro-CT (Micro-CT) image of the high-strength organic-inorganic composite hydrogel prepared in Example 2 after being applied to repair the extreme cranial bone defect of rats.

[0042] Figure 8 It is the result chart of bone volume fraction of the high-strength organic-inorganic composite hydrogel prepared in Example 2 after being applied to repair the extreme cranial bone defect of rats; the data points respectively represent the bone volume fraction results of one sample; 0.677 and 0.104 indicate no significance, and 0.0007 indicates significance.

[0043] Figure 9 It is the Raman spectrum chart of the high-strength organic-inorganic composite hydrogel prepared in Example 2 and the control group. Detailed implementation manners

[0044] The present invention is further described below with reference to examples.

[0045] Example 1

[0046] 1) Weigh 11.76 g of calcium chloride dihydrate and dissolve it in 880 mL of absolute ethanol, and stir evenly for 1 hour;

[0047] After mixing 220 mL of triethylamine solution and 800 mL of absolute ethanol, add 4.18 mL of phosphoric acid solution (volume percentage 85%), and mix evenly for 1 hour;

[0048] Slowly drop the mixed solution of triethylamine, absolute ethanol, and phosphoric acid into the calcium chloride absolute ethanol solution, and continuously stir. It can be seen that the solution gradually changes from transparent and clear to a milky white suspension; continuously stir evenly for 12 hours;

[0049] 2) Leave the solution prepared in step 1) standing at 25 °C for 24 hours to allow the white precipitate to completely settle to the bottom layer of the solution. The upper layer is a clear solvent. Suck off the upper solvent to obtain white ultra-small amorphous calcium phosphate nanoclusters (the average particle size of the nanoparticles therein is 1.7 nm);

[0050] 3) Weigh 0.5 g of sodium alginate and mix it with 100 mL of ultrapure water. Stir at 25 °C for 12 hours to fully dissolve and homogenize the sodium alginate to obtain a 0.5 wt% sodium alginate solution;

[0051] 4) Mix the calcium phosphate nanocluster solution (with a density of 12.5 mg / mL) and the sodium alginate solution in a volume ratio of 0.25∶1, and stir at 25 °C for 3 hours to fully crosslink the ultra-small particle size calcium phosphate and sodium alginate molecules; Pour the mixed solution into a plastic mold and let it dry naturally, and then immerse it in water again to reach the swelling equilibrium, thus obtaining a high-strength organic-inorganic composite hydrogel.

[0052] Example 2

[0053] The previous steps 1-3 are the same as those in Example 1. Mix the calcium phosphate nanocluster solution and the sodium alginate solution in a volume ratio of 0.5∶1, and stir at 25 °C for 3 hours to fully crosslink the ultra-small particle size calcium phosphate and sodium alginate molecules. Pour the mixed solution into a plastic mold and let it dry naturally, and then immerse it in water again to reach the swelling equilibrium, thus obtaining a high-strength organic-inorganic composite hydrogel.

[0054] Example 3

[0055] The previous steps 1-3 are the same as those in Example 1. Mix the calcium phosphate nanocluster solution and the sodium alginate solution in a volume ratio of 1∶1, and stir at 25 °C for 3 hours to fully crosslink the ultra-small particle size calcium phosphate and sodium alginate molecules. Pour the mixed solution into a plastic mold and let it dry naturally, and then immerse it in water again to reach the swelling equilibrium, thus obtaining an ultra-high-strength organic-inorganic composite hydrogel.

[0056] Example 4 Performance Evaluation and Characterization of High-Strength Organic-Inorganic Composite Hydrogel

[0057] Set a control group, and the control group is prepared using 60-80 nm hydroxyapatite particles.

[0058] The appearance diagrams of the high-strength organic-inorganic composite hydrogel prepared in Example 2 and the control group are as Figure 1As shown, the control group showed structural disintegration in water and could not form a hydrogel structure, while the hydrogel material of the present invention exhibited good mechanical properties and could form a high-strength hydrogel structure. At the same time, TEM observation showed that the high-strength organic-inorganic composite hydrogel obtained in Example 2 had a uniform and dense network structure, and the inorganic phase formed a mineralized structure along the organic phase, while the control group could not form a network structure, and there was an obvious boundary between the organic phase and the inorganic phase ( Figure 2 ).

[0059] After that, the proportion of inorganic calcium phosphate in the high-strength organic-inorganic composite hydrogels prepared in Test Examples 1 to 3 of the present invention was tested, and the test results are as Figure 3 shown. The proportion of inorganic calcium phosphate in the high-strength organic-inorganic composite hydrogel prepared in Example 1 was 33%; the proportion of inorganic calcium phosphate in the high-strength organic-inorganic composite hydrogel prepared in Example 2 was 41%; the proportion of inorganic calcium phosphate in the high-strength organic-inorganic composite hydrogel prepared in Example 2 was 54%. Generally speaking, the proportion of inorganic calcium phosphate in the high-strength organic-inorganic composite hydrogel prepared by the present invention was between 33% and 54%.

