Method for determining content of hydroxyapatite in collagen implant containing hydroxyapatite

Abstract

The invention discloses a method for determining the content of hydroxyapatite in a collagen implant containing hydroxyapatite, and relates to the technical field of hydroxyapatite content determination. The determination method comprises the following steps: mixing the collagen implant with an alkali solution or a collagenase solution, treating at the temperature of greater than or equal to 35 DEG C, hydrolyzing collagen in the collagen implant, filtering to obtain a solid, drying the solid, and weighing.

Description

Technical Field

[0001] The present application relates to the technical field of hydroxyapatite content determination, and in particular, to a method for determining the hydroxyapatite content in a collagen implant containing hydroxyapatite. Background Art

[0002] When preparing some compound mixtures, it is usually necessary to test and verify the content of each component added to the product. After preparing a collagen implant containing both hydroxyapatite and collagen, it is necessary to test the content of hydroxyapatite in the collagen implant. However, since hydroxyapatite is mixed with collagen liquid in a tiny powder state and the collagen liquid has a certain viscosity, it is difficult to separate hydroxyapatite from the collagen implant. Therefore, it is difficult to accurately detect the content of hydroxyapatite in the collagen implant containing both hydroxyapatite and collagen. Summary of the invention

[0003] The present application provides a method for determining the content of hydroxyapatite in a collagen implant containing hydroxyapatite, which can more accurately determine the content of hydroxyapatite in an implant containing both hydroxyapatite and collagen.

[0004] This application is implemented as follows:

[0005] The present application provides a method for determining the content of hydroxyapatite in a collagen implant containing hydroxyapatite, comprising the following steps:

[0006] The collagen implant is mixed with an alkaline solution or a collagenase solution, and treated at a temperature greater than or equal to 35° C. to hydrolyze the collagen in the collagen implant, and then filtered to obtain a solid, which is then dried and weighed.

[0007] In a possible embodiment, the alkaline solution includes any one of hydroxide, carbonate, and monohydrogen phosphate.

[0008] In a possible embodiment, the hydroxide includes at least one of NaOH, KOH and ammonia water; and the concentration of the hydroxide is 0.01 to 5 mol / L.

[0009] In one possible embodiment, the carbonate includes at least one of Na2CO3 and K2CO3;

[0010] And / or, the monohydrogen phosphate includes at least one of sodium monohydrogen phosphate and potassium monohydrogen phosphate.

[0011] In a possible embodiment, the collagen implant is mixed with an alkaline solution and then treated at a temperature of 50-70°C.

[0012] In a possible implementation, the concentration of the alkaline solution is 0.1-0.5 mol / L.

[0013] In a possible embodiment, after the collagen implant is mixed with the alkaline solution, the mixture is treated at a temperature of 70°C.

[0014] In a possible implementation, the concentration of the carbonate or the monohydrogen phosphate is 0.1-1 mol / L.

[0015] In a possible embodiment, the collagen implant is mixed with the collagenase solution and then treated at a temperature of 35-40°C.

[0016] In one possible embodiment, the collagen is non-cross-linked collagen.

[0017] The embodiments of the present application have at least the following beneficial effects:

[0018] The method for determining the content of hydroxyapatite in the collagen implant containing hydroxyapatite in the embodiment of the present application is to mix the collagen implant with an alkaline solution or a collagenase solution, and treat it at a temperature greater than or equal to 35°C, so that the collagen in the collagen implant is fully hydrolyzed, while the hydroxyapatite is not dissolved, and then filter to obtain a solid, dry the solid, and weigh it, which is the weight of the hydroxyapatite. It has been verified that the determination method can more accurately determine the content of hydroxyapatite in a collagen implant containing both hydroxyapatite and collagen. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a photo of the hydrolysis results of Example 1 of the present application;

[0020] Figure 2 This is a photo of the hydrolysis results of Example 2 of the present application;

[0021] Figure 3 This is a photo of the hydrolysis results of Example 3 of the present application;

[0022] Figure 4 This is a photo of the hydrolysis results of Example 4 of the present application;

