Hydrogel for repairing soft and hard tissue damage under inflammation as well as preparation method and application of hydrogel

Abstract

The invention discloses hydrogel for repairing soft and hard tissue damage under inflammation as well as a preparation method and application of the hydrogel. The hydrogel is formed by crosslinking acrylic acid modified gelatin and mannose modified hyperbranched polyglycerol (hPG-Man); the ratio of the acrylic acid modified gelatin to the hPG-Man is (3-7): 1. The hydrogel disclosed by the invention can resist inflammation and promote osteogenesis in a double-effect manner, and has a remarkable effect of repairing jaw defects.

Description

Technical Field

[0001] The present invention specifically relates to a hydrogel for repairing soft and hard tissue injuries under inflammation, and a preparation method and use thereof. Background Art

[0002] The repair of soft and hard tissue injuries is of great significance in tissue injury and regenerative medicine, and the tissues involved include bone tissue, tendons, ligaments, skin and other connective tissues. The injuries of these tissues are usually caused by trauma, surgery, chronic diseases (such as diabetes, rheumatic diseases) or infections. Under a high-inflammatory state, the process of tissue repair is usually disturbed, manifested as problems such as poor tissue integration, limited regenerative ability, long-term inflammatory response and postoperative complications, which not only affect the functional recovery of patients, but may also lead to chronic pain and other health problems.

[0003] At present, the treatment means for repairing soft and hard tissue injuries cover tissue engineering strategies such as autologous tissue transplantation, allogeneic / xenogeneic transplantation and biomaterial scaffolds. However, the main focus of these methods is on promoting tissue regeneration in the injured area, and their effects on regulating the inflammatory microenvironment and maintaining immune homeostasis are relatively limited. A high-inflammatory state can lead to abnormal activation of the energy metabolism bypass of macrophages, increasing the production of ROS. At the same time, the intracellular antioxidant system is inhibited, thus reducing the ability to scavenge ROS. The accompanying inflammatory microenvironment can also prolong the local retention of M1 macrophages, significantly delay the expression of M2 macrophages, reduce the activity of osteoblasts, fibroblasts and tendon cells, while enhancing the effects of osteoclasts and matrix metalloproteinases, ultimately affecting the quality of soft and hard tissue repair. Therefore, how to precisely regulate the inflammatory immune microenvironment and achieve the repair of soft and hard tissue injuries under inflammation is an important research direction in the current field of regenerative medicine.

[0004] D-mannose (D-Man) is the C-2 isomer of glucose and is involved in the glycosylation of certain proteins. Research shows that mannose can inhibit the production of IL-1β by inhibiting glycolysis and blocking succinate-mediated HIF-1α activation, thereby inhibiting LPS-induced macrophage activation. In addition, mannose can not only enter mammalian cells through glucose transporters (GLUT), but also specifically bind to mannose receptors (MR) on the macrophage membrane, effectively inducing the transformation of macrophages into the anti-inflammatory M2 phenotype, regulating the secretion of cytokines and growth factors, and thus inhibiting chronic inflammatory responses.

[0005] However, clinical studies have shown that an increase in serum mannose levels is closely associated with insulin resistance, type 2 diabetes (T2D), cardiovascular disease (CVD), and the development of diabetic kidney disease. An increase in intracellular mannose metabolites (such as mannose-6-phosphate) can partially inhibit glycolysis, thereby impairing the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).

[0006] Therefore, constructing a drug containing a mannose structure that is not easily endocytosed by cells and can be applied to the treatment of inflammatory soft and hard tissue injury defects has great application prospects and is expected to promote the repair of soft and hard tissue injuries. Summary of the Invention

[0007] To achieve the above object, the present invention provides a hydrogel with anti-inflammatory and / or repair-promoting (taking osteogenic mineralization as an example) effects, which is crosslinked by acrylic acid-modified gelatin and hPG-Man;

[0008] The ratio of the acrylic acid-modified gelatin to hPG-Man is 3 to 7:1;

[0009] The acrylic acid-modified gelatin is formed by reacting gelatin with methacrylic anhydride in a solvent;

[0010] The hPG-Man is a bioactive hyperbranched polymer molecule with hPG as the main molecule and mannose as the functional group;

[0011] The structural formula of the hPG is:

[0012]

[0013] Further, the ratio of the acrylic acid-modified gelatin to hPG-Man is 5:1.

