Inorganic bone adhesive modified by mussel mucin and preparation method thereof

Abstract

The present invention discloses an inorganic bone adhesive modified with mussel mucin and a preparation method thereof, belonging to the technical field of medical adhesive materials. The inorganic bone adhesive comprises the following raw materials in parts by weight: 20 to 40 parts by weight of an inorganic phosphate compound, 30 to 60 parts by weight of a composite solidifying liquid, and 5 to 10 parts by weight of a buffer solution. The inorganic phosphate compound and the buffer solution are mixed and stirred until the inorganic phosphate compound is completely dissolved, and then the composite solidifying liquid is added and mixed and stirred to form the inorganic bone adhesive. The present invention utilizes the high adhesiveness and water resistance of mussel mucin and combines it with alkaline polysaccharides to modify the phosphate-based bone adhesive, thereby improving the bonding strength and water resistance of the phosphate-based bone adhesive.

Description

Technical Field

[0001] The invention belongs to the technical field of medical adhesive materials, and particularly relates to an inorganic bone adhesive modified by mussel mucin and a preparation method thereof. Background Art

[0002] The traditional method of treating comminuted fractures mainly uses internal fixators such as screws, steel plates and steel needles to fix the bone fragments and allow them to heal and recover on their own. However, there are many disadvantages of traditional treatment. The most obvious one is that these internal fixators need to be removed through surgery after the bone fragments have recovered and healed, which can easily cause secondary damage to the patient and pose a risk of infection. Based on this problem, a new type of medical adhesive material "bone adhesive" has come into the researchers' field of vision. Currently, commercial bone adhesives mainly include acrylate and cyanoacrylate types of bone adhesives, which have the advantages of high bonding strength, good mechanical properties and easy plasticity. However, medical bone adhesives need to meet the requirements of high biocompatibility and good biosafety, and the poor biocompatibility of acrylate and cyanoacrylate types of bone adhesives may cause inflammatory reactions at the fracture site, thereby causing bacterial infection. Therefore, it is of great significance to design a bone adhesive with good biocompatibility.

[0003] Bone cements with excellent biocompatibility include bio-based hydrogel bone cements and inorganic phosphate-based bone cements such as calcium phosphate or magnesium phosphate. Inorganic phosphate-based bone cements are very safe due to their excellent biocompatibility and non-toxicity to cells. However, inorganic phosphate-based bone cements suffer from drawbacks such as poor bond strength and insufficient water resistance. Therefore, research to improve the bond strength and water resistance of inorganic phosphate-based bone cements is of great significance.

[0004] Patent CN114917397B discloses a solid-phase bone adhesive composition and bone adhesive. This invention introduces carboxymethylated polysaccharides, allowing the oxygen atoms in the carboxylic acid structure of the carboxymethylated polysaccharide to bind to the dimethyl calcium ions in tetracalcium phosphate through metal coordination via lone electrons, forming a network structure and thereby improving the adhesive's bond strength. Patent CN105731846B discloses magnesium phosphate bone cement, which enhances the adhesive's bond strength by adding adhesion-promoting materials such as beeswax and hyaluronic acid.

[0005] Mussel mucin is a water-resistant, high-bonding protein secreted by mussel glands. It exhibits excellent biocompatibility, does not induce an immune response in the human body, and has an isoelectric point of 9.5. Mussel mucin's excellent waterproof adhesion properties are primarily due to the presence of DOPA in the protein. DOPA is a post-translational modification of tyrosine produced by tyrosine hydroxylase. DOPA can be oxidized to form DOPAQUINONE. Both DOPA and DOPAQUINONE can form bonded or non-bonded crosslinks on organic, inorganic, and metal surfaces.

[0006] Therefore, it is of great significance to utilize the high adhesiveness and water resistance of mussel mucin to modify phosphate-based bone adhesives, thereby improving the bonding strength and water resistance of phosphate-based bone adhesives. Summary of the Invention

[0007] In view of the shortcomings of the prior art, the present invention prepares a composite curing liquid by mixing modified mussel mucin Mfp3-5 with modified chitosan. The surface of the modified mussel mucin Mfp3-5 carries a large amount of positive charge. On the one hand, it is cross-linked with the negative charge on the surface of the modified chitosan through electrostatic adsorption to form a network structure. On the other hand, the aldehyde group on the modified chitosan and the imino group (-NH-) on the modified mussel mucin Mfp3-5 can be covalently cross-linked to form a -C=N- network structure, which further improves the bonding strength of the composite curing liquid. The composite curing liquid is then mixed with an inorganic phosphate-based bone adhesive material (such as calcium phosphate and magnesium phosphate). Through the carboxylic acid group structure on the modified chitosan, it is cross-linked with the metal ion calcium or metal ion magnesium of the inorganic phosphate-based compound through an anionic and cationic bond to form a network structure. Through the multi-crosslinked network structure, the bonding strength and water resistance of the inorganic phosphate-based bone adhesive are synergistically improved to solve the problems raised in the background technology. Specifically, the technical solution of the present invention includes the following contents:

[0008] An inorganic bone adhesive modified by mussel mucin, comprising the following raw materials in parts by weight:

[0009] 20 to 40 parts by weight of an inorganic phosphate compound, 30 to 60 parts by weight of a composite curing liquid, and 20 to 40 parts by weight of a buffer solution;

[0010] The inorganic phosphate compound includes calcium phosphate, hydroxyapatite or magnesium phosphate;

[0011] The composite solidifying liquid is obtained by mixing modified mussel mucin Mfp3-5 and modified polysaccharide in a mass ratio of 1 to 1, adding ethanol and grinding.

[0012] The buffer solution is prepared by compounding citric acid and sodium citrate in a mass ratio of 1:1.

[0013] Furthermore, the preparation method of the modified mussel mucin Mfp3-5 comprises the following steps:

[0014] The cDNA gene of mussel mucin Mfp3-5 was amplified by PCR and introduced into the vector pET-28a, and then transformed into Escherichia coli DH5α competent cells for induction culture to obtain a bacterial liquid containing recombinant mussel mucin Mfp3-5;

[0015] The bacterial solution was lysed and the precipitate was collected by centrifugation. The precipitate was dissolved in buffer A and then centrifuged to collect the supernatant. The supernatant was eluted with buffer B and buffer C, and then dialyzed with 5% acetic acid to obtain the recombinant mussel mucin Mfp3-5.

[0016] The recombinant mussel mucin Mfp3-5 is treated with 0.1 mol / L phosphate buffer at 25° C. for 6 hours to obtain modified mussel mucin Mfp3-5.

[0017] Furthermore, the nucleotide sequence of the forward primer used in the PCR amplification is 5'-CGCGGATCCGACGACGACGACAAGGGTTATGGTTATTATCCAGG-3', and the nucleotide sequence of the reverse primer used in the PCR amplification is 5'-CCGCTCGAGCTAATACAGATAG-3'.

[0018] Furthermore, the inducer for the induction culture is 0.5 mmol / L IPTG.

[0019] Furthermore, the buffer A consists of 10 mmol / L imidazole, 8 mol / L urea, 0.1 mol / L sodium phosphate and 10 mmol / L Tris-HCl, and the pH of the buffer A is 7.9;

[0020] The buffer solution B is composed of 30 mmol / L imidazole, 8 mol / L urea, 0.1 mol / L sodium phosphate and 10 mmol / L Tris-HCl, and the pH of the buffer solution B is 6.3;

[0021] The buffer C consists of 0.3 mol / L imidazole, 8 mol / L urea, 0.1 mol / L sodium phosphate and 10 mmol / L Tris-HCl, and the pH of the buffer C is 4.5.

[0022] Furthermore, the recombinant mussel mucin Mfp3-5 structure is an inclusion body structure.

[0023] Furthermore, the 0.1 mol / L phosphate buffer contains 50 μg / mL tyrosinase and 25 mmol / L ascorbic acid, and the pH of the 0.1 mol / L phosphate buffer is 7.0.

[0024] Furthermore, the preparation method of the modified polysaccharide comprises the following steps:

[0025] After the alkaline polysaccharide is dissolved in acetic acid in equal parts by weight, 0.2 to 0.5 times the weight of the alkaline polysaccharide is added with sodium periodate, mixed, and reacted at 20 to 30° C. for 3 to 5 hours. After the reaction is completed, deionized water is added and mixed and stirred, and a precipitate is obtained by centrifugation. The precipitate is vacuum dried to obtain the modified polysaccharide.

[0026] Furthermore, the alkaline polysaccharide includes chitosan, and the surface of the alkaline polysaccharide chitosan carries a large amount of negative charge. The negative charge effect on chitosan and the positive charge effect on the surface of the modified mussel mucin Mfp3-5 form electrostatic adsorption, thereby promoting the formation of a cross-linked network structure and improving the bonding strength of the composite curing liquid prepared subsequently.

[0027] A method for preparing an inorganic bone adhesive modified by mussel mucin, the method comprising the following steps:

[0028] The inorganic phosphate compound and the buffer solution are mixed and stirred until the inorganic phosphate compound is completely dissolved, and then the composite solidifying liquid is added and mixed and stirred to form the inorganic bone adhesive.