[0023] Figure 5 This is a photo of the hydrolysis results of Example 5 of the present application;

[0024] Figure 6 This is a photo of the hydrolysis results of Example 6 of the present application;

[0025] Figure 7 This is a photo of the hydrolysis results of Example 7 of the present application;

[0026] Figure 8 This is a photo of the hydrolysis results of Example 8 of the present application;

[0027] Fig. 9 This is a photo of the hydrolysis results of Example 9 of the present application;

[0028] Fig.10 This is a photo of the hydrolysis results of Example 10 of the present application;

[0029] Fig.11 This is a photo of the hydrolysis results of Example 11 of the present application;

[0030] Fig.12 This is a photo of the hydrolysis results of Example 12 of the present application;

[0031] Fig.13 This is a photo of the hydrolysis results of Example 13 of the present application;

[0032] Fig.14 This is a photo of the hydrolysis results of Comparative Example 1 of this application;

[0033] Fig.15 This is a photo of the hydrolysis results of Comparative Example 2 of this application;

[0034] Fig.16 This is a photo of the hydrolysis results of Comparative Example 3 of this application;

[0035] Fig.17 This is a photo of the hydrolysis results of Comparative Example 4 of this application;

[0036] Fig.18 This is a photo of the hydrolysis result of Comparative Example 5 of the present application;

[0037] Fig.19 This is a photo of the hydrolysis results of Comparative Example 6 of this application;

[0038] Fig. 20 This is a photo of the hydrolysis results of Comparative Example 7 of the present application;

[0039] Fig.21 This is a photo of the hydrolysis results of Comparative Example 8 of this application. DETAILED DESCRIPTION

[0040] The embodiments of the present application will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present application and should not be considered as limiting the scope of the present application. In the examples, if specific conditions are not specified, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0041] The following is a specific description of the method for determining the content of hydroxyapatite in the hydroxyapatite-containing collagen implant of the present application embodiment:

[0042] In a first aspect, the present invention provides a method for determining the content of hydroxyapatite in a collagen implant containing hydroxyapatite, the method comprising the following steps:

[0043] The collagen implant is mixed with an alkaline solution or a collagenase solution, and treated at a temperature greater than or equal to 35° C. to hydrolyze the collagen in the collagen implant. The solid is then filtered to obtain a solid. The solid is dried and then weighed.

[0044] The determination method of the present application, by mixing the collagen implant with an alkaline solution or a collagenase solution, treating it at a temperature greater than or equal to 35°C, can make the collagen in the collagen implant be fully hydrolyzed, while the hydroxyapatite will not be dissolved, and then filtering to obtain a solid, and weighing the solid after drying, which is the weight of the hydroxyapatite. The inventors of the present application found in the study that when the temperature is less than 35°C, for example, 25°C, the collagen in the collagen implant cannot be completely hydrolyzed well. Exemplarily, the processing temperature of the present application is any one of 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C and 80°C, or a value between any two of them.

[0045] It has been verified that the determination method of the present application can more accurately determine the content of hydroxyapatite in a collagen implant containing both hydroxyapatite and collagen. It should be noted that the inventors of the present application found in their research that if an acid solution (such as hydrochloric acid) is mixed with a collagen implant containing both hydroxyapatite and collagen, the collagen will be hydrolyzed and the hydroxyapatite will also be dissolved, and the content of hydroxyapatite in the collagen implant containing both hydroxyapatite and collagen cannot be accurately detected.

[0046] In one embodiment, the alkaline solution includes any one of a hydroxide, a carbonate, and a monohydrogen phosphate. For example, the hydroxide includes at least one of NaOH, KOH, and ammonia water; the carbonate includes at least one of Na2CO3 and K2CO3; and the monohydrogen phosphate includes at least one of sodium monohydrogen phosphate and potassium monohydrogen phosphate.

