Hydroxyapatite bone powder for postoperative tissue repair of osteosarcoma and method of making same

Abstract

The application discloses a hydroxyapatite bone powder for osteosarcoma postoperative tissue repair and a preparation method thereof. The hydroxyapatite bone powder is a nanorod-shaped hydroxyapatite bone powder with a length of 150-200 nm and a diameter of 10-15 nm. The hydroxyapatite bone powder is hydrophilic hydroxyapatite bone powder doped with Se and Mg. The preparation method comprises the following steps: taking CaCl2, Na2SeO3, soluble phosphate and MgCl2 as main raw materials, and preparing Se and Mg co-doped hydrophobic hydroxyapatite through two-step sealed hydrothermal reaction; dispersing the Se and Mg co-doped hydrophobic hydroxyapatite in cyclohexane; adding a dextran aqueous solution; stirring; centrifuging; washing; and freeze-drying to obtain the hydroxyapatite bone powder for osteosarcoma postoperative tissue repair. The hydroxyapatite bone powder can effectively realize the inhibition of osteoma cell recurrence and tissue defect repair after osteosarcoma operation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological materials, and particularly relates to hydroxyapatite bone powder for postoperative tissue repair of osteosarcoma and a preparation method thereof. BACKGROUND

[0002] Osteosarcoma is a malignant tumor disease that often occurs in adolescents, and has the characteristics of strong local bone tissue invasiveness, early metastasis, and easy recurrence after surgery. Osteosarcoma is often accompanied by local swelling and persistent pain, which seriously threatens the survival and health of patients. The current clinical treatment strategy is surgical resection, but it is difficult to ensure that the tumor tissue of the patient is completely removed, and the risk of recurrence is still high. Therefore, postoperative radiotherapy and chemotherapy are often combined to minimize tumor metastasis and recurrence. However, the side effects of radiotherapy and chemotherapy, such as hair loss, vomiting, and reduction of white blood cells and platelets, can seriously affect the quality of life of patients. In addition, bone defects caused by osteosarcoma often cannot heal on their own.

[0003] An ideal treatment method for osteosarcoma needs to be able to eliminate residual tumor cells after surgery and inhibit tumor metastasis, and also needs to have good ability to promote bone defect repair. A degradable bone defect repair material with anticancer property is undoubtedly an ideal biological medical material for treating osteosarcoma. SUMMARY

[0004] The technical problem to be solved by the present application is to provide hydroxyapatite bone powder for postoperative tissue repair of osteosarcoma and a preparation method thereof to solve the problems of the prior art. The hydroxyapatite bone powder of the present application is a hydrophilic hydroxyapatite bone powder doped with Se and Mg, and has a nanorod shape with a length of 150-200 nm and a diameter of 10-15 nm, which can effectively inhibit the recurrence of osteosarcoma cells and repair tissue defects after osteosarcoma surgery.

[0005] To solve the above technical problems, the technical solution adopted by the present application is: a hydroxyapatite bone powder for postoperative tissue repair of osteosarcoma, characterized in that the hydroxyapatite bone powder is a nanorod-shaped hydroxyapatite bone powder with a length of 150-200 nm and a diameter of 10-15 nm, and the hydroxyapatite bone powder is a hydrophilic hydroxyapatite bone powder doped with Se and Mg.

[0006] In addition, the present application also provides a method for preparing the above-mentioned hydroxyapatite bone powder for postoperative tissue repair of osteosarcoma, characterized in that it comprises:

[0007] Se and Mg co-doped hydrophobic hydroxyapatite is prepared by two-step sealed hydrothermal reaction using CaCl2, Na2SeO3, soluble phosphate and MgCl2 as main raw materials;

[0008] The Se and Mg co-doped hydrophobic hydroxyapatite is dispersed in cyclohexane, an aqueous dextran solution is added, stirring is carried out for 3-6 hours, centrifugation is carried out, washing is carried out, and freeze-drying is carried out to obtain the hydroxyapatite bone powder for postoperative tissue repair of osteosarcoma.

[0009] The method has the characteristics that CaCl2, Na2SeO3, a soluble phosphate and MgCl2 are used as main raw materials, and Se and Mg co-doped hydrophobic hydroxyapatite is prepared through two-step sealed hydrothermal reaction, and specifically includes the following steps:

[0010] In step 101, CaCl2 aqueous solution, Na2SeO3 aqueous solution and soluble phosphate aqueous solution are sequentially added to a mixed solution of oleic acid and methanol under stirring, and stirring is carried out to obtain a reaction system A.

