Collagen and hydroxyapatite composite bone scaffold as well as preparation method and application thereof

Abstract

The invention discloses a collagen and hydroxyapatite composite bone scaffold as well as a preparation method and application thereof, and relates to the technical field of bone scaffolds. The preparation method of the collagen and hydroxyapatite composite bone scaffold comprises the following steps: dissolving collagen in an acid solution to obtain a collagen solution with the concentration of 10-80 mg / L, mixing the collagen solution with hydroxyapatite, and stirring to obtain a compound; freeze-drying the compound, and cross-linking to obtain a primary product; washing the primary product with water to remove acid residues, and then freeze-drying to obtain the collagen and hydroxyapatite composite bone scaffold. The composite bone scaffold is spongy, has relatively high porosity, is beneficial to bone conduction and bone induction, is not easy to disintegrate and slag when being wetted, and is relatively good in supporting property.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of bone scaffolds, in particular to a collagen and hydroxyapatite composite bone scaffold and a preparation method and application thereof. BACKGROUND

[0002] Bone defect is a disease caused by congenital or acquired reasons, which leads to the destruction of the structural integrity of the bone. The commonly used bone defect repair methods in clinic include autologous bone transplantation, allogeneic bone transplantation and bone tissue engineering technology. However, autologous bone transplantation has limited sources, and allogeneic bone transplantation is prone to immune rejection and other problems. Artificial bone has become the main way for bone defect repair at present.

[0003] Natural bone is a kind of inorganic / organic composite material with multi-level order, in which the organic component is mainly collagen, and the inorganic mineral is mainly hydroxyapatite. Artificial bone materials mainly made of collagen and hydroxyapatite are ideal materials that can be used for artificial bone substitutes. Based on this, the application provides a collagen and hydroxyapatite composite bone scaffold and a preparation method and application thereof. SUMMARY

[0004] The application provides a collagen and hydroxyapatite composite bone scaffold and a preparation method and application thereof. The composite bone scaffold is in the form of sponge, has high porosity, is beneficial to bone conduction and bone induction, and is not easy to crumble and drop slag when wet, and has good support.

[0005] The application is implemented as follows: In a first aspect, the application provides a preparation method of a collagen and hydroxyapatite composite bone scaffold, comprising the following steps: dissolving collagen in acetic acid solution to obtain a collagen solution with a concentration of 10-80 mg / L, and mixing the collagen solution with hydroxyapatite and stirring to obtain a composite; freeze-drying the composite to cross-link to obtain a primary product; washing the primary product to remove acid residues, and then freeze-drying to obtain a collagen and hydroxyapatite composite bone scaffold.

[0006] In a second aspect, the application provides a collagen and hydroxyapatite composite bone scaffold, which is prepared by the preparation method of the first aspect, and the porosity of the composite bone scaffold is greater than or equal to 90%.

[0007] In a third aspect, the application provides a use of the collagen and hydroxyapatite composite bone scaffold according to the second aspect in the preparation of bone tissue repair materials.

[0008] The application has at least the following beneficial effects: The method for preparing a collagen and hydroxyapatite composite bone scaffold disclosed in this application involves dissolving collagen in an acidic solution, then mixing the collagen solution with hydroxyapatite and stirring to ensure thorough and uniform mixing. After freeze-drying the composite, the collagen and hydroxyapatite do not separate into layers. Further cross-linking yields a structurally stable initial product. This initial product, with its stable structure, allows for easy washing to remove acid residue. The resulting freeze-dried composite bone scaffold is spongy with high porosity, beneficial for osteoconduction and bone induction. It is also less prone to fragmentation and flaking upon rehydration, and acid residue is thoroughly removed. The product's pH value meets relevant requirements. Furthermore, the ratio of collagen to hydroxyapatite in the composite bone scaffold can be precisely defined during compounding, making the product's composition more controllable and suitable for mass production. Attached Figure Description

