An injectable high-strength hydrogel for bone tissue repair and its preparation method

Abstract

The present invention relates to an injectable high-strength hydrogel for bone tissue repair and a preparation method thereof. A poly(hydroxyethyl acrylate-methyl acrylate) random copolymer is prepared by solution polymerization. The copolymer is fully swollen in water to obtain a physically cross-linked hydrogel. The hydrogel is injectable and gelled in vivo under shear thinning. The gel exudation is enhanced under physiological conditions. Bioactive glass that can form hydrogen bonds with the copolymer is mixed to prepare the injectable high-strength hydrogel. The hydrogel can not only provide good support for the bone defect site, but also promote the proliferation and differentiation of cells therein. The hydrogel of the present invention is simple to prepare, easy to operate, quick to respond, good in biocompatibility, has good self-repair ability, is injectable, and has little minimally invasive damage. It has good application prospects in the field of irregular bone defect repair.

Description

Technical Field

[0001] The present invention belongs to the intersection field of polymer materials and biomedical materials, and specifically relates to an injectable high-strength hydrogel for bone tissue repair and a preparation method thereof. Background Art

[0002] Bone defects caused by aging, trauma, tumors, etc. have a serious impact on human life. The most common treatment method to solve this problem is autologous or allogeneic bone transplantation. More than 2 million bone transplant operations are performed worldwide each year. However, bone transplantation has problems such as donor shortage, easy immune rejection reaction and insufficient transplant materials. Therefore, alternative materials such as metals, bioceramics, and polymer hydrogels have been tried in the field of bone repair. Metal scaffolds can cause inflammation and allergic reactions, bioceramic scaffolds are non-degradable, and the processing conditions are harsh. Due to its three-dimensional porous network structure, polymer hydrogels can absorb large amounts of water or body fluids. It is similar to the structure of the natural extracellular matrix (ECM), which makes it easy to deliver nutrients and can load growth factors or bone cells. It has become an important candidate material in the field of bone tissue repair.

[0003] Natural polymers generally have good biocompatibility, but their mechanical properties and processability are mostly poor, and their biodegradation rate is too fast, which limits their application in the field of bone repair. In contrast, synthetic polymer hydrogels can be obtained through molecular and structural design to obtain multifunctional high-performance hydrogels, but most synthetic polymers and their gels have poor biocompatibility, which limits their application in tissue repair. In addition, hydrogel implantation into organisms usually requires secondary or multiple open surgeries. During the treatment process, repeated open surgeries increase medical risks and patient pain. Injectable hydrogels can use minimally invasive surgery to fill geometrically complex spaces, allowing the gel to be injected into irregular bone defects and reducing damage to surrounding tissues.

[0004] To address the above-mentioned issues, the present invention provides an injectable, high-strength hydrogel for bone tissue repair. This polymer is a copolymer of the hydrophilic structural unit hydroxyethyl acrylate and the hydrophobic structural unit methyl acrylate, resulting in groups capable of both hydrogen bonding with water and coordination with salt ions. By incorporating bioactive glass particles, the gelation time is effectively shortened. Furthermore, the phase transition of the responsive polymer, in response to changes in the chemical and physical environment both in vivo and in vitro, allows for dehydration and strengthening of the gel before and after injection. This results in a novel injectable bone tissue repair hydrogel with multiple responses, excellent biocompatibility, and high strength. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide an injectable high-strength hydrogel for bone tissue repair and a preparation method thereof.

[0006] An injectable high-strength hydrogel for bone tissue repair comprises a linear thermosensitive polymer, a functional filler, and a solvent. The mass ratio of the linear thermosensitive polymer to the solvent is 1:(1-9); the mass ratio of the functional filler to the linear thermosensitive polymer is 1:(2.33-19). The linear thermosensitive polymer has a significant lower critical solution temperature (LCST) in aqueous solution, and its LCST is adjustable within the range of 10-70°C. The linear thermosensitive polymer comprises a hydrophilic unit and an unsaturated hydrophobic unit. The molar ratio of the hydrophilic unit to the functional unit is 1:(0.1-9). The hydrophilic unit is selected from one or more of acrylamide, hydroxyethyl acrylate, N-vinyl pyrrolidone, and acrylic acid. The unsaturated hydrophobic unit is selected from one or more of alkyl acrylates, alkyl methacrylates, N-alkyl acrylamides, and polyoxyethylene alkyl acrylates. The polymer is prepared by solution polymerization. The functional filler is selected from one or more of hydroxyapatite, bioactive glass, and growth factors. The solvent is selected from one or more of pure water, ethanol, and phosphate buffered saline.