[0011] In step 102, the reaction system A is reacted for 5-15 hours under sealed conditions at 100-180 ℃.

[0012] In step 103, the post-reaction system A is cooled to room temperature, MgCl2 aqueous solution is added under stirring, and stirring is continuously carried out for 10-30 min to obtain a reaction system B.

[0013] In step 104, the reaction system B is reacted for 5-15 hours under sealed conditions at 100-180 ℃, and is cooled to room temperature, and a precipitate is collected.

[0014] In step 105, the precipitate is dispersed and centrifuged to obtain Se and Mg co-doped hydrophobic hydroxyapatite.

[0015] The method has the characteristics that in step 101, the volume of methanol is 3-5 times the volume of oleic acid; in step 101, the stirring time is 10-30 min; and in step 101, the volume of the CaCl2 aqueous solution is 1-7 times the volume of oleic acid.

[0016] The method has the characteristics that in step 101, the molar ratio of Ca / (P+Se) is 1.67, and the molar ratio of Se / (P+Se) is 0.005-0.2.

[0017] The method has the characteristics that in step 101, the soluble phosphate is Na2HPO4, NaH2PO4, Na3PO4·12H2O, K2HPO4 or KH2PO4.

[0018] The method has the characteristics that in step 103, the molar ratio of Mg / Ca is 0.01-0.4.

[0019] The method has the advantages that the volume of the cyclohexane is 10-100 times the mass of the Se and Mg co-doped hydrophobic hydroxyapatite, the volume of the cyclohexane is in mL, and the mass of the Se and Mg co-doped hydrophobic hydroxyapatite is in g.

[0020] The method has the advantages that the volume of the cyclohexane is 10-100 times the mass of the Se and Mg co-doped hydrophobic hydroxyapatite, the volume of the cyclohexane is in mL, and the mass of the Se and Mg co-doped hydrophobic hydroxyapatite is in g.

[0021] The method has the advantages that the volume of the cyclohexane is 10-100 times the mass of the Se and Mg co-doped hydrophobic hydroxyapatite, the volume of the cyclohexane is in mL, and the mass of the Se and Mg co-doped hydrophobic hydroxyapatite is in g.

[0022] The method has the advantages that the volume of the cyclohexane is 10-100 times the mass of the Se and Mg co-doped hydrophobic hydroxyapatite, the volume of the cyclohexane is in mL, and the mass of the Se and Mg co-doped hydrophobic hydroxyapatite is in g.

[0023] 1、The hydroxyapatite bone powder of the application is a Se and Mg doped hydrophilic hydroxyapatite bone powder with a nanorod length of 150-200 nm and a diameter of 10-15 nm, and can effectively inhibit the recurrence of osteosarcoma cells and repair tissue defects after osteosarcoma surgery.

[0024] 2、The preparation method of the hydroxyapatite bone powder of the application comprises the following steps: taking CaCl2 aqueous solution, Na2SeO3 aqueous solution, soluble phosphate and MgCl2 aqueous solution as main raw materials, and preparing Se and Mg co-doped hydrophobic hydroxyapatite through two-step closed hydrothermal reaction; and then dispersing the Se and Mg co-doped hydrophobic hydroxyapatite in a system containing cyclohexane and dextran, and stirring to obtain Se and Mg co-doped hydrophilic hydroxyapatite bone powder, which can effectively realize the exchange and substitution of phosphate, hydroxyl and calcium ions in the hexagonal crystal lattice of hydroxyapatite with selenium and magnesium, and can play the performance of metal-doped hydroxyapatite in inducing cell apoptosis, inhibiting the proliferation and metastasis of various cancer cells, promoting the osteogenic differentiation and mineralization of bone marrow mesenchymal stem cells, and improving the osteogenic function.

[0025] 3、The bone powder obtained by the preparation method of the hydroxyapatite bone powder of the application has stable performance and remarkable biological performance, and can meet the postoperative treatment requirements of osteosarcoma.

[0026] 4、The preparation method of the hydroxyapatite bone powder of the application has the advantages of cheap and easily available raw materials, environmentally friendly process, easy to scale up, and good market application value.

[0027] The technical solutions of the application will be further described in detail below in combination with the drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 It is a TEM image of the hydroxyapatite bone powder of Example 1 of the application.

[0029] Figure 2 The XRD pattern of the hydroxyapatite bone powder of Example 1 of the present application.