[0014] Further, the introduction rate of mannose in the hPG-Man is greater than 0%, preferably 10% 。

[0015] Further, the hPG-Man is prepared by the following method:

[0016] Step 1):

[0017]

[0018] The glycidol is polymerized into hPG in a solvent;

[0019] Step 2): Modify hPG

[0020] The obtained hPG in step 1) reacts with p-toluenesulfonyl chloride TsCl or methanesulfonyl chloride MsCl in pyridine to form intermediate 2. Intermediate 2 reacts with sodium azide NaN3 in DMF to form intermediate 3. Intermediate 3 reacts with isocyanatoethyl methacrylate under the action of a catalyst to form intermediate 4;

[0021] Step 3):

[0022]

[0023] The mannose reacts with acetic anhydride under the catalysis of iodine to form compound 6. Compound 6 reacts with 2-[2-(2-propynyloxy)ethoxy]ethanol in CH2Cl2 to form compound 7. Compound 7 reacts with CH3ONa in CH3OH to form compound 8;

[0024] Step 4): Prepare hPG-Man

[0025] Take the obtained intermediate 4 in step 2) and compound 8 obtained in step 3) and react them in a solvent to form hPG-Man.

[0026] Furthermore, the solvent in step 1) is the solution obtained by removing tert-butanol after the reaction of trimethylolpropane and potassium tert-butoxide;

[0027] The ratio of glycidol to trimethylolpropane and potassium tert-butoxide is 200 - 500 mmol:5 - 1 mmol:1 mmol, preferably 355 mmol:2.5 mmol:1 mmol;

[0028] The temperature of the reaction is 60 - 70 °C, and the time is more than 1 h;

[0029] The temperature during polymerization is 110 - 130 °C, preferably 120 °C.

[0030] Furthermore, the molar ratio of TsCl or MsCl to the hydroxyl group in hPG in step 2) is 0.01 - 1.2:1, preferably 0.1:1; the reaction conditions are in an argon atmosphere, the temperature is 0 - 4 °C, and the time is 10 - 20 h;

[0031] And / or: The molar ratio of the p-toluenesulfonyl group in intermediate 2 to NaN3 is 1:2 - 4, preferably 1:3, the reaction temperature is 70 - 90 °C, and the time is 2 - 4 days;

[0032] And / or: The molar ratio of isocyanatoethyl methacrylate, the catalyst and the hydroxyl group in intermediate 3 is 0.05 - 0.3:0.001 - 0.01:1, preferably 0.1:0.05:1; the catalyst is tin laurate.

[0033] Further, the mass-volume ratio of iodine, mannose, and acetic anhydride in step 3) is 0.2 - 0.4 g: 4 - 6 g: 40 - 60 mL, preferably 0.25 g: 5.0 g: 50 mL, and the reaction conditions are: stirring at room temperature for 20 - 60 minutes;

[0034] And / or: the mass ratio of compound 6 to 2-[2-(2-propynyloxy)ethoxy]ethanol is 3 - 5 g: 2 - 4 g, preferably 3.90 g: 2.16 g; each 50 ml of CH2Cl2 contains 4 - 5 g of catalyst, preferably 4.38 g of catalyst; the catalyst is BF3·OEt2; the reaction conditions are: stirring at room temperature for 12 - 36 h;

[0035] And / or: the mass-volume ratio of compound 7 to CH3ONa is 1 - 3 g: 160 - 170 mg, preferably 1.5 g: 167.4 mg, and the reaction conditions are stirring at room temperature for 1 - 3 h.

[0036] Further, the molar ratio of the azide group in compound 8 and intermediate 4 in step 4) is 1 - 2:1;

[0037] The solvent used is DMF;

[0038] Cu is dissolved in the DMF 2+ , sodium ascorbate, and N,N-diisopropylethylamine, where the molar ratio of Cu 2+ to sodium ascorbate, N,N-diisopropylethylamine, and the azide group is 0.08 - 0.12: 0.1 - 0.3: 1.3 - 1.9: 1.

[0039] Further, the acrylic acid-modified gelatin is obtained by dissolving gelatin in DPBS and reacting it with methacrylic anhydride;

[0040] The mass-volume ratio of gelatin to methacrylic anhydride is 5 g: 1 - 3 mL;

[0041] The reaction temperature is 40 - 60 °C and the time is 1 - 5 h.