[0047] Exemplarily, when the alkaline solution is a hydroxide, the concentration of the hydroxide is 0.01 to 5 mol / L, for example, the concentration of the hydroxide is 0.01 mol / L, 0.05 mol / L, 0.1 mol / L, 0.3 mol / L, 0.5 mol / L, 0.7 mol / L, 1 mol / L, 1.5 mol / L, 2 mol / L, 2.5 mol / L, 3 mol / L, 3.5 mol / L, 4 mol / L, 4.5 mol / L and 5 mol / L, or any value between two of them. When the hydroxide concentration is within the range of 0.01 to 5 mol / L and the temperature is greater than or equal to 35°C, the collagen in the collagen implant can be completely hydrolyzed.

[0048] When carbonate or monohydrogen phosphate is selected as the alkaline solution, the collagen implant is mixed with the alkaline solution and then treated at a temperature of 50 to 70°C. The inventors of the present application have found in their research that when carbonate or monohydrogen phosphate is selected as the alkaline solution, the collagen implant is mixed with the alkaline solution and then treated at a temperature of 50 to 70°C, which can make it easier to completely dissolve the collagen in the collagen implant. If the temperature is less than 50°C, the collagen in the collagen implant will not be completely dissolved for a long time.

[0049] In addition, when the alkaline solution is selected as carbonate or monohydrogen phosphate, and the collagen implant is mixed with the alkaline solution and treated at a temperature of 50 to 70°C. Exemplarily, the concentration of the alkaline solution is selected to be 0.1 to 0.5 mol / L, for example, any one of 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L and 0.5 mol / L or a value between any two of them. The inventors of the present application found in the study that when treated at a temperature of 50°C, when the concentration of carbonate or monohydrogen phosphate is greater than 0.5 mol / L (for example, 1 mol / L), the collagen in the collagen implant cannot be completely hydrolyzed. This may be because a high concentration of carbonate or monohydrogen phosphate can cause the collagen protein to precipitate easily and cannot be completely hydrolyzed, so that the hydroxyapatite cannot be completely filtered out, which leads to the inability to accurately detect the content of hydroxyapatite in the collagen implant.

[0050] In one embodiment, the collagen implant is mixed with the alkaline solution and then treated at a temperature of 70° C. When treated at a temperature of 70° C., whether the alkaline solution is a hydroxide, a carbonate or a monohydrogen phosphate, the collagen in the collagen implant can be completely hydrolyzed in a relatively short period of time.

[0051] For example, after the collagen implant is mixed with the alkaline solution, when the alkaline solution is treated at a temperature of 70°C, carbonate or monohydrogen phosphate is selected, and the concentration of carbonate or monohydrogen phosphate is 0.1 to 1 mol / L. When the temperature condition is 70°C, a higher concentration of carbonate or monohydrogen phosphate is selected, such as 1 mol / L, which can also completely hydrolyze the collagen in the collagen implant.

[0052] In another embodiment, the collagen implant is mixed with the collagenase solution and then treated at a temperature of 35-40° C., and the collagen is non-cross-linked collagen.

[0053] The inventors of the present application have found in their research that when the collagen in the collagen implant is non-cross-linked collagen, the collagen in the collagen implant can be completely hydrolyzed by utilizing the activity of collagenase at 35-40°C. For example, after the collagen implant is mixed with the collagenase solution, it is treated at temperatures of 35°C, 36°C, 37°C, 38°C, 39°C and 40°C. In addition, if the collagen is cross-linked collagen, the cross-linked collagen cannot be completely hydrolyzed, and the content of hydroxyapatite in the collagen implant cannot be detected more accurately.

[0054] The method for determining the content of hydroxyapatite in the hydroxyapatite-containing collagen implant of the present application is further described in detail below in conjunction with the examples.

[0055] Example 1

[0056] This embodiment provides a method for determining the content of hydroxyapatite in a collagen implant containing hydroxyapatite. The collagen implant is compounded with 150 mg of hydroxyapatite powder and 850 mg of collagen solution. The determination method comprises the following steps:

[0057] The collagen implant was mixed with 1 mol / L NaOH at a ratio of 1 g:5 mL and treated at 70°C for 3 min. The collagen in the collagen implant was completely hydrolyzed (refer to Figure 1 ). Then filter to obtain solid matter, dry the solid matter and weigh it. The results are recorded in Table 1.