[0030] Figure 3 The EDS pattern of the hydroxyapatite bone powder of Example 1 of the present application.

[0031] Figure 4 The photo of the hydroxyapatite bone powder of Example 1 of the present application.

[0032] Figure 5 The cytotoxicity diagram of the hydroxyapatite bone powder of Example 1 of the present application to hBMSCs.

[0033] Figure 6 The cytotoxicity diagram of the hydroxyapatite bone powder of Example 1 of the present application to MG-63. DETAILED DESCRIPTION

[0034] The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0035] Example 1

[0036] The present embodiment provides a method for preparing a hydroxyapatite bone powder for osteosarcoma postoperative tissue repair, comprising:

[0037] Step one, providing Se and Mg co-doped hydrophobic hydroxyapatite, specifically comprising:

[0038] Step 101, 3mL of oleic acid and 12mL of methanol are mixed to obtain a mixed solution, 10mL of CaCl2 aqueous solution, 1mL of Na2SeO3 aqueous solution and 9mL of K2HPO4 aqueous solution are sequentially added to the mixed solution under stirring conditions, and stirring is performed for 20 minutes to obtain a reaction system A; the concentration of the CaCl2 aqueous solution is 0.25mol / L, the concentration of the Na2SeO3 aqueous solution is 0.15mol / L, and the concentration of the K2HPO4 aqueous solution is 0.15mol / L;

[0039] Step 102, the reaction system A is transferred into a polytetrafluoroethylene reaction kettle, sealed, and reacted at 140℃ for 10 hours;

[0040] Step 103, the post-reaction system A is cooled to room temperature, 2.5mL of MgCl2 aqueous solution is added under stirring conditions, and stirring is continued for 20 minutes to obtain a reaction system B; the concentration of the MgCl2 aqueous solution is 0.25mol / L;

[0041] Step 104, the reaction system B is placed in a polytetrafluoroethylene reaction kettle, sealed, reacted at 140℃ for 10 hours, cooled to room temperature, and the precipitate is collected;

[0042] Step 105, the precipitate is dispersed in cyclohexane, anhydrous ethanol is added, centrifuged, and Se and Mg co-doped hydrophobic hydroxyapatite is obtained;

[0043] Step two, providing hydroxyapatite bone powder for osteosarcoma postoperative tissue repair

[0044] Step 201, 0.3g of the Se and Mg co-doped hydrophobic hydroxyapatite is dispersed in 5mL of cyclohexane, 125mL of a 0.1g / mL aqueous dextran solution is added, and stirred at room temperature for 4.5 hours; the molecular weight of the dextran is 1500Da;

[0045] Step 202, the stirred system is centrifuged, the precipitate is collected, washed with water and anhydrous ethanol, and freeze-dried to obtain Se and Mg co-doped hydrophilic hydroxyapatite, which is hydroxyapatite bone powder.

[0046] Example 2

[0047] The present embodiment provides a method for preparing hydroxyapatite bone powder for osteosarcoma postoperative tissue repair, comprising:

[0048] Step one, providing Se and Mg co-doped hydrophobic hydroxyapatite, specifically comprising:

[0049] Step 101, 3mL of oleic acid and 9mL of methanol are mixed to obtain a mixed solution, 5mL of CaCl2 aqueous solution, 0.025mL of Na2SeO3 aqueous solution and 4.975mL of K2HPO4 aqueous solution are sequentially added to the mixed solution under stirring conditions, and stirred for 10 minutes to obtain reaction system A; the concentration of the CaCl2 aqueous solution is 0.25mol / L, the concentration of the Na2SeO3 aqueous solution is 0.15mol / L, and the concentration of the K2HPO4 aqueous solution is 0.15mol / L;

[0050] Step 102, the reaction system A is transferred into a polytetrafluoroethylene reaction kettle, sealed, and reacted at 100℃ for 5 hours;

[0051] Step 103, the reaction system A is cooled to room temperature, 0.05mL of MgCl2 aqueous solution is added under stirring conditions, and stirring is continued for 10 minutes to obtain reaction system B; the concentration of the MgCl2 aqueous solution is 0.25mol / L;

[0052] Step 104, the reaction system B is placed in a polytetrafluoroethylene reaction kettle, sealed, reacted at 100℃ for 5 hours, cooled to room temperature, and the precipitate is collected;

[0053] Step 105, the precipitate is dispersed in cyclohexane, anhydrous ethanol is added, centrifuged, and Se and Mg co-doped hydrophobic hydroxyapatite is obtained;

[0054] Step two, providing hydroxyapatite bone powder for osteosarcoma postoperative tissue repair

[0055] Step 201, 0.05g of the Se and Mg co-doped hydrophobic hydroxyapatite is dispersed in 5mL of cyclohexane, 75mL of aqueous dextran solution with a concentration of 0.05g / mL is added, and stirred at room temperature for 3 hours; the molecular weight of the dextran is 1500Da;

[0056] Step 202, the stirred system is centrifuged, the precipitate is collected, washed with water and anhydrous ethanol, and freeze-dried to obtain Se and Mg co-doped hydrophilic hydroxyapatite, which is hydroxyapatite bone powder.