[0009] Figure 1 This is a photograph of the composite bone scaffold of Embodiment 1 of this application; Figure 2 Here is a SEM image of the composite bone scaffold of Embodiment 1 of this application; Figure 3 A photograph of the composite bone scaffold of Embodiment 13 of this application; Figure 4 Here is a SEM image of the composite bone scaffold of Embodiment 13 of this application; Figure 5 This is a photograph of the composite bone scaffold of Embodiment 1 of this application after rehydration; Figure 6 This is a photograph of the composite bone scaffold of Embodiment 5 of this application after rehydration; Figure 7 This is a photograph of the composite bone scaffold of Example 23 of this application after rehydration; Figure 8 This is a photograph of the composite bone scaffold of Example 24 of this application after rehydration; Figure 9 This is a photograph of the lyophilized composite of Comparative Example 1 of this application; Figure 10 This is a photograph of the lyophilized composite of Comparative Example 2 of this application; Figure 11 This is a photograph of the lyophilized composite of Comparative Example 3 of this application; Figure 12 This is a photograph of the complex of Comparative Example 5 of this application after lyophilization and rehydration. Figure 13 This is a photograph of the composite of Comparative Example 7 of this application after lyophilization and immersion in a crosslinking agent solution. Figure 14 This is a photograph of the composite of Example 1 of this application after it has been freeze-dried and then immersed in a crosslinking agent solution. Detailed Implementation

[0010] The embodiments of the present application will be described in detail below with examples, but those skilled in the art will understand that the following examples are only for illustration of the present application and should not be regarded as limiting the scope of the present application. The specific conditions are not specified in the examples, and the conventional conditions or the conditions recommended by the manufacturer are used. The reagents or instruments used are not specified by the manufacturer, and are conventional products that can be obtained by commercial purchase.

[0011] The following will be specifically described for the preparation of the composite bone scaffold of collagen and hydroxyapatite according to the embodiments of the present application: In a first aspect, the embodiments of the present application provide a preparation method of a composite bone scaffold of collagen and hydroxyapatite, which comprises the following steps: (1) Dissolve collagen in an acid solution to obtain a collagen solution with a concentration of 10-80 mg / L, and mix the collagen solution with hydroxyapatite to obtain a composite after stirring. By dissolving collagen in an acid solution and then mixing the collagen solution with hydroxyapatite and stirring, the collagen and hydroxyapatite can be fully mixed and uniform, and the freeze-dried product is not prone to delamination. Exemplarily, the collagen is provided in the form of collagen sponge or collagen solution to be dissolved in acetic acid solution.

[0012] The inventors of the present application found in the research that the process of preparing the collagen solution has an important influence on the subsequent forming of the composite bone scaffold. The inventors of the present application tried to swell the collagen sponge in water and then add hydroxyapatite to stir and mix. Since the collagen sponge swells in water to form a gel, the collagen and hydroxyapatite are difficult to fully mix and uniform, and the freeze-dried product will delaminate obviously, and will become soft and spread after rehydration. In addition, if anhydrous ethanol is used to dissolve the collagen sponge, the collagen sponge is not soluble in anhydrous ethanol; if the collagen sponge is swelled in 50% ethanol, the collagen is partially swelled and forms a gel, and the collagen and hydroxyapatite are also difficult to fully mix and uniform after adding hydroxyapatite to stir and mix, and the freeze-dried product will spread after rehydration, and has poor hydrophobicity.

[0013] In addition, the inventors of the present application found in the research that if a phosphate solution (for example, containing monosodium phosphate and potassium dihydrogen phosphate) is used to mix with collagen, the phosphate solution contains sodium ions and potassium ions, and in order to meet the subsequent requirements for the bone scaffold product, it is necessary to remove the sodium ions and potassium ions, which increases the process and difficulty.

[0014] In the technical scheme of the present application, collagen is dissolved in an acid solution, mixed with hydroxyapatite to form a compound, and then the compound is freeze-dried, cross-linked, washed with water to remove acid residues, and freeze-dried again to obtain a composite bone scaffold of collagen and hydroxyapatite, which is not easy to disperse after rehydration. The proportion of collagen and hydroxyapatite in the composite bone scaffold can be defined during compounding, and the proportion of product components of the composite bone scaffold is more controllable, which is suitable for batch production of products.