[0029] Compared with the prior art, the beneficial effects of the present invention include the following steps:

[0030] (1) The yield of mussel mucin obtained by natural extraction is extremely low (about 1 mg can only be extracted from 10,000 living mussels), and the price is expensive. The present invention uses gene recombination technology and the properties of protein inclusion bodies (insoluble and easy to separate after centrifugation) to design specific forward and reverse primers based on the cDNA structure of mussel mucin Mfp3-5. Then, after PCR amplification and synthesis, the primers follow the vector into the recipient cells and are treated with inducers to obtain a large amount of bacterial liquid containing recombinant mussel mucin Mfp3-5. At this time, after the bacterial liquid is lysed, the dissolved protein appears in the form of inclusion bodies, which is conducive to separation; the DOPA content in the recombinant mussel mucin Mfp3-5 obtained by recombinant technology is low (0.25ng / μg), it is difficult to effectively exert the adhesion effect. Therefore, the present invention uses a phosphate buffer system composed of tyrosinase and ascorbic acid to modify the recombinant mussel mucin Mfp3-5, so that the modified mussel mucin Mfp3-5 has a high content of DOPA (3.05ng / μg). On the one hand, the yield of the recombinant mussel mucin Mfp3-5 is increased by genetic recombination technology. On the other hand, by modifying the recombinant mussel mucin Mfp3-5, the DOPA content is effectively improved, which facilitates the increase in the number of cross-linking systems formed subsequently with the modified chitosan, so that the prepared composite solidifying liquid has good adhesion performance and water resistance.

[0031] (2) The adhesive formed by hydrogen bonding is susceptible to competitive bonding with water molecules, thereby weakening its water resistance. The present invention utilizes the electrostatic adsorption between the large amount of negative charges on the surface of the alkaline polysaccharide chitosan and the positive charges on the modified mussel mucin Mfp3-5 to form a cross-linked network system. On the other hand, by oxidative modification of chitosan, the chitosan obtains aldehyde and carboxyl structures. The aldehyde groups and the imino groups (-NH-) on the modified mussel mucin Mfp3-5 are covalently cross-linked to form a -C=N- network structure. The carboxyl structure and the metal ions calcium or magnesium on the inorganic phosphate compound are cross-linked by anionic and cationic bonds to form a network structure. Through the multiple cross-linked network structure, the bonding strength and water resistance of the inorganic phosphate bone adhesive are synergistically improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the induced expression of the recombinant mussel mucin Mfp3-5 in Preparation Example 1 of the present invention at different times.

[0033] Figure 2 This is the Tris-Tris ne-SDS-PAGE detection of the purified product of the recombinant mussel mucin Mfp3-5 in Preparation Example 1 of the present invention.

[0034] Figure 3This is the HPLC separation and purification of the recombinant mussel mucin Mfp3-5 in Preparation Example 1 of the present invention.

[0035] Figure 4 This is the molecular weight analysis and identification of the recombinant mussel mucin Mfp3-5 in Preparation Example 1 of the present invention.

[0036] Figure 5 This is the C-terminal sequence analysis and identification of the recombinant mussel mucin Mfp3-5 in Preparation Example 1 of the present invention.

[0037] Figure 6 This is the DOPA content determination of the modified mussel mucin Mfp3-5 in Preparation Example 1 of the present invention. In the figure, "1" represents the DOPA content of natural mussel mucin, "2" represents the DOPA content of recombinant mussel mucin Mfp3-5, and "3" represents the DOPA content of the modified mussel mucin Mfp3-5 of the present invention.

[0038] Figure 7 This is a test of the protein adsorption capacity in Preparation Example 1 of the present invention. In the figure, "1" represents the negative control adsorption capacity of BSA, "2" represents the adsorption capacity of the recombinant mussel mucin Mfp3-5, "3" represents the adsorption capacity of the modified mussel mucin Mfp3-5 of the present invention, and "4" represents the adsorption capacity of the natural mussel mucin.

[0039] Figure 8 This is the pixel density analysis of the protein staining spots in Preparation Example 1 of the present invention. In the figure: "1" represents the pixel density analysis of the negative control staining spots of BSA, "2" represents the pixel density analysis of the staining spots of the recombinant mussel mucin Mfp3-5, "3" represents the pixel density analysis of the staining spots of the modified mussel mucin Mfp3-5 of the present invention, and "4" represents the pixel density analysis of the staining spots of the natural mussel mucin.

[0040] Figure 9 This is the quartz crystal microbalance (QCM) test in Preparation Example 1 of the present invention. In the figure, "1" represents the negative control QCM test result of BSA, "2" represents the QCM test result of the recombinant mussel mucin Mfp3-5, "3" represents the QCM test result of the modified mussel mucin Mfp3-5 of the present invention, and "4" represents the QCM test result of the natural mussel mucin. DETAILED DESCRIPTION

[0041] The following will clearly and completely describe the technical solutions of the present invention through the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making any creative efforts shall fall within the scope of protection of the present invention.

[0042] Unless otherwise specified, the raw materials and reagents used in the present invention are commercially available or can be prepared by known methods.