[0057] The physicochemical properties of the hydroxyapatite bone powder of the example are similar to those of Example 1.

[0058] Example 3

[0059] The example provides a method for preparing hydroxyapatite bone powder for osteosarcoma postoperative tissue repair, comprising:

[0060] Step one, providing Se and Mg co-doped hydrophobic hydroxyapatite, specifically comprising:

[0061] Step 101, 3mL of oleic acid and 15mL of methanol are mixed to obtain a mixed solution, 20mL of CaCl2 aqueous solution, 4mL of Na2SeO3 aqueous solution and 16mL of K2HPO4 aqueous solution are sequentially added to the mixed solution under stirring conditions, and stirred for 30 minutes to obtain reaction system A; the concentration of the CaCl2 aqueous solution is 0.25mol / L, the concentration of the Na2SeO3 aqueous solution is 0.15mol / L, and the concentration of the K2HPO4 aqueous solution is 0.15mol / L;

[0062] Step 102, the reaction system A is transferred into a polytetrafluoroethylene reaction kettle, sealed, and reacted at 180℃ for 15 hours;

[0063] Step 103, the reaction system A is cooled to room temperature, 8mL of MgCl2 aqueous solution is added under stirring conditions, and stirring is continued for 30 minutes to obtain reaction system B; the concentration of the MgCl2 aqueous solution is 0.25mol / L;

[0064] Step 104, the reaction system B is placed in a polytetrafluoroethylene reactor, sealed, reacted at 180℃ for 15 hours, cooled to room temperature, and the precipitate is collected;

[0065] Step 105, the precipitate is dispersed in cyclohexane, anhydrous ethanol is added, centrifuged, and Se and Mg co-doped hydrophobic hydroxyapatite is obtained;

[0066] Step two, providing hydroxyapatite bone powder for osteosarcoma postoperative tissue repair

[0067] Step 201, 0.5g of the Se and Mg co-doped hydrophobic hydroxyapatite precipitate is dispersed in 5mL of cyclohexane, 175mL of aqueous dextran solution with a concentration of 0.15g / mL is added, and stirred at room temperature for 6 hours; the molecular weight of the dextran is 1500Da;

[0068] Step 202, the stirred system is centrifuged, the precipitate is collected, washed with water and anhydrous ethanol, and freeze-dried to obtain Se and Mg co-doped hydrophilic hydroxyapatite, which is hydroxyapatite bone powder.

[0069] The physicochemical properties of the hydroxyapatite bone powder of the present example are similar to those of Example 1.

[0070] Example 4

[0071] The present example provides a method for preparing hydroxyapatite bone powder for osteosarcoma postoperative tissue repair, comprising:

[0072] Step one, providing Se and Mg co-doped hydrophobic hydroxyapatite, specifically comprising:

[0073] Step 101, 3mL of oleic acid and 12mL of methanol are mixed to obtain a mixed solution, 10mL of CaCl2 aqueous solution, 1mL of Na2SeO3 aqueous solution and 9mL of Na3PO4·12H2O aqueous solution are sequentially added to the mixed solution under stirring conditions, and stirred for 20 minutes to obtain reaction system A; the concentration of the CaCl2 aqueous solution is 0.25mol / L, the concentration of the Na2SeO3 aqueous solution is 0.15mol / L, and the concentration of Na3PO4 in the Na3PO4·12H2O aqueous solution is 0.15mol / L;

[0074] Step 102, the reaction system A is transferred into a polytetrafluoroethylene reactor, sealed, and reacted at 140℃ for 10 hours;

[0075] Step 103, the reaction system A is cooled to room temperature, 2.5mL of MgCl2 aqueous solution is added under stirring conditions, and stirring is continued for 20 minutes to obtain reaction system B; the concentration of the MgCl2 aqueous solution is 0.25mol / L;