[0015] Exemplarily, the acid solution includes any one or both of acetic acid solution and hydrochloric acid solution. Optionally, the concentration of the acetic acid solution is 0.1-2 mol / L, for example, any one of 0.1 mol / L, 0.2 mol / L, 0.5 mol / L, 0.8 mol / L, 1.0 mol / L, 1.2 mol / L, 1.5 mol / L, 1.8 mol / L and 2 mol / L or a value between any two of them.

[0016] Exemplarily, the concentration of the collagen solution is any one of 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, 50 mg / L, 60 mg / L, 70 mg / L and 80 mg / L or a value between any two of them.

[0017] Exemplarily, the particle size of the hydroxyapatite is microns, and optionally, the particle size of the hydroxyapatite is 10-100 μm, for example, any one of 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm and 100 μm or a value between any two of them.

[0018] In some embodiments, the mass ratio of collagen to hydroxyapatite in the compound is 0.5-3:1. The inventors of the present application found in research that if the collagen content in the compound is too low, the final composite bone scaffold will be prone to falling off, and the mass ratio of collagen to hydroxyapatite in the compound of the present application is 0.5-3:1, so that the prepared composite bone scaffold is not prone to falling off. Exemplarily, the mass ratio of collagen to hydroxyapatite in the compound is any one of 0.5:1, 1:1, 2:1 and 3:1 or a value between any two of them.

[0019] (2) The initial product is obtained by freeze-drying and cross-linking the compound.

[0020] The inventors of the present application found in research that different cross-linking methods also have an impact on the morphological stability of the composite bone scaffold, and the cross-linking methods include any one of cross-linking with a cross-linking agent solution and thermal cross-linking.

[0021] In some embodiments, the cross-linking method employs heat cross-linking, and the temperature of the heat cross-linking is exemplarily 110-150°C, and the treatment time of the heat cross-linking is exemplarily 20-48h. If the temperature is too low or the treatment time is too short, the composite bone scaffold will be scattered in water when employing the heat cross-linking method. In addition, the heat cross-linking method is relatively safer than the cross-linking method employing a cross-linking agent, and there is no problem of residual cross-linking agent.

[0022] Optionally, the temperature of the heat cross-linking is any one of 110°C, 120°C, 130°C, 140°C and 150°C or a value between any two of them. Optionally, the treatment time of the heat cross-linking is any one of 20h, 24h, 28h, 32h, 36h, 40h, 44h and 48h or a value between any two of them.

[0023] Further, the inventors of the present application found in research that when the cross-linking method employs heat cross-linking, increasing the step of immersing the initial product in an ethanol solution after heat cross-linking can promote the stability of the three-dimensional structure of the scaffold, and the composite bone scaffold obtained after subsequent water washing and freeze-drying is less likely to crumble and fall apart when rehydrated.

[0024] Optionally, the concentration of the ethanol solution is 30-60%, for example, 30%, 40%, 50% or 60%. Exemplarily, the temperature condition in the step of immersing the initial product in the ethanol solution is 2-10°C, and the time is 18-36h. Optionally, the temperature condition is any one of 2°C, 4°C, 6°C, 8°C and 10°C or a value between any two of them. Optionally, the immersion time is any one of 18h, 21h, 24h, 26h, 28h, 30h, 32h, 34h and 36h or a value between any two of them.

[0025] In another embodiment, the cross-linking method employs a cross-linking agent solution to cross-link the initial product. Optionally, the cross-linking agent solution contains 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (abbreviated as EDC) and N-hydroxysuccinimide (abbreviated as NHS), and the solvent of the cross-linking agent solution is an ethanol solution. The inventors of the present application found in research that the solvent of the cross-linking agent also affects the morphology of the composite bone scaffold. When water is used as the solvent of the cross-linking agent, the freeze-dried composite will partially dissolve, resulting in a paste-shaped composite that is not uniform, and subsequent water washing to remove acetic acid residues will be inconvenient and will easily cause uneven distribution of collagen and hydroxyapatite in the prepared composite bone scaffold, affecting the mechanical properties. When the solvent of the cross-linking agent is the ethanol solution of the present application, the freeze-dried composite still maintains its shape, facilitating subsequent water washing to remove acetic acid, and the obtained composite bone scaffold is less likely to crumble and fall apart after rehydration.