[0007] The hydrogel absorbs water and swells at room temperature (25° C.), the modulus decreases, and the injectability is good. After the temperature reaches 37° C. after being injected into the body, the polymer hydrogel with LCST characteristics has enhanced water exudation and increased modulus.

[0008] The hydrogel has good injectability when containing a small amount of ethanol, and the strength of the gel increases as the ethanol is lost through metabolism in the body.

[0009] The present invention also provides a method for preparing the hydrogel:

[0010] The hydrogel is prepared by physical blending.

[0011] The preparation method of the hydrogel is as follows: a hydrophilic monomer and an unsaturated hydrophobic monomer are dissolved in a solvent to prepare a solution with a monomer concentration of 5wt% to 30wt%, the initiation temperature is controlled at 10 to 80°C, nitrogen is passed through to deoxygenate, and an initiator is added to initiate polymerization for 3 to 8 hours to obtain a polymer solution; the polymer solution is transferred to a dialysis bag, and deionized water is used to dialyze to remove small molecules whose molecular weight does not meet pre-set requirements, and the supernatant is taken out and freeze-dried to obtain a copolymer; the copolymer is dissolved in water, and a functional filler is added thereto, and mixed to obtain an injectable hydrogel, and the hydrogel is obtained by shear thinning to obtain a hydrogel for bone defect repair.

[0012] The molar ratio of the unsaturated hydrophobic monomer to the hydrophilic monomer in the solution is 1:(0.25-9); the molecular weight distribution of the polymer is 2.1-10, and the molecular weight is 100,000-1,000,000; the content of the polymer is 10-50 wt%; the content of the functional filler is 1-20 wt%; the initiator can be any initiator well known to those skilled in the art and is not particularly limited.

[0013] The present invention also provides application of the hydrogel in the field of bone tissue repair.

[0014] The present invention has the following beneficial effects:

[0015] The present invention provides an injectable high-strength hydrogel for bone tissue repair and a preparation method thereof. The high-strength hydrogel is obtained by physically blending a linear thermosensitive polymer, a functional filler and a solvent. The linear thermosensitive polymer has a significant lower critical solution temperature (LCST) property in an aqueous solution, so that the hydrogel absorbs water and swells at low temperatures and has good injectability. After being injected into the body, the temperature rises and the gel excretes water more strongly. The hydrogel has the following beneficial effects: (1) The injectable hydrogel system of the present invention has good biocompatibility, and has the advantages of mild reaction conditions, moderate reaction rate, no need to add small molecule cross-linking agents with bio- / cytotoxicity, and low biotoxicity; (2) The hydrogel of the present invention has good shear thinning properties and can be directly injected into the bone defect site through a medical syringe to achieve rapid filling of the bone defect, avoid secondary damage caused by surgery, and is easy to operate; (3) The hydrogel of the present invention has LCST in water or saline. By rationally designing the molecular structure and screening suitable monomers and proportions, the hydrogel can absorb water and swell at 25°C (before injection) and has good injectability. At 37°C (after injection), the gel exudes water more strongly, providing good support for the damaged site; (4) The hydrogel of the present invention has a tendency to promote new bone formation without the addition of growth factors. After being mixed with functional fillers, it can effectively and quickly promote the repair of bone tissue. The functional fillers can form strong interactions with the polymer, which not only increases the cross-linking degree of the hydrogel network, but also improves the mechanical properties of the hydrogel, making it suitable for the repair of bone defects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 These are scanning electron microscope images and EDS mapping images of the injectable hydrogel obtained in Example 1 of the present invention, wherein Figure (a) is an electron microscope image of the pure hydrogel, Figure (b) is an electron microscope image of the bioactive glass hydrogel, Figure (c) is an electron microscope image of the bioactive glass hydrogel after being immersed in PBS solution, and Figures (d) to (g) are EDS mapping images of the sodium, calcium, silicon, and phosphorus elements at Figure (b);