[0076] Step 104, the reaction system B is placed in a polytetrafluoroethylene reaction kettle, sealed, reacted at 140℃ for 10 hours, cooled to room temperature, and the precipitate is collected;

[0077] Step 105, the precipitate is dispersed in cyclohexane, anhydrous ethanol is added, centrifuged, and Se and Mg co-doped hydrophobic hydroxyapatite is obtained;

[0078] Step two, providing hydroxyapatite bone powder for osteosarcoma postoperative tissue repair

[0079] Step 201, 0.3g of the Se and Mg co-doped hydrophobic hydroxyapatite is dispersed in 5mL of cyclohexane, 125mL of aqueous dextran solution with a concentration of 0.1g / mL is added, and stirred at room temperature for 4.5 hours; the molecular weight of the dextran is 1500Da;

[0080] Step 202, the stirred system is centrifuged, the precipitate is collected, washed with water and anhydrous ethanol, and freeze-dried to obtain Se and Mg co-doped hydrophilic hydroxyapatite, which is hydroxyapatite bone powder.

[0081] Example 5

[0082] The embodiment provides a method for preparing hydroxyapatite bone powder for osteosarcoma postoperative tissue repair, comprising:

[0083] Step one, providing Se and Mg co-doped hydrophobic hydroxyapatite, specifically comprising:

[0084] Step 101, 3mL of oleic acid and 9mL of methanol are mixed to obtain a mixed solution, 5mL of CaCl2 aqueous solution, 0.025mL of Na2SeO3 aqueous solution and 4.975mL of NaH2PO4 aqueous solution are sequentially added to the mixed solution under stirring conditions, and stirred for 10 minutes to obtain a reaction system A; the concentration of the CaCl2 aqueous solution is 0.25mol / L, the concentration of the Na2SeO3 aqueous solution is 0.15mol / L, and the concentration of the NaH2PO4 aqueous solution is 0.15mol / L;

[0085] Step 102, the reaction system A is transferred into a polytetrafluoroethylene reaction kettle, sealed, and reacted at 100℃ for 5 hours;

[0086] Step 103, the reaction system A is cooled to room temperature, 0.05mL of MgCl2 aqueous solution is added under stirring conditions, and stirring is continued for 10 minutes to obtain a reaction system B; the concentration of the MgCl2 aqueous solution is 0.25mol / L;

[0087] Step 104, the reaction system B is placed in a polytetrafluoroethylene reaction kettle, sealed, reacted at 100℃ for 5 hours, cooled to room temperature, and the precipitate is collected;

[0088] Step 105, the precipitate is dispersed in cyclohexane, anhydrous ethanol is added, centrifuged, and Se and Mg co-doped hydrophobic hydroxyapatite is obtained;

[0089] Step two, providing hydroxyapatite bone powder for osteosarcoma postoperative tissue repair

[0090] Step 201, 0.05g of the Se and Mg co-doped hydrophobic hydroxyapatite is dispersed in 5mL of cyclohexane, 75mL of aqueous dextran solution with a concentration of 0.05g / mL is added, and stirred at room temperature for 3 hours; the molecular weight of the dextran is 1500Da;

[0091] Step 202, the stirred system is centrifuged, the precipitate is collected, washed with water and anhydrous ethanol, and freeze-dried to obtain Se and Mg co-doped hydrophilic hydroxyapatite, which is hydroxyapatite bone powder.

[0092] The physicochemical properties of the hydroxyapatite bone powder of the example are similar to those of Example 1.

[0093] Example 6

[0094] The example provides a method for preparing hydroxyapatite bone powder for osteosarcoma postoperative tissue repair, comprising:

[0095] Step one, providing Se and Mg co-doped hydrophobic hydroxyapatite, specifically comprising:

[0096] Step 101, 3mL of oleic acid and 15mL of methanol are mixed to obtain a mixed solution, 20mL of CaCl2 aqueous solution, 4mL of Na2SeO3 aqueous solution and 16mL of KH2PO4 aqueous solution are sequentially added to the mixed solution under stirring conditions, and stirred for 30 minutes to obtain reaction system A; the concentration of the CaCl2 aqueous solution is 0.25mol / L, the concentration of the Na2SeO3 aqueous solution is 0.15mol / L, and the concentration of the KH2PO4 aqueous solution is 0.15mol / L;

[0097] Step 102, the reaction system A is transferred into a polytetrafluoroethylene reaction kettle, sealed, and reacted at 180℃ for 15 hours;