[0042] The present invention also provides a method for preparing the aforementioned hydrogel, comprising the following steps:

[0043] Taking acrylic acid-modified gelatin, hPG-Man, and LAP according to the ratio, and crosslinking under ultraviolet light irradiation to obtain it;

[0044] The dosage of LAP accounts for 0.2 - 0.8% of the mixture, preferably 0.3%.

[0045] The present invention also provides the use of the aforementioned hydrogel in the preparation of a drug for repairing hard and soft tissue injuries under inflammation, where the hard and soft tissue injuries include bone defects; the bone defects include jaw bone defects.

[0046] The introduction rate described in the present invention refers to the ratio between the number of functional groups or specific structural units successfully introduced onto the polymer molecular chain and the theoretical maximum introduction amount.

[0047] In the present invention, through a series of chemical reactions, the metabolic small molecule D-mannose (D-Man) is materialized, and then gelatin methacrylate (GelMA) is bonded by molecular bonds to form a double-network hydrogel, namely Gel-hPG-Man hydrogel. Compared with the Gel-Man hydrogel without materialized D-Man and the Gel-hPG hydrogel during the materialization process of D-Man, this hydrogel has a more significant anti-inflammatory effect, showing a synergistic effect in anti-inflammation, and can be used for the treatment of inflammatory diseases.

[0048] It is determined through cell experiments that when the Gel-hPG-Man hydrogel of the present invention is implanted or locally injected into the bone injury area, the D-mannose end groups in the gel system bind to the mannose receptors on the macrophage cell membrane, regulating the activity of T cells and simultaneously inducing macrophage polarization, and expressing and secreting corresponding anti-inflammatory and repair-promoting factors, thereby promoting the efficient repair and regeneration of damaged bone under inflammatory pathological conditions. It also has a more significant effect in repairing bone defects accompanied by inflammation compared with other gel products, such as the Gel-hPG hydrogel formed by mixing hPG and GelMa, and the Gel-Man hydrogel formed by mixing D-Man and GelMa, and has practical application value for promotion.

[0049] Obviously, based on the above content of the present invention, according to the common general technical knowledge and conventional means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions or changes can be made.

[0050] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be understood as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention. Description of the Drawings

[0051] Figure 1 Expression of inflammation-related genes under different groups (qPCR experiment)

[0052] Figure 2 Alizarin red staining photos

[0053] Figure 3 Immunofluorescence staining of macrophages Detailed Description of the Invention

[0054] The raw materials, reagents, and equipment used in the specific implementation mode of the present invention are obtained through commercial purchase. The structural formula of the hyperbranched polyglycidol (hPG) prepared in the examples is as follows: Abbreviated as where R represents

[0055] Example 1. Preparation of the hydrogel of the present invention

[0056] 1. Synthesis of hPG homopolymer

[0057] Using trimethylolpropane as the initiator and glycidol as the monomer, anionic ring-opening polymerization was carried out in NMP solvent to prepare hyperbranched polyglycidol (hPG) by a one-step method. The specific synthesis route is as follows:

[0058]

[0059] The specific method is as follows:

[0060] ① Add trimethylolpropane (TMP) and potassium tert-butoxide (K-t-OBU) to a dry reaction vessel, set the temperature to 65 °C, and ensure that the reaction system is dry and oxygen-free. After magnetic stirring for 1 h, remove the by-product tert-butanol in the reaction system, raise the temperature to 120 °C, and use a micro-injection pump to drop pure glycidol at a rate of 0.75 mL / min. The ratio of glycidol, trimethylolpropane (TMP), and potassium tert-butoxide (K-t-OBU) is 355 mmol: 2.5 mmol: 1 mmol

[0061] ② After the addition of glycidol in step ① is completed, continue stirring for 3 h. Dissolve the reaction product with excessive anhydrous methanol, transfer the solution to 10 times the volume of acetone for precipitation, then dissolve the precipitated viscous oil with methanol, and remove methanol at 45 °C using a rotary evaporator to obtain a yellow transparent viscous liquid. Dialyze the liquid through a dialysis bag (cut-off molecular weight of 3500) in methanol to collect hPG.