[0017] Figure 2These are the rheological property test results of the injectable hydrogel obtained in Example 1 of the present invention. Figure (a) shows the viscosity of the pure hydrogel as a function of shear rate, Figure (b) shows the viscosity of the hydrogel after mixing with bioactive glass as a function of shear rate, Figure (c) shows the viscosity of the water / ethanol gel as a function of shear rate, and Figure (d) shows the viscosity of the water / ethanol gel as a function of shear rate after mixing with bioactive glass.

[0018] Figure 3 This is a graph of an alternating large and small strain scanning experiment of the injectable hydrogel obtained in Example 1 of the present invention;

[0019] Figure 4 This is the cytotoxicity result of the injectable hydrogel obtained in Example 1 of the present invention on rBMSCs cells;

[0020] Figure 5 1 is a general view of the specimen after the injectable hydrogel obtained in Example 1 of the present invention was injected into the rat skull, wherein Figure (a) is a blank group, Figure (b) is a pure hydrogel group, and Figure (c) is a group mixed with bioactive glass hydrogel;

[0021] Figure 6 These are Micro-CT scans and three-dimensional reconstruction images of the injectable hydrogel obtained in Example 1 of the present invention injected into the rat skull 4 and 8 weeks later. DETAILED DESCRIPTION

[0022] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0023] The present invention provides an injectable high-strength hydrogel for bone tissue repair. The high-strength hydrogel is obtained by physically blending a linear thermosensitive polymer, a functional filler, and a solvent. The mass ratio of the linear thermosensitive polymer to the solvent is 1:(1-9); the mass ratio of the functional filler to the linear thermosensitive polymer is 1:(2.33-19). The linear thermosensitive polymer has a significant lower critical solution temperature (LCST) in aqueous solution, and the LCST is adjustable within the range of 10-70°C.

[0024] The linear thermosensitive polymer comprises a hydrophilic unit and an unsaturated hydrophobic unit; wherein the molar ratio of the hydrophilic unit to the functional unit is 1:(0.1-9), preferably 1:(0.4-4), and most preferably 1:(1-3.5). The hydrophilic unit is selected from one or more of acrylamide, hydroxyethyl acrylate, N-vinyl pyrrolidone, and acrylic acid, preferably hydroxyethyl acrylate and N-vinyl pyrrolidone, and most preferably hydroxyethyl acrylate. The unsaturated hydrophobic unit is selected from one or more of alkyl acrylate, alkyl methacrylate, N-alkyl acrylamide, and polyoxyethylene alkyl acrylate, preferably alkyl acrylate and alkyl methacrylate, more preferably alkyl acrylate, and most preferably methyl acrylate. The linear thermosensitive polymer is prepared by solution polymerization.

[0025] The functional filler is selected from one or more of hydroxyapatite, bioactive glass and growth factors, preferably hydroxyapatite and bioactive glass, and most preferably bioactive glass.

[0026] The solvent is selected from one or more of pure water, ethanol and phosphate buffered saline solution.

[0027] The present invention provides an injectable, high-strength hydrogel for bone tissue repair and a preparation method thereof. A thermosensitive linear polymer is prepared using hydrophilic and hydrophobic monomers as primary raw materials. This polymer is then dissolved in deionized water at a specific ratio to produce an injectable hydrogel with shear-thinning properties. A specific proportion of functional fillers is then added to the hydrogel to produce an injectable hydrogel for bone tissue repair with a controllable gelation rate and high strength. The shear-thinning injectable hydrogel of the present invention exhibits excellent injectability, in vivo formability, biocompatibility, and self-repairing properties, and has great potential for application in the repair of irregular bone lesions.