[0098] Step 103, the reaction system A is cooled to room temperature, 8mL of MgCl2 aqueous solution is added under stirring conditions, and stirring is continued for 30 minutes to obtain reaction system B; the concentration of the MgCl2 aqueous solution is 0.25mol / L;

[0099] Step 104, the reaction system B is placed in a polytetrafluoroethylene reaction kettle, sealed, reacted at 180℃ for 15 hours, cooled to room temperature, and the precipitate is collected;

[0100] Step 105, the precipitate is dispersed in cyclohexane, anhydrous ethanol is added, centrifuged, and Se and Mg co-doped hydrophobic hydroxyapatite is obtained;

[0101] Step two, providing hydroxyapatite bone powder for osteosarcoma postoperative tissue repair

[0102] Step 201, 0.5g of the Se and Mg co-doped hydrophobic hydroxyapatite precipitate is dispersed in 5mL of cyclohexane, 175mL of aqueous dextran solution with a concentration of 0.15g / mL is added, and stirred at room temperature for 6 hours; the molecular weight of the dextran is 1500Da;

[0103] Step 202, the stirred system is centrifuged, the precipitate is collected, washed with water and anhydrous ethanol, and freeze-dried to obtain Se and Mg co-doped hydrophilic hydroxyapatite, which is hydroxyapatite bone powder.

[0104] The physicochemical properties of the hydroxyapatite bone powder of the example are similar to those of Example 1.

[0105] Example 7

[0106] The example provides a method for preparing hydroxyapatite bone powder for osteosarcoma postoperative tissue repair, comprising:

[0107] Step one, providing Se and Mg co-doped hydrophobic hydroxyapatite, specifically comprising:

[0108] Step 101, 3mL of oleic acid and 9mL of methanol are mixed to obtain a mixed solution, 5mL of CaCl2 aqueous solution, 0.025mL of Na2SeO3 aqueous solution and 4.975mL of Na2HPO4 aqueous solution are sequentially added to the mixed solution under stirring conditions, and stirred for 10 minutes to obtain reaction system A; the concentration of the CaCl2 aqueous solution is 0.25mol / L, the concentration of the Na2SeO3 aqueous solution is 0.15mol / L, and the concentration of the Na2HPO4 aqueous solution is 0.15mol / L;

[0109] Step 102, the reaction system A is transferred into a polytetrafluoroethylene reaction kettle, sealed, and reacted at 100℃ for 5 hours;

[0110] Step 103, the reaction system A is cooled to room temperature, 0.05mL of MgCl2 aqueous solution is added under stirring conditions, and stirring is continued for 10 minutes to obtain reaction system B; the concentration of the MgCl2 aqueous solution is 0.25mol / L;

[0111] Step 104, the reaction system B was placed in a polytetrafluoroethylene reactor, sealed, reacted at 100°C for 5 hours, cooled to room temperature, and the precipitate was collected;

[0112] Step 105, the precipitate was dispersed in cyclohexane, anhydrous ethanol was added, centrifuged, and Se and Mg co-doped hydrophobic hydroxyapatite was obtained;

[0113] Step two, providing hydroxyapatite bone powder for osteosarcoma postoperative tissue repair

[0114] Step 201, 0.05 g of the Se and Mg co-doped hydrophobic hydroxyapatite was dispersed in 5 mL of cyclohexane, 75 mL of a 0.05 g / mL aqueous dextran solution was added, and stirred at room temperature for 3 hours; the molecular weight of the dextran was 1500 Da;

[0115] Step 202, the stirred system was centrifuged, the precipitate was collected, washed with water and anhydrous ethanol, and freeze-dried to obtain Se and Mg co-doped hydrophilic hydroxyapatite, which was hydroxyapatite bone powder.

[0116] The physicochemical properties of the hydroxyapatite bone powder of the example were similar to those of Example 1.

[0117] Performance evaluation

[0118] The TEM image of the hydroxyapatite bone powder of Example 1 is shown in Figure 1 According to Figure 1 it can be seen that the hydroxyapatite bone powder is in the shape of nanorods, with a length of 150-200 nm and a diameter of 10-15 nm.

[0119] The XRD pattern of the hydroxyapatite bone powder of Example 1 is shown in Figure 2 Compared with the hydroxyapatite standard card JCPDS no. 74-0565, it can be seen that the product is hydroxyapatite.

[0120] The EDS detection results of the hydroxyapatite bone powder of Example 1 are shown in Figure 3 According to Figure 3 it can be seen that Se and Mg are successfully doped.