[0062] 2. Group modification of hPG

[0063] The group modification of hPG is completed through the following synthetic schematic route, so that the hydroxyl groups with high reactivity in hPG are replaced by azide groups, enabling it to participate in subsequent reactions; and by introducing isocyanatoethyl methacrylate, a double bond is introduced to form a complete double-network gel system with the subsequent GelMa hydrogel, rather than a simple doping and fusion.

[0064]

[0065] The specific method for group modification of hPG is as follows:

[0066] ③ Dissolve the hPG obtained in step ② in a Schlenk flask containing pyridine. Cool the solution to 0 °C in an ice bath to obtain an hPG solution. Drop a pyridine solution containing p-toluenesulfonyl chloride (TsCl) into the cooled hPG solution. After stirring for 16 h under an argon atmosphere, ensure an anhydrous and anaerobic environment. Evaporate the solvent by rotary evaporation. Redissolve the residue in methanol and dialyze it in methanol for 24 h. The obtained brown honey-like product is hPG-OTs (intermediate 2).

[0067] ④ In a single-necked flask equipped with a reflux condenser, dissolve the hPG-OTs obtained in step ③ in a DMF solution by ultrasonic wave. After adding NaN3, heat the resulting suspension to 80 °C and react for 3 days. After cooling, filter the mixture to remove the excess NaN3. Concentrate the filtrate under vacuum conditions below 40 °C, and then dialyze the residue in water for 24 h for further purification to obtain a brown paste-like compound (intermediate 3). Then, isocyanatoethyl methacrylate is used as a double-bond precursor, and tin laurate is used as a catalyst. Stir with the brown paste-like compound in a container for 4 h to modify the double bond in the hPG molecular chain. After the reaction, use methanol as a solvent for dialysis and rotary evaporation to obtain a viscous liquid (intermediate 4).

[0068] The intermediate 4 obtained by the above method has some hydroxyl groups in hPG substituted by azide groups and isocyanatoethyl methacrylate is introduced. The introduction rate of the azide group varies with the ratio of hydroxyl groups in hPG to TsCl and NaN3. When the introduction rate of the azide group is 10%, the feeding reaction is carried out according to the following ratio:

[0069] Take the hPG obtained in step 1) (10.0 g, 135 mmol OH-groups) and dissolve it in 50 mL of pyridine. Cool the solution to 0 °C in an ice bath to obtain an hPG solution. Drop a pyridine (10 mL) solution containing p-toluenesulfonyl chloride (TsCl) (16.2 mmol, 0.12 eq.) into the cooled hPG solution. After stirring for 16 h under an argon atmosphere, ensure an anhydrous and anaerobic environment. Evaporate the solvent by rotary evaporation. Redissolve the residue in methanol and dialyze it in methanol for 24 h. The obtained brown honey-like product is hPG-OTs.

[0070] In a single-necked flask equipped with a reflux condenser, the obtained hPG-OTs was dissolved in 40 mL of DMF solution by ultrasound. After adding NaN3 in a molar ratio of 3 times that of the p-toluoyl chloride group -OTs, the mixture was heated to 80 °C and reacted for 3 days. After cooling, the mixture was filtered to remove the excess NaN3. The filtrate was concentrated under vacuum conditions below 40 °C, and then the residue was dialyzed in water for 24 h to obtain a brown paste-like compound. Then, according to the molar ratio of isocyanatoethyl methacrylate, catalyst, and hydroxyl groups in the brown paste-like compound of 0.1:0.05:1, it was placed in a container and stirred for 4 h. After the reaction, dialysis was carried out using methanol as a solvent, and rotary evaporation was performed to obtain a viscous liquid.

[0071] 3. Modification of D-Mannose Group

[0072] D-Mannose was modified according to the following synthetic route so that the hydroxyl groups in D-mannose were modified by the introduced 2-[2-(2-propynyloxy)ethoxy]ethanol:

[0073]

[0074] The specific method is as follows:

[0075] ⑤ Iodine (0.25 g, 0.035 equivalent) was added to a 50 mL acetic anhydride solution containing mannose (5.0 g, 1.0 equivalent). The solution was stirred at room temperature for 30 minutes, and then an excess of sodium thiosulfate solution was added to quench the reaction. The product was extracted three times with dichloromethane DCM. Saturated NaHCO3 solution was added to the organic layer until no bubbles were generated. The organic layer was separated, washed with saturated NaCl aqueous solution and concentrated to obtain the desired product, acetate-protected mannose (Compound 6).