[0058] Example 2

[0059] This embodiment provides a method for determining the content of hydroxyapatite in a collagen implant containing hydroxyapatite, which is different from the embodiment 1 only in that the concentration of NaOH in the embodiment 2 is 0.1 mol / L.

[0060] Example 3

[0061] This embodiment provides a method for determining the content of hydroxyapatite in a collagen implant containing hydroxyapatite. The only difference is that in this embodiment, NaOH in Example 1 is replaced by Na2CO3 with a concentration of 1 mol / L, and the collagen implant is mixed with Na2CO3 and treated at a temperature of 70°C for 6 minutes.

[0062] Example 4

[0063] This embodiment provides a method for determining the content of hydroxyapatite in a collagen implant containing hydroxyapatite, the only difference being that in this embodiment, NaOH in Example 1 is replaced with Na2CO3 having a concentration of 0.1 mol / L.

[0064] Example 5

[0065] This embodiment provides a method for determining the content of hydroxyapatite in a collagen implant containing hydroxyapatite. The only difference is that in this embodiment, NaOH in Example 1 is replaced by Na2HPO4 with a concentration of 1 mol / L, and the collagen implant is mixed with Na2HPO4 and then treated at a temperature of 70°C for 30 minutes.

[0066] Example 6

[0067] This embodiment provides a method for determining the content of hydroxyapatite in a collagen implant containing hydroxyapatite, the only difference being that in this embodiment, NaOH in Example 1 is replaced with Na2HPO4 with a concentration of 0.1 mol / L.

[0068] Example 7

[0069] This embodiment provides a method for determining the content of hydroxyapatite in a collagen implant containing hydroxyapatite. The collagen implant is compounded with 150 mg of hydroxyapatite powder and 850 mg of collagen solution. The determination method comprises the following steps:

[0070] The collagen implant was mixed with 1 mol / L NaOH at a ratio of 1 g:5 mL and treated at 50°C for 5 min. The collagen in the collagen implant was completely hydrolyzed (refer to Figure 7 ). Then filter to obtain solid matter, dry the solid matter and weigh it. The results are recorded in Table 1.

[0071] Example 8

[0072] This embodiment provides a method for determining the content of hydroxyapatite in a collagen implant containing hydroxyapatite, which is different from Embodiment 7 only in that the concentration of NaOH in Embodiment 8 is 0.1 mol / L.

[0073] Example 9

[0074] This embodiment provides a method for determining the content of hydroxyapatite in a collagen implant containing hydroxyapatite. The only difference is that in this embodiment, NaOH in Example 7 is replaced by Na2CO3 with a concentration of 0.1 mol / L, and the collagen implant is mixed with Na2CO3 and then treated at a temperature of 50°C for 10 minutes.

[0075] Example 10

[0076] This embodiment provides a method for determining the content of hydroxyapatite in a collagen implant containing hydroxyapatite. The only difference is that in this embodiment, NaOH in Example 7 is replaced by Na2HPO4 with a concentration of 0.1 mol / L, and the collagen implant is mixed with Na2HPO4 and then treated at a temperature of 50°C for 10 minutes.

[0077] Embodiment 11

[0078] This embodiment provides a method for determining the content of hydroxyapatite in a collagen implant containing hydroxyapatite. The collagen implant is compounded with 150 mg of hydroxyapatite powder and 850 mg of collagen solution. The determination method comprises the following steps:

[0079] The collagen implant was mixed with 1 mol / L NaOH at a ratio of 1 g:5 mL and treated at 35°C for 40 min. The collagen in the collagen implant was completely hydrolyzed (refer to Fig.11 ). Then filter to obtain solid matter, dry the solid matter and weigh it. The results are recorded in Table 1.

[0080] Example 12

[0081] This embodiment provides a method for determining the content of hydroxyapatite in a collagen implant containing hydroxyapatite, which is different from Example 11 only in that the concentration of NaOH in Example 12 is 0.1 mol / L, and after the collagen implant is mixed with NaOH, it is treated at a temperature of 35° C. for 23 h.