[0121] The morphology of the hydroxyapatite bone powder of Example 1 is shown in Figure 4 According to Figure 4 it can be seen that it is in the form of fine powder, which can be directly applied or mixed with a small amount of physiological saline and implanted into the osteosarcoma site or bone defect repair site by surgical method.

[0122] The cytology experiment of the hydroxyapatite bone powder is shown in Figure 5 and Figure 6As shown, this study investigated the efficacy of hydroxyapatite bone powder against normal cells (hBMSCs) and cancer cells (MG-63). The testing methods included:

[0123] (1) Preparation of DMEM extract of hydroxyapatite bone powder

[0124] ① Preparation of PBS solution: Dissolve 0.005g KCl, 0.2g NaCl, 0.005g K2HPO4 and 0.085g Na2HPO4 in 500mL deionized water, sterilize at 121℃ for 30 minutes, seal and store at 4℃ for later use to obtain PBS solution;

[0125] ② Preparation of trypsin: Place 50 mg of trypsin in 20 mL of PBS solution, filter through a 0.22 μm filter membrane to obtain a trypsin solution with a concentration of 2.5 mg / mL; seal and store at 4℃ for later use;

[0126] ③ Preparation of DMEM complete culture medium: Add 50 mL of fetal bovine serum and 5 mL of penicillin and streptomycin to 500 mL of DMEM basal culture medium, seal and store at 4℃ for later use to obtain DMEM complete culture medium; the penicillin and streptomycin are used.

[0127] ④ Preparation of DMEM extract of hydroxyapatite bone powder: The hydroxyapatite bone powder prepared in Example 1 was dispersed in DMEM complete culture medium and soaked for 3 days (37℃) to obtain hydroxyapatite bone powder extracts of different concentrations (4, 8, 16, 32, 64, 128, 256, 512 μg / mL). The extracts were sealed and stored in a refrigerator at 4℃ for later use.

[0128] (2) Cytotoxicity test of hydroxyapatite bone powder on hBMSCs: hBMSCs were used as normal cells, and MG-63 cells were used as cancer cell models. Cells were cultured in DMEM extract of hydroxyapatite bone powder to investigate the effect of the extract on cell growth. Specifically, the following was included:

[0129] ① Cell culture: hBMSCs and MG-63 cells were cultured in DMEM complete culture medium and placed in an incubator containing 5% CO2 at 37°C. The cells were passaged every 36-48 hours when the cell density was 80%-95%.

[0130] ② Cell plating: Once the cells are stable and viable, perform cell plating, which includes: removing the culture medium for both types of cells separately, infiltrating with PBS solution to remove dead cells, removing the PBS, digesting with trypsin, dispersing the cells with DMEM complete culture medium at a concentration of 1×10⁻⁶. 4The cell density of each well is 100 μL of the cell dispersion liquid is added to each well of a 96-well plate, and the plate is placed in an incubator (37°C, 5% CO2) for 24 hours;

[0131] ③Detection of cytotoxicity: the 96-well plate after the culturing in step ② is taken out, and the original DMEM complete culture solution is aspirated; 5 replicate wells are set for each concentration, and the wells are replaced with different concentrations of the hydroxyapatite bone powder extract solution, and the plate is placed in an incubator (37°C, 5% CO2) for 24 hours; 50 μL of MTT is added to each well after 24 hours, and the plate is placed in an incubator for 2-4 hours; then the liquid in the wells is aspirated, 150 μL of DMSO is added to each well, and the absorbance (OD) value of each well is detected at 490 nm by using an enzyme-labeled instrument; the cell survival rate is calculated according to the OD value; and the blank group is cultured by using the DMEM complete culture solution;

[0132] The experimental results show that Figure 5 and Figure 6 as shown in the following table, wherein Figure 5 is a schematic diagram of the cytotoxicity of the hBMSCs cultured by the DMEM extract solution of the hydroxyapatite bone powder, Figure 6 is a schematic diagram of the cytotoxicity of the MG-63 cells cultured by the DMEM extract solution of the hydroxyapatite bone powder. The results show that the cell survival rate of the hBMSCs cultured by the extract solution of the hydroxyapatite bone powder at different concentrations is higher than 100% when the culturing time is 24 hours or 48 hours, which indicates that the prepared hydroxyapatite bone powder has the ability to promote the proliferation of hBMSCs; and the proliferation of the MG-63 cells is inhibited by all concentrations of the extract solution, and the inhibition is more significant as the concentration of the extract solution increases. Figure 5 and Figure 6 It can be seen that the hydroxyapatite bone powder has the effect of promoting the proliferation of normal cells but inhibiting the proliferation of tumor cells. Through the cell experiment, the hydroxyapatite bone powder prepared by the application can effectively realize the repair treatment after the osteosarcoma surgery, and has the characteristics of inhibiting the growth of residual osteosarcoma cells, resisting tumors, and promoting the generation of normal bones.