[0076] ⑥ A 50 mL flask was taken, and acetate-protected mannose (3.90 g, 10 mmol) and 2-[2-(2-propynyloxy)ethoxy]ethanol (2.16 g, 15.0 mmol) were dissolved in dry CH2Cl2 (30 mL). Then, under a nitrogen atmosphere at 0 °C, the catalyst BF3·OEt2 (4.38 g, 25.0 mmol) was added to CH2Cl2 (20 mL) and then titrated into the above solution. The resulting mixture was stirred at room temperature for 24 h. The reaction mixture was diluted with saturated NaHCO3 solution, extracted with CH2Cl2, and the organic phase was washed with brine and water, and then dried over MgSO4. The crude oil obtained was purified by silica gel column chromatography to obtain a pale yellow oily substance (Compound 7).

[0077] ⑦Compound 7 (1.5 g, 3.1 mmol) was added with CH3ONa (167.4 mg, 3.1 mmol) in a dry MeOH (10 mL) solution. The reaction mixture was stirred at room temperature for 2 h, and neutral resin IR120 H+ was added to neutralize the reaction mixture to pH = 6. Then the resin was filtered off, and the filtrate was dried under vacuum to obtain a white solid (780 mg, compound 8).

[0078] 4. Synthesis of D-mannose-functionalized hPG - hPG-Man

[0079] Using copper-catalyzed azide-alkyne cycloaddition, the azide-modified hPG and the alkyne-modified Mannose were reacted through the following synthetic schematic route:

[0080]

[0081] The specific method is as follows:

[0082] Take the intermediate 4 obtained in step ④, compound 8 obtained in step ⑦, sodium ascorbate and N,N-diisopropylethylamine and mix them, then add them to a DMF solution containing Cu 2+ and react for 24 h. After dialysis, rotary evaporation is carried out to collect the viscous liquid, and hPG-Man is obtained.

[0083] hPG-Man is a hyperbranched polymer with hPG as the main molecule and mannose as the modified functional group. The introduction rate of mannose in the polymer varies with the ratio of azide groups to mannose in intermediate 4. When the introduction rate of mannose is 10%, the feeding reaction is carried out according to the following ratio:

[0084] Take the hPG with a 10% azide substitution rate obtained in step ④ (intermediate 4, 10.0 g, 13.5 mmol N3-groups), the alkyne-modified D-mannose obtained in step ⑦ (compound 8, 6 g, 20.25 mmol), sodium ascorbate and N,N-diisopropylethylamine are 0.518 g (0.2 eqv) and 2.7 g (1.6 eqv) respectively, and add them to 50 ml of a DMF solution containing 0.026 mol / L Cu 2+ (CuSO4·5H2O, 0.1 eqv) and react for 24 h. After dialysis, rotary evaporation is carried out to collect the viscous liquid, and hPG-Man is obtained.

[0085] 5. Synthesis of acrylic acid-modified gelatin (GelMa)

[0086] 5 g of gelatin was dissolved in 40 mL of DPBS and stirred at 60 °C. 1 - 3 mL of methacrylic anhydride was added dropwise thereto, and the mixture was stirred and reacted at 50 °C for 2 h. 100 mL of preheated PBS (40 °C) was added, and dialysis was carried out with a molecular weight cut-off of 7 kDa for 7 days at 40 °C. It was freeze-dried and stored at -20 °C for later use.

[0087] 6. Synthesis of hPG-Man functionalized GelMa (Gel-hPG-Man)

[0088] The hPG-Man obtained in step 4 and the GelMa obtained in step 5 with a mass ratio of 1:5 were combined, and 0.5% of lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) was added. Under ultraviolet light irradiation, the double bonds in the hydrogel were activated to form a double-network bioactive hydrogel Gel-hPG-Man.