[0060] In order to explore the interaction between the inorganic phase and the organic phase in the high-strength organic-inorganic composite hydrogel of the present invention, the high-strength organic-inorganic composite hydrogel prepared in Example 2 and the control group were analyzed for chemical composition and bonding state using a Raman spectrometer and compared. The results are as Figure 9 shown. In the Raman spectrum, the P-O band of the control group was at 426.3 cm -1 , while the P-O band of the Example 2 group was at 408.5 cm -1 , indicating that there was an ionic bond interaction between calcium phosphate nanoclusters and sodium alginate molecules.

[0061] In addition, the anti-deformation ability of the high-strength organic-inorganic composite hydrogel of the present invention was tested to evaluate the mechanical properties of the material. The comparison of the tensile stress-strain results and the comparison of the elastic modulus results of the high-strength organic-inorganic hydrogels obtained in Examples 1 to 3 of the present invention are as Figure 4 shown. It can be seen that the strain range of the high-strength organic-inorganic composite hydrogel prepared by the present invention was 33% - 66%, the maximum tensile strength range was 30 - 200 kPa, and the elastic modulus range was 34 - 154 kPa.

[0062] Example 5 Degradability of the organic-inorganic composite hydrogel

[0063] The organic-inorganic composite hydrogels prepared in Examples 1 to 3 were immersed in PBS buffer (37°C, pH = 7.4), the liquid was changed every day, and the samples were collected at preset time points, dried in an oven at 40°C and weighed. The results are as Figure 5As shown, the organic-inorganic composite hydrogels prepared in Examples 1 to 3 can be degraded by at least 20% within 1 week.

[0064] The high-strength organic-inorganic composite hydrogel prepared in Example 2 was embedded subcutaneously in 6-week-old male rats. One week after surgery, the samples were taken and embedded for HE staining. The results were as follows: Figure 6 As shown, the number of inflammatory cells in the implanted tissue was low and the hydrogel was gradually degrading.

[0065] Example 6 Experimental study on the effect of high-strength organic-inorganic composite hydrogel in skull defect repair

[0066] Application of high-strength organic-inorganic composite hydrogel: Rats were anesthetized with 0.3% sodium pentobarbital, and 4% articaine (Bilan) injection was injected locally for anesthesia and analgesia. The skin was prepared with iodine disinfection, the skin and periosteum of the rat's skull were cut, the skull was fully exposed, and a circular bone drill with a diameter of 8 mm was used to drill a defect on the top of the rat's skull to avoid damaging the meninges. The high-strength organic-inorganic composite hydrogel prepared in Example 2 was applied to the 8 mm extreme skull defect model of rats, and the repair effect within 4 weeks was significantly better than that of autologous bone transplantation ( Figure 7 , Figure 8 ). The 3D reconstruction results of Micro-CT scans showed that compared with the blank group, the hydrogel can significantly promote the repair of skull defects, the range of bone defects is significantly reduced, new bone tissue appears in the center of the defect within 4 weeks, and the new bone density reaches a level better than that of autologous bone treatment.

[0067] The above-described embodiment is only a preferred solution of the present invention, but it is not intended to limit the present invention. A person skilled in the relevant technical field may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. Use of a high-strength, high inorganic ratio and suitable degradability organic-inorganic composite hydrogel as a bionic periosteum in the preparation of medical materials for repairing skulls; The degradation characteristic of the organic-inorganic composite hydrogel is that it can degrade at least 20% within one week; The strain range of the organic-inorganic composite hydrogel is 33% - 66%, the maximum tensile strength range is 30 - 200 kPa, and the elastic modulus range is 34 - 154 kPa; The preparation method of the organic-inorganic composite hydrogel is as follows: (1) Add triethylamine as a stabilizer, calcium salt as a calcium source, and phosphoric acid as a phosphorus source into an organic solvent to generate a calcium phosphate nanocluster solution; (2) Blend the calcium phosphate nanocluster solution with an aqueous sodium alginate solution to form a uniform emulsion; The blending volume ratio of the calcium phosphate nanocluster solution to the sodium alginate solution is 0.25 - 1:1; the mass fraction of the sodium alginate solution is 0.5 wt%, and the viscosity is 200 ± 20 mPa·s; (3) Evaporate and dry the emulsion to obtain a composite film; (4) Immerse the composite film prepared in step (3) in water until swelling equilibrium is reached to obtain the high-strength, high inorganic ratio and suitable degradability organic-inorganic composite hydrogel with an ordered structure.

2. The application according to claim 1, characterized in that The damage diameter of the skull is ≤ 8 mm.

3. The application according to claim 1, wherein In step (1), the density of the calcium phosphate nanocluster solution is 12.5 mg / mL, and the average size of the nanoparticles in the calcium phosphate nanocluster solution is 1.7 nm; In step (2), the blending conditions are: the stirring speed is 500 rpm, and the stirring time is 3 hours.

4. The application according to claim 1, characterized in that In step (3), pour the emulsion into a cell culture dish with a diameter of 10 cm, and naturally evaporate and dry at 25°C for 4 - 7 days, where the volume of the emulsion is 50 - 100 mL; In step (4), the thickness of the organic-inorganic composite hydrogel is 0.2 - 1.0 mm.

5. The application according to claim 1, characterized in that, The organic-inorganic composite hydrogel contains 30 - 70 wt% of calcium phosphate nanoclusters and 30 - 70 wt% of sodium alginate.

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