[0043] Preparation Example 1:

[0044] The preparation method of modified mussel mucin Mfp3-5 specifically includes the following steps:

[0045] (1) Construction of recombinant expression vector of mussel mucin Mfp3-5

[0046] Forward and reverse primers were designed based on the mature peptide sequence of the mussel mucin Mfp3-5 cDNA gene (GenBank database number: GU321204.1). The forward primer Mfp3-5-F had a protective base and an enzyme cleavage site added to the 5' end of the sequence; the reverse primer Mfp3-5-R had a protective base and an enzyme cleavage site added to the 5' end of the sequence; the nucleotide sequence of the forward primer Mfp3-5-F was: 5'-CGCGGATCCGACGACGACGACAAGGGTTATGGTTATTATCCAGG-3'; the nucleotide sequence of the reverse primer Mfp3-5-R was: 5'-CCGCTCGAGCTAATACAGATAG-3';

[0047] The PCR amplification reaction cycle conditions were as follows: denaturation at 94°C for 30 s → annealing at 52°C for 30 s → extension at 72°C for 30 s, cycled 30 times. The amplified product was purified using a PCR product purification kit. The purified product and the expression vector pET-28a were cut with endonucleases, ligated with T4-DNA ligase, and transformed into Escherichia coli DH5α competent cells. The cells were plated on LB plates containing resistance screening and cultured overnight at 37°C.

[0048] (2) Expression and purification of recombinant mussel mucin Mfp3-5:

[0049] The identified recombinant pET-28a / Mfp3-5 monoclonal colonies were picked and cultured overnight in LB medium. The bacterial solution was expanded at a ratio of 1:1000. When the bacterial solution A value was 0.5-0.7, the inducer IPTG (0.5 mmol / L) was added for induction. After 4 hours of induction, the bacterial solution was collected and centrifuged at 5000×g for 20 minutes, washed with pre-cooled PBS, resuspended in bacterial lysis buffer, broken by ultrasonication in an ice bath, and the inclusion body structure protein was collected by centrifugation; the inclusion body structure protein was solubilized with buffer A (composed of 10 mmol / L imidazole, 8 mol / L urea, 0.1 mol / L sodium phosphate and 10 mmol / L Tris-HCl, pH adjusted to 7.9), and the supernatant was collected by centrifugation after ice bath ultrasonication. After 0.45 μm microfiltration, it was added to a nickel column equilibrated with buffer A and washed with buffer B (composed of 30 mmol / L imidazole, 8 mol / L urea, 0.1 mol / L sodium phosphate and 10 mmol / L Tris-HCl, pH adjusted to 7.9). The column was washed with 1% urea, 0.1 mol / L sodium phosphate and 10 mmol / L Tris-HCl, pH adjusted to 6.3) and the target protein was eluted with buffer C (composed of 0.3 mol / L imidazole, 8 mol / L urea, 0.1 mol / L sodium phosphate and 10 mmol / L Tris-HCl, pH adjusted to 4.5), and dialyzed against 5% acetic acid at 4°C overnight; reversed-phase high-performance liquid separation was performed using a semi-preparative Waters 600E high-pressure chromatograph and an analytical C18 column (4.6 mm × 250 mm) for reversed-phase chromatography; the detector was a Waters 2487 UV detector; the mobile phases were water (solution A) containing 0.1% trifluoroacetic acid (TFA) and acetonitrile (solution B), respectively, with an elution gradient of 5% to 60% solution B over 40 min; the main elution peaks were collected, freeze-dried, and then subjected to Tris elution. s-Tr ici ne-SDS-PAGE, Western blot analysis, and mass spectrometry identification;

[0050] The bacterial suspension at different time periods after induction was subjected to Tris-Tris ine-SDS-PAGE detection. The expression level of recombinant mussel mucin Mfp3-5 reached the highest peak 4 hours after induction. Figure 1 After 4 hours of induction, the bacterial solution was ultrasonically disrupted, and the supernatant and precipitate were subjected to Tris-Tris ne-SDS-PAGE detection. The results were as follows: Figure 2 , Figure 2The results showed that the recombinant mussel mucin Mfp3-5 was mainly expressed in the form of inclusion bodies. The cells were treated with a lysis solution containing 8 mol / L urea, the inclusion body proteins were collected, and after redissolution with 8 mol / L urea, the samples were loaded onto a nickel column for affinity chromatography. The target protein was collected and further loaded onto a C18 reverse phase chromatography column for high performance liquid phase separation. After high performance liquid phase separation, the main peak data were collected. Figure 3 , and protein identification was performed after freeze-drying.