[0026] Optionally, the concentration of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide in the crosslinking agent solution is 1-8 mg / mL, for example, any one of 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL and 8 mg / mL or a value between any two of them. Optionally, the molar ratio of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide to N-hydroxysuccinimide is 1-4:1, for example, 1:1, 2:1, 3:1 or 4:1. Optionally, the concentration of the ethanol solvent used in the crosslinking agent solution is 50%-80%, for example, any one of 50%, 60%, 70% and 80% or a value between any two of them.

[0027] In addition, the inventors of the present application have also found in research that if the composite is crosslinked with a crosslinking agent first and then freeze-dried, the addition of the crosslinking agent solution to the composite solution will cause the composite solution to become dilute, and after freeze-drying, a larger pore size will occur, and the support is poor.

[0028] In a second aspect, the present application provides a collagen and hydroxyapatite composite bone scaffold, which is prepared by the preparation method of the first aspect. The porosity of the composite bone scaffold is ≥90%, which is beneficial to bone conduction and bone induction, and can be used in bone tissue repair materials.

[0029] In a third aspect, the present application provides the use of the collagen and hydroxyapatite composite bone scaffold of the second aspect in the preparation of bone tissue repair materials.

[0030] The collagen and hydroxyapatite composite bone scaffold and the preparation method and use thereof of the present application are described in further detail below in conjunction with examples.

[0031] Example 1 The present example provides a preparation method of a collagen and hydroxyapatite composite bone scaffold, which comprises the following steps: The collagen sponge is dissolved in an acetic acid solution with a concentration of 1 mol / L to obtain a collagen solution with a concentration of 40 mg / mL. Hydroxyapatite is added to the collagen solution for mixing and stirring according to a mass ratio of hydroxyapatite to collagen of 1:1 to obtain a composite. The particle size of the hydroxyapatite is 10-100 μm.

[0032] The composite is freeze-dried and then crosslinked with a crosslinking agent solution to obtain a preliminary product. The crosslinking agent solution contains 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (abbreviated as EDC) and N-hydroxysuccinimide (abbreviated as NHS), and the solvent in the crosslinking agent solution is an ethanol solution with a concentration of 70%. The content of EDC in the crosslinking agent solution is 4 mg / mL, and the molar ratio of EDC:NHS is 2:1. The state of the composite after freeze-drying and soaking in the crosslinking agent solution is as follows:Figure 14 as shown.

[0033] The primary product is washed with water to remove acetic acid residues, and then freeze-dried to obtain a collagen and hydroxyapatite composite bone scaffold (as shown in Figure 1

[0034] Example 2 The present example provides a method for preparing a collagen and hydroxyapatite composite bone scaffold, which differs from Example 1 only in that the acetic acid solution in Example 1 is replaced with a hydrochloric acid solution.

[0035] Example 3 The present example provides a method for preparing a collagen and hydroxyapatite composite bone scaffold, which differs from Example 1 only in that the concentration of the acetic acid solution in the present example is 0.5 mol / L.

[0036] Example 4 The present example provides a method for preparing a collagen and hydroxyapatite composite bone scaffold, which differs from Example 1 only in that the concentration of the acetic acid solution in the present example is 2 mol / L.

[0037] Example 5 The present example provides a method for preparing a collagen and hydroxyapatite composite bone scaffold, which differs from Example 1 only in that the mass ratio of collagen to hydroxyapatite in the composite is 3:2.

[0038] Example 6 The present example provides a method for preparing a collagen and hydroxyapatite composite bone scaffold, which differs from Example 1 only in that the mass ratio of collagen to hydroxyapatite in the composite is 3:1.

[0039] Example 7 The present example provides a method for preparing a collagen and hydroxyapatite composite bone scaffold, which differs from Example 1 only in that the mass ratio of collagen to hydroxyapatite in the composite is 1:2.

[0040] Example 8 The present example provides a method for preparing a collagen and hydroxyapatite composite bone scaffold, which differs from Example 1 only in that the content of EDC in the crosslinking agent solution of the present example is 2 mg / mL.

[0041] Example 9 The present example provides a method for preparing a collagen and hydroxyapatite composite bone scaffold, which differs from Example 1 only in that the content of EDC in the crosslinking agent solution of the present example is 1 mg / mL.