[0089] The beneficial effects of the present invention are further illustrated by the following experimental examples:

[0090] Experimental Example 1 Study on the osteogenic and anti-inflammatory effects of the hydrogel of the present invention

[0091] I. Effects of hydrogels made of different components on osteogenic and anti-inflammatory effects

[0092] 1. Method

[0093] RAW264.7 cells were inoculated into cell culture dishes, and 10 mL of DMEM medium (containing 10% FBS, 100 U / mL penicillin, and 100 U / mL streptomycin) was added. The cells were cultured in a cell culture incubator at 37 °C, 5% CO2, and saturated humidity, and the cell culture medium was changed every other day. The cells grew adherently. The cell suspension was taken and added to the lower layer of the transwell well plate to grow. Under the action of different media (the Control group was normal medium, and other groups were all added with 200 ng / mL of lipopolysaccharide, LPS) and hydrogels, the cells were incubated for 24 h, and the inflammation-related genes of RAW cells were detected by qPCR. The specific detection steps were as follows: lysing the cells to obtain RNA, and detecting the gene expression after reverse transcription, that is, detecting the inflammatory factor TNF-α and the markers Arg-1 and CD206 of M2 macrophages (anti-inflammatory phenotype). The hydrogels used were as follows:

[0094] Hydrogel Gel-hPG-Man: The hydrogel Gel-hPG-Man with a mannose functional group introduction rate of 10% prepared according to Example 1;

[0095] Hydrogels Gel-hPG and Gel-Man: hPG and D-Man in the same amounts as those in the hydrogel Gel-hPG-Man with a mannose functional group introduction rate of 10% were respectively mixed with GelMa to form Gel-hPG and Gel-Man. Specifically, hPG obtained in step ② of Example 1 and GelMa obtained in step 5 with a mass ratio of 0.875:5 were stirred and mixed, and 0.5% LAP was added. Under ultraviolet light irradiation, the double bonds in GelMa were activated to form the double-network bioactive hydrogel Gel-hPG. D-Man with a mass ratio of 0.0286:5 and GelMa obtained in step 5 of Example 1 were stirred and mixed, and 0.5% LAP was added. Under ultraviolet light irradiation, the double bonds of GelMa were activated to form the double-network bioactive hydrogel Gel-Man.

[0096] 2. Results

[0097] TNF-α is tumor necrosis factor-α, which is closely related to inflammation. The higher its expression level, the stronger the inflammatory response; Arg-1 and CD206 are marker genes of macrophage M2 type, representing an anti-inflammatory phenotype. The higher the expression, the more significant the anti-inflammatory state; Results of qPCR experiment Figure 1 Show that: compared with the LPS group, there were no obvious changes in the expression levels of TNF-α, Arg-1, and CD206 in RAW264.7 cells in the LPS+Gel-hPG group, indicating that the hydrogel Gel-hPG made of hPG and GelMa has no anti-inflammatory effect; the expression level of TNF-α in RAW264.7 cells in the LPS+Gel-Man group decreased to a certain extent, the expression level of Arg-1 increased, and the expression of CD206 increased, but it was not as good as the LPS+Gel-hPG-Man group, indicating that the hydrogel Gel-Man made of D-Man and GelMa has a certain anti-inflammatory effect, but the effect is not as significant as Gel-hPG-Man; In contrast, the LPS+Gel-hPG-Man group significantly inhibited the expression of the inflammation-related factor TNF-α in RAW264.7 cells and promoted the expression of the anti-inflammatory factors Arg-1 and CD206, indicating that the hydrogel Gel-hPG-Man has a significant anti-inflammatory effect.

[0098] Comparing the anti-inflammatory effects of each group, it can be seen that hPG and GelMa in the hydrogel cannot endow it with anti-inflammatory function. However, when hPG is functionalized with D-Man, the hydrogel prepared with GelMa can produce a stronger anti-inflammatory effect than the hydrogel prepared with D-Man alone. Since osteogenic activities are inhibited under inflammatory conditions, the regulation of inflammation is crucial for bone repair. Meanwhile, M2 macrophages secrete osteoblast cytokines during the repair period to promote bone formation. Therefore, in theory, the hydrogel Gel-hPG-Man can promote bone repair through inflammation regulation.