[0051] (3) Identification of recombinant mussel Mfp3-5 protein:

[0052] Mass spectrometry analysis The precise molecular weight of the recombinant protein was analyzed and identified using a matrix-assisted laser desorption time-of-flight (MALD I-TOF) mass spectrometer (Voyager DETM STR, AB Company, USA). The results are shown in Figure 4 , Figure 4 The results showed that the molecular weight of recombinant mussel mucin Mfp3-5 was 9.18 kD, which was only 0.03 kD different from the theoretical molecular weight (9.15 kD). The partial amino acid sequence of recombinant mussel mucin Mfp3-5 was further analyzed by 4700 tandem time-of-flight mass spectrometer (4700 Proteomics Analyzer TOF / TOFTM, Applied Biosystems, USA). The fragment sequence of recombinant mussel mucin Mfp3-5 after trypsin digestion was analyzed. The results are shown in the figure. Figure 5 , Figure 5 The results showed that the amino acid sequence of its C-terminal fragment (m / z 1597.67) was completely consistent with the theoretical C-terminal sequence of recombinant mussel mucin Mfp3-5 (GYLYLEHHHHHH); Figure 4 and Figure 5 The results showed that the recombinant mussel mucin Mfp3-5 was successfully expressed in Escherichia coli with high purity.

[0053] (4) Tyrosinase catalysis of recombinant mussel mucin Mfp3-5 and determination of DOPA content in modified mussel mucin Mfp3-5:

[0054] After purification, the recombinant mussel mucin Mfp3-5 was dissolved in 0.1 mol / L phosphate buffer (containing 50 μg / mL tyrosinase and 25 mmol / L ascorbic acid, pH adjusted to 7.0) and modified at 25°C in the dark for 6 h. The modified sample solution was loaded onto a high-pressure molecular sieve column (Ultrahydrogel™ 500, 7.8 mm × 300 mm, Waters, USA) for separation and eluted with 5% acetic acid. The modified mussel mucin Mfp3-5 was collected and freeze-dried for DOPA content analysis and adhesion function test. The test results are shown in Figure 2. Figure 6 , Figure 6 The results showed that the DOPA content of the modified mussel mucin Mfp3-5 after modification was 3.05 ng / μg, which was much higher than the DOPA content of 0.25 ng / μg before modification; while the DOPA content of the natural mussel mucin Mfp3-5 protein was 4.32 ng / μg; the above results showed that after tyrosinase-catalyzed modification, part of the tyrosine in the recombinant mussel mucin Mfp3-5 protein was successfully oxidized to DOPA, thereby greatly increasing the DOPA content.

[0055] (5) Adhesion function test of modified mussel mucin Mfp3-5 (glass coating test and quartz crystal microbalance method were used to detect the adhesion function of modified mussel mucin Mfp3-5):

[0056] Bovine serum albumin (BSA) was used as a negative control, and mussel mucin Mfp3-5 was used as a positive control. The sample concentration was 0.4 mg / mL. 10 μL of the sample was added to the cleaned glass surface, incubated at 25°C for 12 hours, rinsed with deionized water for 2 hours, and dried before staining with Coomassie Brilliant Blue. The stained images were acquired with a scanner, and the pixel density of the stained spots was analyzed using PDQUest software. The adsorption capacity of the sample protein on the glass was determined based on the pixel density value.

[0057] A quartz crystal microbalance (QCM) was used to measure the adsorption capacity of the modified mussel mucin Mfp3-5 on a gold surface. The modified mussel mucin Mfp3-5 was dissolved in 5% acetic acid at a concentration of 0.1 mg / mL. An AT-cut quartz crystal with a diameter of 12.5 mm and a resonant fundamental frequency of 9 MHz was used. Gold electrodes on both sides of the crystal had a diameter of 6 mm. The quartz crystal was connected to a research quartz crystal microbalance (RQCM) (Maxtek, USA) via a thin wire. The amount of sample protein adsorbed on the gold surface was determined by monitoring changes in the crystal's resonant frequency.

[0058] Glass coating experiment Figure 7 The results showed that the unmodified recombinant mussel mucin Mfp3-5 had a certain adsorption capacity, while the modified mussel mucin Mfp3-5 had a stronger adsorption capacity on glass; PDQest software analysis see Figure 8 The results showed that the modified mussel mucin Mfp3-5 was adsorbed on glass and stained, and its spot pixel density increased by 2 times compared with that before modification. The modified mussel mucin Mfp3-5 has a strong adsorption capacity.

[0059] The QCM experiment shows Figure 9 The results showed that the frequency change value brought about by the deposition of recombinant mussel mucin Mfp3-5 on the gold surface after tyrosinase catalysis was significantly improved compared with before modification. Since the frequency change of the quartz chip is positively correlated with the amount of protein adsorbed on its surface, the result that the modified mussel mucin Mfp3-5 obtained after tyrosinase catalysis has stronger adsorption capacity is consistent with the results of the glass coating experiment.