[0042] Example 10 ​The present example provides a preparation method of a composite bone scaffold of collagen and hydroxyapatite, which is different from that of Example 1 only in that the content of EDC in the crosslinking agent solution of the present example is 6 mg / mL.

[0043] Example 11 The present example provides a preparation method of a composite bone scaffold of collagen and hydroxyapatite, which is different from that of Example 1 only in that the content of EDC in the crosslinking agent solution of the present example is 8 mg / mL.

[0044] Example 12 The present example provides a preparation method of a composite bone scaffold of collagen and hydroxyapatite, which is different from that of Example 1 only in that the crosslinking agent solution uses ethanol with a concentration of 50% as a solvent.

[0045] Example 13 The present example provides a preparation method of a composite bone scaffold of collagen and hydroxyapatite, which is different from that of Example 1 only in that the present example uses a heat crosslinking method for crosslinking, and the initial product is obtained by treating at a temperature of 120°C for 24 h, then the initial product is soaked in an ethanol solution with a concentration of 40% and treated at a temperature of 4°C for 24 h, and then washed with water. The composite bone scaffold prepared in the present example is shown in FIG. 13. Figure 3

[0046] Example 14 The present example provides a preparation method of a composite bone scaffold of collagen and hydroxyapatite, which is different from that of Example 13 only in that the initial product is soaked in an ethanol solution with a concentration of 60% and treated at a temperature of 8°C for 20 h.

[0047] Example 15 The present example provides a preparation method of a composite bone scaffold of collagen and hydroxyapatite, which is different from that of Example 13 only in that the heat crosslinking in the present example is treated at a temperature of 120°C for 48 h.

[0048] Example 16 The present example provides a preparation method of a composite bone scaffold of collagen and hydroxyapatite, which is different from that of Example 13 only in that the heat crosslinking in the present example is treated at a temperature of 110°C for 48 h.

[0049] Example 17 The present example provides a preparation method of a composite bone scaffold of collagen and hydroxyapatite, which is different from that of Example 13 only in that the heat crosslinking in the present example is treated at a temperature of 130°C for 20 h.

[0050] Example 18 ​The present example provides a method for preparing a composite bone scaffold of collagen and hydroxyapatite, which differs from Example 13 in that the heat crosslinking in the present example is performed at a temperature of 100°C for 12h.

[0051] Example 19 The present example provides a method for preparing a composite bone scaffold of collagen and hydroxyapatite, which differs from Example 1 only in that the concentration of the collagen solution in the present example is 30mg / mL.

[0052] Example 20 The present example provides a method for preparing a composite bone scaffold of collagen and hydroxyapatite, which differs from Example 1 only in that the concentration of the collagen solution in the present example is 15mg / mL.

[0053] Example 21 The present example provides a method for preparing a composite bone scaffold of collagen and hydroxyapatite, which differs from Example 1 only in that the concentration of the collagen solution in the present example is 60mg / mL.

[0054] Example 22 The present example provides a method for preparing a composite bone scaffold of collagen and hydroxyapatite, which differs from Example 1 only in that the concentration of the collagen solution in the present example is 80mg / mL.

[0055] Example 23 Example 23 provides a method for preparing a composite bone scaffold of collagen and hydroxyapatite, which differs from Example 1 only in that the mass ratio of collagen to hydroxyapatite in the composite is 3:7.

[0056] Example 24 Example 24 provides a method for preparing a composite bone scaffold of collagen and hydroxyapatite, which differs from Example 1 only in that the mass ratio of collagen to hydroxyapatite in the composite is 1:4.

[0057] Example 25 Example 25 provides a method for preparing a composite bone scaffold of collagen and hydroxyapatite, which differs from Example 1 only in that the collagen sponge in Example 1 is replaced with a collagen solution in the present example.

[0058] Example 26 Example 26 provides a method for preparing a composite bone scaffold of collagen and hydroxyapatite, which differs from Example 13 only in that the step of immersing the initial product in an ethanol solution with a concentration of 40% and treating at a temperature of 4°C for 24h is removed in the present example.