[0028] The injectable hydrogel is prepared as follows: a solution with a monomer concentration of 5% to 30% by weight is prepared, the initiation temperature is controlled between 10°C and 80°C, nitrogen is passed through to remove oxygen, and then an initiator is added to initiate polymerization for 3 to 8 hours. The resulting polymer solution is transferred to a dialysis bag, dialyzed with deionized water to remove small molecules, and the supernatant is removed and freeze-dried to obtain a polymer. The polymer is dissolved in deionized water, a functional filler is added, and the mixture is mixed to obtain an injectable hydrogel with shear-thinning properties for bone tissue repair.

[0029] The monomers are hydroxyethyl acrylate and methyl acrylate, the functional filler is bioactive glass, and the initiator is any initiator well known to those skilled in the art without any special limitation.

[0030] The multi-responsive injectable hydrogel with bone defect repairing function of the present invention is further described below through examples.

[0031] The reagents used in the following examples are all commercially available.

[0032] Example 1

[0033] Methyl acrylate and hydroxyethyl acrylate were dissolved in N,N-dimethylformamide at a molar ratio of 1:(0.25-9) to prepare a 20 wt% monomer concentration. The polymerization temperature was controlled at 70°C, nitrogen was purged, and then the initiator azobisisobutyronitrile was added to initiate polymerization for 8 hours. The polymer solution was transferred to a dialysis bag with a molecular weight cutoff of 14,000 and dialyzed against deionized water for 5 days (to remove small molecules with a molecular weight less than 14,000). After freeze-drying, the resulting poly(methyl acrylate-hydroxyethyl acrylate) copolymer was obtained. The polymers were dissolved in deionized water to produce injectable hydrogels with varying water contents (50 wt% to 70 wt%). Bioactive glass particles were dispersed in deionized water, and the polymers were then added and mixed thoroughly to produce the injectable hydrogels for bone tissue repair.

[0034] Example 2

[0035] The hydrogel obtained in Example 1 was freeze-dried and then fractured. The cross-sectional morphology of the hydrogel was observed using a scanning electron microscope, and the distribution of the bioactive glass in the hydrogel was analyzed using an energy dispersive spectrometer. The accelerating voltage range of the scanning electron microscope was 0.2-30 kV, continuously adjustable in 10 V steps.

[0036] Figure 1 (a) to (c) are morphological images of the injectable hydrogel in Example 1 characterized by scanning electron microscopy. The results show that the hydrogel has a through-hole structure, which is conducive to the exchange of nutrients. Figure 1 (d) to (g) show the positional distribution of sodium, calcium, silicon, and phosphorus elements in the bioactive glass within the gel. The results show that the bioactive glass particles are mixed into the hydrogel and are evenly dispersed.

[0037] Example 3

[0038] The injectable hydrogel sample obtained in Example 1 was placed on the stage of a torque rheometer to investigate the rheological behavior of the gel. The hydrogel sample was a disc with a thickness of 1 mm and a diameter of 8 mm. The edges of the gel were sealed with silicone oil to prevent water evaporation during the test. In the injectable experiment, the test frequency was 1 Hz, the strain was 1%, the temperature was 25°C, and the shear rate test range was 0.1 s -1 to 1000s -1 In the self-healing experiment, the test frequency was 1 Hz, the temperature was 37°C, and alternating large and small strain sweeps (large strain: 100%, small strain: 1%) were used.

[0039] Figure 2 and Figure 3 This is the rheological test result of the injectable hydrogel obtained in Example 1. Figure 2 As shown in Figure 2, at 25°C, the viscosity of the hydrogel decreases significantly with the increase of shear rate, showing obvious shear thinning characteristics, indicating that the hydrogel is injectable. Figure 3 As shown in the figure, at 37°C, the storage modulus (G′) of the hydrogel was always greater than the loss modulus (G″) regardless of whether it was under large or small strain, and the modulus was recoverable, indicating that the gel had self-healing properties.