[0060] Preparation Example 2:

[0061] The specific process of preparation of modified polysaccharide is as follows:

[0062] Weigh 100g of chitosan and 100g of acetic acid solution and mix and dissolve them. After they are completely dissolved, add 20g of sodium periodate and mix them. Then place the entire reaction system in a water bath environment, control the temperature at 20°C, and react for 3 hours. Then, add excess deionized water and mix and stir. After centrifugation at a speed of 1000r / min for 3 minutes, remove the supernatant, retain the bottom precipitate, and place the precipitate in a vacuum drying oven and dry it at a low temperature of 60°C for 10 to 15 hours to obtain a modified polysaccharide.

[0063] Preparation Example 3:

[0064] The specific process of preparation of modified polysaccharide is as follows:

[0065] Weigh 100g of chitosan and 100g of acetic acid solution and mix and dissolve them. After they are completely dissolved, add 30g of sodium periodate and mix them. Then place the entire reaction system in a water bath environment, control the temperature to 25°C, and time the reaction for 3 hours. Then, add excess deionized water and mix and stir. After centrifugation at a speed of 1000r / min for 5 minutes, remove the supernatant, retain the bottom precipitate, and place the precipitate in a vacuum drying oven and dry it at a low temperature of 60°C for 10 to 15 hours to obtain a modified polysaccharide.

[0066] Preparation Example 4:

[0067] The specific process of preparation of modified polysaccharide is as follows:

[0068] Weigh 100g of chitosan and 100g of acetic acid solution and mix and dissolve them. After they are completely dissolved, add 40g of sodium periodate and mix them. Then place the entire reaction system in a water bath environment, control the temperature to 25°C, and time the reaction for 4 hours. Then, add excess deionized water and mix and stir. After centrifugation at a speed of 1000r / min for 5 minutes, remove the supernatant, retain the bottom precipitate, and place the precipitate in a vacuum drying oven and dry it at a low temperature of 60°C for 10 to 15 hours to obtain a modified polysaccharide.

[0069] Preparation Example 5:

[0070] The specific process of preparation of modified polysaccharide is as follows:

[0071] Weigh 100g of chitosan and 100g of acetic acid solution and mix and dissolve them. After they are completely dissolved, add 40g of sodium periodate and mix them. Then place the entire reaction system in a water bath environment, control the temperature to 30°C, and react for 5 hours. Then, add excess deionized water and mix and stir. After centrifugation at a speed of 1200r / min for 5 minutes, remove the supernatant, retain the bottom precipitate, and place the precipitate in a vacuum drying oven and dry it at a low temperature of 60°C for 10 to 15 hours to obtain a modified polysaccharide.

[0072] Example 1:

[0073] A method for preparing an inorganic bone adhesive modified by mussel mucin, specifically comprising the following steps:

[0074] 20 g of the modified mussel mucin Mfp3-5 prepared in Preparation Example 1 and 20 g of the modified polysaccharide prepared in Preparation Example 2 were mixed, and 40 g of 0.9% physiological saline was added and stirred to obtain a composite solidified liquid;

[0075] Weigh 20 g of calcium phosphate and 20 g of buffer solution (obtained by mixing 10 ml of citric acid and 10 ml of sodium citrate) and mix them. After the calcium phosphate is completely dissolved, weigh 30 g of the composite curing liquid prepared above and mix them with the calcium phosphate and buffer solution for 5 minutes to obtain an inorganic bone adhesive.

[0076] Example 2:

[0077] A method for preparing an inorganic bone adhesive modified by mussel mucin, specifically comprising the following steps:

[0078] 20 g of the modified mussel mucin Mfp3-5 prepared in Preparation Example 1 and 20 g of the modified polysaccharide prepared in Preparation Example 2 were mixed, and 40 g of 0.9% physiological saline was added and stirred to obtain a composite solidified liquid;

[0079] Weigh 20 g of calcium phosphate and 30 g of buffer solution (obtained by mixing 15 ml of citric acid and 15 ml of sodium citrate) and mix them. After the calcium phosphate is completely dissolved, weigh 30 g of the composite curing liquid prepared above and mix them with the calcium phosphate and buffer solution for 5 minutes to obtain an inorganic bone adhesive.

[0080] Example 3:

[0081] A method for preparing an inorganic bone adhesive modified by mussel mucin, specifically comprising the following steps:

[0082] 20 g of the modified mussel mucin Mfp3-5 prepared in Preparation Example 1 and 20 g of the modified polysaccharide prepared in Preparation Example 3 were mixed, and 40 g of 0.9% physiological saline was added and stirred to obtain a composite solidified liquid;

[0083] Weigh 30 g of hydroxyapatite and 30 g of buffer solution (obtained by mixing 15 ml of citric acid and 15 ml of sodium citrate) and mix them. After the hydroxyapatite is completely dissolved, weigh 40 g of the composite curing liquid prepared above and mix them with the hydroxyapatite and buffer solution for 5 minutes to obtain an inorganic bone adhesive.