[0099] II. Verification of the osteogenic and anti-inflammatory effects of the hydrogel of the present invention

[0100] 1. Method

[0101] 1.1 Test for osteogenic mineralization ability

[0102] Bone mesenchymal stem cells (BMSCs) were used for cell culture to detect the ability of the hydrogel to promote cell osteogenic mineralization. BMSC cells were seeded in a 6-well Transwell plate at a density of 2×10 5 cells per well. After culturing for one day, the GelMa hydrogel (Gel) prepared in step 5 of Example 1 or the Gel-hPG-Man hydrogel with a mannose functional group introduction rate of 10% prepared in step 6 was placed on the upper layer respectively. The lower layer was replaced with normal induction medium or induction medium containing simulated inflammation (containing lipopolysaccharide, LPS) for culture. The groups were divided into a blank gel control group (Gel), an LPS-stimulated group + Gel hydrogel group, and an LPS-stimulated group + Gel-hPG-Man hydrogel group. The induction medium was changed every 3 days, and the cells were cultured for 14 days. The culture medium in the wells was aspirated, and the cells were rinsed 3 times with PBS for 5 minutes each time; fixed with 4% paraformaldehyde at room temperature for 30 minutes, and an appropriate amount of alizarin red staining solution was added to each well and incubated at room temperature in the dark for 30 minutes; the staining solution was aspirated, and the cells were washed 2-3 times with double-distilled water to remove the unbound dye completely. Observation and photography were carried out under an inverted microscope.

[0103] 1.2 Test for anti-inflammatory effect

[0104] Add the RAW264.7 cell suspension to the transwell plate with coverslips, and add complete medium or high-glucose + LPS medium and hydrogel according to the groups. After culturing for 24 h, perform immunofluorescence staining. CD206 (1:5000, 26903-1-AP, Proteintech) and INOS antibody (1:5000, 53-3697-82, Thermo Fisher) are incubated overnight at 4 °C. Anti-rabbit antibody (594 nm, SA00013-4, Proteintech, against CD206) and goat secondary antibody (488 nm, Proteintech, against INOS) are treated at room temperature for 2 h. The cell nuclei are stained with DAPI. Sample imaging is performed using an LSM980 laser scanning confocal microscope. Among them, CD206 represents the marker of M2 macrophages (anti-inflammatory phenotype), and the stronger the expression, the better the anti-inflammatory effect. iNOS represents M1 macrophages (inflammatory phenotype), and the stronger the expression, the more severe the inflammation. The specific grouping and hydrogel use are the same as in "1.1 Osteogenic mineralization ability test".

[0105] 2. Results

[0106] 2.1 Results of osteogenic mineralization ability

[0107] The specific results are shown in Figure 2 . From Figure 2 It can be seen that alizarin red will form red calcium nodules with mineralized substances. After using lipopolysaccharide (LPS) to simulate the inflammatory environment, the osteogenic effect will be inhibited. However, the GelMA hydrogel modified with hPG-Man (Gel-hPG-Man) can not only reverse the inhibition of osteogenesis in its inflammatory environment, but also promote cell remineralization.

[0108] 2.2 Test results of anti-inflammatory effect

[0109] The specific results are shown in Figure 3 , from Figure 3 It can be seen that when macrophages are cultured, it can be seen that under the inflammatory environment, the inflammatory stress expression of iNOS increases, and after using the Gel-hPG-Man hydrogel, this situation can be effectively improved.

[0110] In summary, the Gel-hPG-Man hydrogel of the present invention promotes the efficient repair and regeneration of damaged bone under inflammatory pathological conditions by inducing macrophage polarization and expressing and secreting corresponding anti-inflammatory and repair-promoting factors. Cell experiments have proved that the gel system of the present invention has a more significant effect in repairing bone defects accompanied by inflammation compared with other gel products, such as Gel-hPG and Gel-Man hydrogels formed by mixing hPG and D-Man with GelMa respectively.

Claims

1. A hydrogel with anti-inflammatory and / or osteogenic mineralization effects, characterized in that: It is crosslinked by acrylic acid modified gelatin and hPG-Man; The ratio of the acrylic acid modified gelatin to hPG-Man is 3-7:1; The acrylic acid modified gelatin is formed by reacting gelatin with methacrylic anhydride in a solvent; The hPG-Man is a functionalized hyperbranched polymer with hPG as the main molecule and mannose as the functional group; The structural formula of the hPG is:

2. The hydrogel according to claim 1, characterized in that: The ratio of the acrylic acid modified gelatin to hPG-Man is 5:

1.