[0082] Embodiment 13

[0083] This embodiment provides a method for determining the content of hydroxyapatite in a collagen implant containing hydroxyapatite. The collagen implant is compounded with 150 mg of hydroxyapatite powder and 850 mg of collagen solution. The determination method comprises the following steps:

[0084] The collagen implant was mixed with collagenase at a concentration of 100 U / mL at a ratio of 1 g:5 mL and treated at a constant temperature of 37°C for 8 h. The collagen in the collagen implant was completely hydrolyzed (refer to Fig.13 ). Then filter to obtain solid matter, dry the solid matter and weigh it. The results are recorded in Table 1.

[0085] Embodiment 14

[0086] This embodiment provides a method for determining the content of hydroxyapatite in a collagen implant containing hydroxyapatite, which is different from Embodiment 1 only in that the concentration of NaOH in Embodiment 14 is 0.01 mol / L.

[0087] Embodiment 15

[0088] This embodiment provides a method for determining the content of hydroxyapatite in a collagen implant containing hydroxyapatite, which is different from Embodiment 1 only in that the concentration of NaOH in Embodiment 15 is 3 mol / L.

[0089] Comparative Example 1

[0090] Comparative Example 1 provides a method for determining the content of hydroxyapatite in a collagen implant containing hydroxyapatite, which is different from Example 9 only in that the concentration of Na2CO3 in Comparative Example 1 is 1 mol / L, and the collagen implant is mixed with Na2CO3 and treated at a temperature of 50°C for 23 hours. The experimental results show that the collagen in the collagen implant is not completely hydrolyzed.

[0091] Comparative Example 2

[0092] Comparative Example 2 provides a method for determining the content of hydroxyapatite in a collagen implant containing hydroxyapatite, which is different from Example 10 only in that the concentration of Na2HPO4 in Comparative Example 2 is 1 mol / L, and the collagen implant is mixed with Na2HPO4 and treated at a temperature of 50°C for 23 hours. The experimental results show that the collagen in the collagen implant is not completely hydrolyzed.

[0093] Comparative Example 3

[0094] Comparative Example 3 provides a method for determining the content of hydroxyapatite in a collagen implant containing hydroxyapatite, the only difference being that in Comparative Example 3, NaOH in Example 11 is replaced with Na2CO3 having a concentration of 1 mol / L, and the collagen implant is mixed with Na2CO3 and treated at a temperature of 35° C. for 23 h. The experimental results show that the collagen in the collagen implant is not completely hydrolyzed.

[0095] Comparative Example 4

[0096] Comparative Example 4 provides a method for determining the content of hydroxyapatite in a collagen implant containing hydroxyapatite, the only difference being that in Comparative Example 4, NaOH in Example 11 is replaced with Na2CO3 having a concentration of 0.1 mol / L, and the collagen implant is mixed with Na2CO3 and treated at a temperature of 35° C. for 23 h. The experimental results show that the collagen in the collagen implant is not completely hydrolyzed.

[0097] Comparative Example 5

[0098] Comparative Example 5 provides a method for determining the content of hydroxyapatite in a collagen implant containing hydroxyapatite, the only difference being that in Comparative Example 5, NaOH in Example 11 is replaced with Na2HPO4 having a concentration of 1 mol / L, and the collagen implant is mixed with Na2HPO4 and treated at a temperature of 35° C. for 23 h. The experimental results show that the collagen in the collagen implant is not completely hydrolyzed.

[0099] Comparative Example 6

[0100] Comparative Example 6 provides a method for determining the content of hydroxyapatite in a collagen implant containing hydroxyapatite, the only difference being that in Comparative Example 6, NaOH in Example 11 is replaced with Na2HPO4 having a concentration of 0.1 mol / L, and the collagen implant is mixed with Na2HPO4 and treated at a temperature of 35° C. for 23 h. The experimental results show that the collagen in the collagen implant is not completely hydrolyzed.