[0084] Example 4:

[0085] A method for preparing an inorganic bone adhesive modified by mussel mucin, specifically comprising the following steps:

[0086] 20 g of the modified mussel mucin Mfp3-5 prepared in Preparation Example 1 and 20 g of the modified polysaccharide prepared in Preparation Example 3 were mixed, and 40 g of 0.9% physiological saline was added and stirred to obtain a composite solidified liquid;

[0087] 30 g of hydroxyapatite and 30 g of buffer solution (obtained by mixing 15 ml of citric acid and 15 ml of sodium citrate) were weighed and mixed. After the hydroxyapatite was completely dissolved, 50 g of the composite curing liquid prepared above was weighed and mixed with the hydroxyapatite and buffer solution for 5 minutes to obtain an inorganic bone adhesive.

[0088] Example 5:

[0089] A method for preparing an inorganic bone adhesive modified by mussel mucin, specifically comprising the following steps:

[0090] 20 g of the modified mussel mucin Mfp3-5 prepared in Preparation Example 1 and 20 g of the modified polysaccharide prepared in Preparation Example 4 were mixed, and 40 g of 0.9% physiological saline was added and stirred to obtain a composite solidified liquid;

[0091] Weigh 40 g of magnesium phosphate and 40 g of buffer solution (obtained by mixing 20 ml of citric acid and 20 ml of sodium citrate) and mix them. After the magnesium phosphate is completely dissolved, weigh 50 g of the composite curing liquid prepared above and mix them with the magnesium phosphate and buffer solution for 5 minutes to obtain an inorganic bone adhesive.

[0092] Example 6:

[0093] A method for preparing an inorganic bone adhesive modified by mussel mucin, specifically comprising the following steps:

[0094] 20 g of the modified mussel mucin Mfp3-5 prepared in Preparation Example 1 and 20 g of the modified polysaccharide prepared in Preparation Example 5 were mixed, and 40 g of 0.9% physiological saline was added and stirred to obtain a composite solidified liquid;

[0095] Weigh 40 g of magnesium phosphate and 40 g of buffer solution (obtained by mixing 20 ml of citric acid and 20 ml of sodium citrate) and mix them. After the magnesium phosphate is completely dissolved, weigh 60 g of the composite curing liquid prepared above and mix them with the magnesium phosphate and buffer solution for 5 minutes to obtain an inorganic bone adhesive.

[0096] Comparative Example 1:

[0097] A method for preparing an inorganic bone adhesive modified by mussel mucin, specifically comprising the following steps:

[0098] 20 g of the modified mussel mucin Mfp3-5 prepared in Preparation Example 1 and 20 g of unmodified chitosan were mixed, and 40 g of 0.9% physiological saline was added and stirred to obtain a composite solidified liquid;

[0099] Weigh 40 g of magnesium phosphate and 40 g of buffer solution (obtained by mixing 20 ml of citric acid and 20 ml of sodium citrate) and mix them. After the magnesium phosphate is completely dissolved, weigh 60 g of the composite curing liquid prepared above and mix them with the magnesium phosphate and buffer solution for 5 minutes to obtain an inorganic bone adhesive.

[0100] Comparative Example 2:

[0101] A method for preparing an inorganic bone adhesive modified by mussel mucin, specifically comprising the following steps:

[0102] 20 g of the recombinant mussel mucin Mfp3-5 prepared in Preparation Example 1 and 20 g of the modified polysaccharide prepared in Preparation Example 5 were mixed, and 40 g of 0.9% physiological saline was added and stirred to obtain a composite solidified liquid;

[0103] Weigh 40 g of magnesium phosphate and 40 g of buffer solution (obtained by mixing 20 ml of citric acid and 20 ml of sodium citrate) and mix them. After the magnesium phosphate is completely dissolved, weigh 60 g of the composite curing liquid prepared above and mix them with the magnesium phosphate and buffer solution for 5 minutes to obtain an inorganic bone adhesive.

[0104] The inorganic bone adhesives obtained in Examples 1 to 6 and Comparative Examples 1 to 2 were tested for their bonding properties:

[0105] Eight fresh beef bones measuring 4.5 cm × 2.5 cm × 1.5 cm were prepared. After cleaning, each bone was cut into two sections. Inorganic bone adhesive was then applied to the fracture of one of the bones and the corresponding fractured bone was bonded to the other. The bones were then dried in a 37°C oven for 30 minutes, simulating body temperature, and tested at a speed of 15 mm / min. The bonding data is shown in the following table:

[0106] Inorganic bone adhesive sources Bond strength (kPa) Example 1 90.42 Example 2 92.08 Example 3 97.25 Example 4 98.63 Example 5 98.82 Example 6 105.14 Comparative Example 1 73.02 Comparative Example 2 51.37

[0107] The inorganic bone adhesives obtained in Examples 1 to 6 and Comparative Examples 1 to 2 were tested for water resistance:

[0108] The inorganic bone adhesives of Examples 1-6 and Comparative Examples 1-2 were completely immersed in simulated body fluid (SBF) having a pH of 7.3-7.4, and the temperature of the simulated body fluid was controlled at 40° C. After treatment for one week, the surface of the inorganic bone adhesive was rinsed and then dried in a vacuum drying oven at 50° C. for 24 hours. The weight loss rates of the inorganic bone adhesives of Examples 1-6 and Comparative Examples 1-2 were calculated as shown in the following table:

[0109] Inorganic bone adhesive sources Weight loss rate (%) Example 1 3.51 Example 2 3.26 Example 3 1.05 Example 4 0.97 Example 5 0.51 Example 6 0.47 Comparative Example 1 11.05 Comparative Example 2 15.27

[0110] The above-described embodiments are described in detail in comparison with the technical solutions and beneficial effects of the present invention. It should be understood that the above is only a specific embodiment of the present invention and is not intended to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, including optimization of reaction conditions and adjustment of reaction ratio, and these changes and improvements all fall within the scope of the present invention claimed.

Claims

1. An inorganic bone adhesive modified by mussel mucin, characterized in that: The inorganic bone adhesive comprises the following raw materials in parts by weight: 20-40 parts by weight of an inorganic phosphate compound, 30-60 parts by weight of a composite curing liquid, and 20-40 parts by weight of a buffer solution; The inorganic phosphate compound includes calcium phosphate, hydroxyapatite or magnesium phosphate; The composite solidifying liquid is prepared by mixing the modified mussel mucin Mfp3-5 and the modified polysaccharide in a mass ratio of 1:1, adding physiological saline equal to the total mass of the modified mussel mucin Mfp3-5 and the modified polysaccharide, and stirring; The buffer solution is prepared by mixing citric acid and sodium citrate in a mass ratio of 1:1; The preparation method of the modified mussel mucin Mfp3-5 comprises the following steps: The cDNA gene of mussel mucin Mfp3-5 was amplified by PCR and introduced into the vector pET-28a, and then transformed into Escherichia coli DH5α competent cells for induction culture to obtain a bacterial liquid containing recombinant mussel mucin Mfp3-5; The bacterial liquid was lysed and the precipitate was collected by centrifugation. The precipitate was dissolved in buffer A and then centrifuged to collect the supernatant. The supernatant was eluted with buffer B and buffer C, and then dialyzed with 5% acetic acid to obtain the recombinant mussel mucin Mfp3-5. The recombinant mussel mucin Mfp3-5 is treated with 0.1 mol / L phosphate buffer at 25° C. for 6 hours to obtain modified mussel mucin Mfp3-5; The preparation method of the modified polysaccharide comprises the following steps: After the alkaline polysaccharide is dissolved in equal parts of acetic acid, 0.2 to 0.5 times the weight of the alkaline polysaccharide is added with sodium periodate, mixed, and reacted at 20 to 30° C. for 3 to 5 hours. After the reaction is completed, deionized water is added and mixed, and the mixture is stirred and centrifuged to obtain a precipitate. The precipitate is vacuum dried to obtain the modified polysaccharide; The 0.1 mol / L phosphate buffer contains 50 μg / mL tyrosinase and 25 mmol / L ascorbic acid, and the pH value of the 0.1 mol / L phosphate buffer is 7.

0.

2. The inorganic bone adhesive modified by mussel mucin according to claim 1, characterized in that: The inducer for the induction culture is 0.5 mmol / L IPTG.

3. The inorganic bone adhesive modified by mussel mucin according to claim 1, characterized in that: The buffer A is composed of 10 mmol / L imidazole, 8 mol / L urea, 0.1 mol / L sodium phosphate and 10 mmol / L Tris-HCl, and the pH of the buffer A is 7.9; The buffer B is composed of 30 mmol / L imidazole, 8 mol / L urea, 0.1 mol / L sodium phosphate and 10 mmol / L Tris-HCl, and the pH of the buffer B is 6.3; The buffer C consists of 0.3 mol / L imidazole, 8 mol / L urea, 0.1 mol / L sodium phosphate and 10 mmol / L Tris-HCl, and the pH of the buffer C is 4.

5.

4. The inorganic bone adhesive modified by mussel mucin according to claim 1, characterized in that: The recombinant mussel mucin Mfp3-5 structure is an inclusion body structure.

5. The inorganic bone adhesive modified by mussel mucin according to claim 1, characterized in that: The alkaline polysaccharide includes chitosan.

6. A method for preparing the inorganic bone adhesive modified by mussel mucin according to any one of claims 1 to 5, characterized in that: The preparation method comprises the following steps: The inorganic phosphate compound and the buffer solution are mixed and stirred until the inorganic phosphate compound is completely dissolved, and then the composite solidifying liquid is added and mixed and stirred to form the inorganic bone adhesive.

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