[0133] The above is only a preferred embodiment of the application, and does not limit the application in any way. Any simple modification, change, and equivalent structural change made according to the technical essence of the application to the above embodiment are still within the protection scope of the technical solution of the application.

Claims

1. A method for preparing hydroxyapatite bone powder for tissue repair after osteosarcoma surgery, characterized in that, The hydroxyapatite bone powder is a nanorod-shaped hydroxyapatite bone powder with a length of 150-200 nm and a diameter of 10-15 nm. The hydroxyapatite bone powder is a hydrophilic hydroxyapatite bone powder doped with Se and Mg. The preparation method of the hydroxyapatite bone powder includes: Step 1: Using CaCl2, Na2SeO3, soluble phosphate, and MgCl2 as main raw materials, a two-step sealed hydrothermal reaction is conducted to prepare Se and Mg co-doped hydrophobic hydroxyapatite; specifically including: Step 101: Under stirring conditions, add CaCl2 aqueous solution, Na2SeO3 aqueous solution and soluble phosphate aqueous solution to the mixed solution of oleic acid and methanol in sequence, stir, and obtain reaction system A; Step 102: Under sealed conditions at 100–180°C, react the reaction system A for 5–15 hours; Step 103: Cool the reaction system A to room temperature, add MgCl2 aqueous solution under stirring, and continue stirring for 10-30 min to obtain reaction system B; Step 104: Under sealed conditions at 100-180°C, react the reaction system B for 5-15 hours, cool to room temperature, and collect the precipitate; Step 105: After dispersing the precipitate, centrifuge to obtain Se and Mg co-doped hydrophobic hydroxyapatite; Step 2: Disperse the Se and Mg co-doped hydrophobic hydroxyapatite in cyclohexane, add dextran aqueous solution, stir for 3-6 hours, centrifuge, wash, freeze dry to obtain hydroxyapatite bone powder for tissue repair after osteosarcoma surgery.

2. The method for preparing hydroxyapatite bone powder for postoperative tissue repair in osteosarcoma according to claim 1, characterized in that, In step 101, the volume of methanol is 3 to 5 times the volume of oleic acid; in step 101, the stirring time is 10 to 30 minutes; in step 101, the volume of the CaCl2 aqueous solution is 1 to 7 times the volume of oleic acid.

3. The method for preparing hydroxyapatite bone powder for postoperative tissue repair in osteosarcoma according to claim 1, characterized in that, In step 101, the molar ratio of Ca / (P+Se) is 1.67, and the molar ratio of Se / (P+Se) is 0.005 to 0.

2.

4. The method for preparing hydroxyapatite bone powder for postoperative tissue repair in osteosarcoma according to claim 1, characterized in that, In step 101, the soluble phosphate is Na2HPO4, NaH2PO4, Na3PO4·12H2O, K2HPO4, or KH2PO4.

5. The method for preparing hydroxyapatite bone powder for postoperative tissue repair in osteosarcoma according to claim 1, characterized in that, In step 103, the molar ratio of Mg / Ca is 0.01 to 0.

4.

6. The method for preparing hydroxyapatite bone powder for postoperative tissue repair in osteosarcoma according to claim 1, characterized in that, The volume of the cyclohexane is 10 to 100 times the mass of the Se and Mg co-doped hydrophobic hydroxyapatite, the volume of the cyclohexane is in mL, and the mass of the Se and Mg co-doped hydrophobic hydroxyapatite is in g.

7. The method for preparing hydroxyapatite bone powder for postoperative tissue repair in osteosarcoma according to claim 1, characterized in that, The mass of the dextran is 40 to 75 times the mass of the Se and Mg co-doped hydrophobic hydroxyapatite.

8. The method for preparing hydroxyapatite bone powder for postoperative tissue repair in osteosarcoma according to claim 1, characterized in that, The concentration of the dextran aqueous solution is 0.05–0.15 g / mL.

9. A hydroxyapatite bone powder for postoperative tissue repair in osteosarcoma, prepared by the method described in claim 1.

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