[0040] Example 4

[0041] Mouse bone marrow mesenchymal stem cells (BMSCs) were used to investigate the cytocompatibility of the hydrogel obtained in Example 1 using live / dead cell staining. Live cells, which exhibited green fluorescence, and dead cells, which exhibited red fluorescence, were observed under an inverted fluorescence microscope at 490 nm excitation light, and 545 nm excitation light, respectively. The experimental results were photographed and recorded.

[0042] Figure 4 The results of the cell compatibility test of the hydrogel obtained in Example 1 are shown in Figure 1. Green represents living cells and red represents dead cells. Figure 4 In (a), (b) and (c), a large number of living cells stained with Calcein-AM green can be seen, while only a few dead cells stained with red can be seen. Figure 4 As shown in (d), (e) and (f). Figure 4 (g), (h), and (i) are the superimposed results of live and dead cell staining images, which clearly show the live / dead cell staining. The above phenomenon shows that the injectable hydrogel of the present invention has good biocompatibility and is promising for application in bone tissue repair.

[0043] Example 5

[0044] The injectable hydrogel obtained in Example 1 was injected into the defective tissue of the rat skull, disinfected, and raised as usual. The rats were euthanized after four and eight weeks, respectively. The muscles and fascia of the rats were removed, and the skull was retained. After being soaked in 4% paraformaldehyde for 24 hours, it was transferred to new paraformaldehyde for fixation and placed in preparation for testing. Micro-CT was used to detect the repair of the rat skull defect. The test scanning parameters were: scanning energy intensity was 70KVp, 114μA, filter was 0.5Al, and CT value was calibrated with 1200mg HA / ccm.

[0045] Figure 5 This is a gross specimen obtained 4 weeks after the in vivo injection of the injectable hydrogel obtained in Example 1. Figure 6Micro-CT scanning and 3D reconstruction were used to examine the formation of new bone tissue 4 and 8 weeks after hydrogel implantation. A scalpel was first used to separate the skin fascia, exposing the skull. A defect model was then created in the skull. The hydrogel was injected into the defect site in rats, and the skin of the rat heads was then sutured and disinfected. The skulls of rats in the blank group remained untreated. CT scans revealed that 4 weeks after surgery, new bone tissue had formed around the hydrogel, primarily at both ends and in the surrounding area. 8 weeks after surgery, even more new bone tissue had formed around the hydrogel, demonstrating significant bone repair capabilities compared to the blank group.

[0046] The above-described embodiments merely express the implementation methods of the present invention, but should not be understood as limiting the scope of the patent 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, and these all fall within the scope of protection of the present invention.

Claims

1. An injectable high-strength hydrogel for bone tissue repair, characterized in that: The hydrogel comprises a linear thermosensitive polymer, a functional filler and a solvent; the mass ratio of the linear thermosensitive polymer to the solvent is 1:(1-9); the mass ratio of the functional filler to the linear thermosensitive polymer is 1:(2.33-19); wherein the linear thermosensitive polymer has a significant lower critical solution temperature (LCST) in aqueous solution, and its LCST is adjustable within the range of 10-70°C; the linear thermosensitive polymer comprises a hydrophilic unit and an unsaturated hydrophobic unit; the molar ratio of the hydrophilic unit to the functional unit is 1:(0.1-9); the hydrophilic unit is selected from acrylamide, hydroxyethyl acrylate, N-vinyl pyrrolidone and One or more of acrylic acid; the unsaturated hydrophobic unit is selected from one or more of alkyl acrylate, alkyl methacrylate, N-alkyl acrylamide and polyoxyethylene alkyl acrylate, and the polymer is prepared by solution polymerization; the functional filler is bioactive glass; the solvent is pure water and ethanol; the hydrogel absorbs water and swells at 25°C, the modulus decreases, and the injectability is good. After the temperature reaches 37°C after injection into the body, the polymer hydrogel with LCST characteristics has enhanced water excretion and increased modulus; the molecular weight distribution of the polymer in the gel is 2.1-10, and the molecular weight is 100,000-1,000,000; the content of the polymer in the gel is 10-50 wt%, and the content of the functional filler is 1-20 wt%.

2. Use of the hydrogel according to claim 1 in preparing materials in the field of bone tissue repair.

Images