3. The hydrogel according to claim 2, wherein: The hPG-Man is prepared by the following method: Step 1): The glycidol is polymerized into hPG in a solvent; Step 2): Modify hPG Take the hPG obtained in Step 1) and react it with p-toluenesulfonyl chloride TsCl or methanesulfonyl chloride MsCl in pyridine to form intermediate 2. Intermediate 2 reacts with sodium azide NaN3 in DMF to form intermediate 3. Intermediate 3 reacts with isocyanatoethyl methacrylate under the action of a catalyst to form intermediate 4; Step 3): The mannose reacts with acetic anhydride under the catalysis of iodine to form compound 6. Compound 6 reacts with 2-[2-(2-propynyloxy)ethoxy]ethanol in CH2Cl2 to form compound 7. Compound 7 reacts with CH3ONa in CH3OH to form compound 8; Step 4): Prepare hPG-Man Take the intermediate 4 obtained in Step 2) and react it with compound 8 obtained in Step 3) in a solvent to form hPG-Man.

4. The hydrogel according to claim 3, characterized in that: The solvent described in Step 1) is the solution obtained by reacting trimethylolpropane and potassium tert-butoxide and removing tert-butanol; The ratio of the glycidol to trimethylolpropane and potassium tert-butoxide is 200-500 mmol:5-1 mmol:1 mmol, preferably 355 mmol:2.5 mmol:1 mmol; The temperature of the reaction is 60-70 °C and the time is more than 1 h; The temperature during polymerization is 110-130 °C, preferably 120 °C.

5. The hydrogel according to claim 3, characterized in that: The molar ratio of TsCl or MsCl to the hydroxyl group in hPG in Step 2) is 0.01-1.2:1, preferably 0.1:1; the reaction conditions are in an argon atmosphere, the temperature is 0-4 °C, and the time is 10-20 h; And / or: The molar ratio of the p-toluenesulfonyl group to NaN3 in intermediate 2 is 1:2-4, preferably 1:3, the reaction temperature is 70-90 °C, and the time is 2-4 days; And / or: The molar ratio of isocyanatoethyl methacrylate, the catalyst and the hydroxyl group in intermediate 3 is 0.05-0.3:0.001-0.01:1, preferably 0.1:0.05:1; the catalyst is preferably tin laurate.

6. The hydrogel according to claim 3, characterized in that: The mass-volume ratio of iodine, mannose and acetic anhydride in Step 3) is 0.2-0.4 g:4-6 g:40-60 mL, preferably 0.25 g:5.0 g:50 mL, and the reaction conditions are; stir at room temperature for 20-60 minutes; And / or: The mass ratio of the compound 6 to 2-[2-(2-propynyloxy)ethoxy]ethanol is 3-5 g: 2-4 g, preferably 3.90 g: 2.16 g; Each 50 ml of the CH2Cl2 contains 4-5 g of the catalyst, preferably 4.38 g of the catalyst; The catalyst is BF3·OEt2; The reaction conditions are: stirring at room temperature for 12-36 h; And / or: The mass-volume ratio of the compound 7 to CH3ONa is 1-3 g: 160-170 mg, preferably 1.5 g: 167.4 mg, and the reaction conditions are stirring at room temperature for 1-3 h.

7. The hydrogel according to claim 3, wherein: In step 4), the molar ratio of the azide group in the compound 8 to the intermediate 4 is 1-2:1; The solvent used is DMF; Cu is dissolved in the DMF 2+ , sodium ascorbate and N,N-diisopropylethylamine, wherein the molar ratio of Cu 2+ , sodium ascorbate, N,N-diisopropylethylamine to the azide group is 0.08 to 0.12:0.1 to 0.3:1.3 to 1.9:

1.

8. The hydrogel according to claim 1, characterized in that: The acrylic acid-modified gelatin is obtained by dissolving gelatin in DPBS and reacting with methacrylic anhydride; The mass-volume ratio of the gelatin to the methacrylic anhydride is 5 g: 1-3 mL; The reaction temperature is 40-60 °C and the time is 1-5 h.

9. A method for preparing the hydrogel according to any one of claims 1 to 8, characterized in that: Comprising the following steps: Taking the acrylic acid-modified gelatin, hPG-Man and a photoinitiator in proportion, mixing, and crosslinking under ultraviolet light irradiation to obtain the product; The dosage of the photoinitiator accounts for 0.2-0.8% of the mixture, preferably 0.3%.

10. Use of the hydrogel according to any one of claims 1 to 8 in the preparation of a medicament for repairing hard and soft tissue injuries under inflammation, characterized in that: The soft and hard tissue defects include bone defects; The bone defects include jaw bone defects.

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