[0101] Comparative Example 7

[0102] Comparative Example 7 provides a method for determining the content of hydroxyapatite in a collagen implant containing hydroxyapatite, which is different from Example 1 only in that in Comparative Example 7, the collagen implant and NaOH are mixed and then treated at a temperature of 25° C. for 23 hours. The experimental results show that the collagen in the collagen implant is not completely hydrolyzed.

[0103] Comparative Example 8

[0104] Comparative Example 8 provides a method for determining the content of hydroxyapatite in a collagen implant containing hydroxyapatite, which is different from Example 2 only in that in Comparative Example 8, the collagen implant and NaOH are mixed and then treated at a temperature of 25° C. for 23 hours. The experimental results show that the collagen in the collagen implant is not completely hydrolyzed.

[0105] The collagen in the collagen implants of Examples 1 to 15 was completely hydrolyzed. The photos of the collagen in the collagen implants of Examples 1 to 13 were as follows: Figures 1 to 13 The collagen in the collagen implants of Comparative Examples 1 to 8 was not completely hydrolyzed, as shown in the photographs. Figures 14 to 21 In addition, the content of hydroxyapatite added to the collagen implant during compounding is 150 mg, and the hydroxyapatite detected by the determination method of the examples and comparative examples of the present application is recorded as M0, and the results are recorded in Table 1.

[0106] Table 1. Results of hydroxyapatite content determination

[0107]

[0108]

[0109] From the results in Table 1, it can be seen that the content of hydroxyapatite determined by the determination method of the present application is very close to or has no difference with the content of hydroxyapatite added during compounding in the collagen implant, which indicates that the determination method of the embodiment of the present application can more accurately determine the content of hydroxyapatite in the collagen implant containing both hydroxyapatite and collagen.

[0110] The above are only specific embodiments of the present application and are not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for determining the content of hydroxyapatite in a collagen implant containing hydroxyapatite, characterized in that: The following steps are involved: The collagen implant is mixed with an alkaline solution or a collagenase solution, and treated at a temperature greater than or equal to 35° C. so that the collagen in the collagen implant is hydrolyzed, and then filtered to obtain a solid, and the solid is dried and weighed.

2. The method for determining the content of hydroxyapatite in the collagen implant containing hydroxyapatite according to claim 1, characterized in that: The alkaline solution includes any one of hydroxide, carbonate, and monohydrogen phosphate.

3. The method for determining the content of hydroxyapatite in the collagen implant containing hydroxyapatite according to claim 2, characterized in that: The hydroxide includes at least one of NaOH, KOH and ammonia water; the concentration of the hydroxide is 0.01-5 mol / L.

4. The method for determining the content of hydroxyapatite in the collagen implant containing hydroxyapatite according to claim 2, characterized in that: The carbonate includes at least one of Na2CO3 and K2CO3; And / or, the monohydrogen phosphate includes at least one of sodium monohydrogen phosphate and potassium monohydrogen phosphate.

5. The method for determining the content of hydroxyapatite in the collagen implant containing hydroxyapatite according to claim 4, characterized in that: After the collagen implant is mixed with the alkaline solution, it is treated at a temperature of 50 to 70°C.

6. The method for determining the content of hydroxyapatite in the collagen implant containing hydroxyapatite according to claim 5, characterized in that: The concentration of the alkaline solution is 0.1-0.5 mol / L.

7. The method for determining the content of hydroxyapatite in the collagen implant containing hydroxyapatite according to claim 2, characterized in that: The collagen implant is mixed with an alkaline solution and then treated at a temperature of 70°C.

8. The method for determining the content of hydroxyapatite in the collagen implant containing hydroxyapatite according to claim 7, characterized in that: The concentration of the alkaline solution is 0.1-1 mol / L.

9. The method for determining the content of hydroxyapatite in the collagen implant containing hydroxyapatite according to claim 1, characterized in that: The collagen implant and the collagenase solution are mixed and then treated at a temperature of 35 to 40°C.

10. The method for determining the content of hydroxyapatite in the collagen implant containing hydroxyapatite according to claim 9, characterized in that: The collagen is non-cross-linked collagen.

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