[0059] Comparative Example 1 Comparative Example 1 provides a method for preparing a composite bone scaffold of collagen and hydroxyapatite, which comprises the following steps: The collagen sponge in Example 1 was swelled in water to obtain a collagen solution with a concentration of 10 mg / mL, and hydroxyapatite was added in the collagen solution for mixing and stirring according to a mass ratio of hydroxyapatite to collagen of 1:1 to obtain a composite. The hydroxyapatite used herein was the same as that in Example 1. After the composite was freeze-dried, the hydroxyapatite and the collagen were layered, and obvious sponge-like objects were visible in the upper part of the centrifuge tube, and the hydroxyapatite in the lower part was shrunk after freeze-drying and had a large hardness (as shown in Figure 9 ).

[0060] Comparative Example 2 Comparative Example 2 provides a method for preparing a composite bone scaffold of collagen and hydroxyapatite, which is different from Comparative Example 1 only in that the concentration of the collagen solution in Comparative Example 2 is 20 mg / mL. The results show that after the composite is freeze-dried, the hydroxyapatite and the collagen are layered, and obvious sponge-like objects are visible in the upper part of the centrifuge tube, and the hydroxyapatite in the lower part is shrunk after freeze-drying and has a large hardness (as shown in Figure 10 ).

[0061] Comparative Example 3 Comparative Example 3 provides a method for preparing a composite bone scaffold of collagen and hydroxyapatite, which is different from Comparative Example 1 only in that the concentration of the collagen solution in Comparative Example 3 is 30 mg / mL. The results show that after the composite is freeze-dried, the hydroxyapatite and the collagen are layered, and obvious sponge-like objects are visible in the upper part of the centrifuge tube, and the lower part is the hydroxyapatite with a large hardness (as shown in Figure 11 ).

[0062] Comparative Example 4 Comparative Example 4 provides a method for preparing a composite bone scaffold of collagen and hydroxyapatite, which is different from Comparative Example 1 only in that the concentration of the collagen solution in Comparative Example 4 is 48 mg / mL. The results show that the hydroxyapatite and the collagen are difficult to mix uniformly.

[0063] Comparative Example 5 Comparative Example 5 provides a method for preparing a composite bone scaffold of collagen and hydroxyapatite, which is different from Example 1 only in that the collagen sponge in Example 1 is swelled in ethanol with a concentration of 50%. The collagen and the hydroxyapatite in the composite cannot be mixed uniformly, and the composite is scattered after rehydration after freeze-drying, and has a poor hydrophobicity (as shown in Figure 12 ).

[0064] Comparative Example 6 Comparative Example 6 provides a method for preparing a composite bone scaffold of collagen and hydroxyapatite, which is different from Example 1 only in that the composite is first crosslinked with a crosslinking agent solution and then freeze-dried. The freeze-drying conditions and the crosslinking agent solution are the same as in Example 1. The sponge pores of the composite bone scaffold are relatively large, and the support is poor.

[0065] Comparative Example 7 Comparative Example 7 provides a method for preparing a composite bone scaffold of collagen and hydroxyapatite, which is different from Example 1 only in that the solvent of the crosslinking agent in Comparative Example 7 is water. After the freeze-dried composite is soaked in the crosslinking agent, the composite is partially dissolved, resulting in the formed composite being mixed into an uneven paste (as shown in Figure 13 ).

[0066] Test Example 1 The composite bone scaffolds of collagen and hydroxyapatite prepared in some embodiments of the present application were subjected to porosity determination by mercury intrusion method, and the results are shown in Table 1.

[0067]

[0068] As can be seen from the results in Table 1, the composite bone scaffolds of collagen and hydroxyapatite prepared in the embodiments of the present application have a high porosity, all greater than 90%, which is conducive to bone conduction and bone induction.

[0069] Test Example 2 The composite bone scaffolds of collagen and hydroxyapatite prepared in Examples 1-12 were placed in centrifuge tubes containing water, and then subjected to shaking at 37°C and 120 rpm for 48 h, and the results showed that the composite bone scaffolds did not have a collapse phenomenon; after 72 h of shaking, the edges of the composite bone scaffolds were slightly scattered.

[0070] The composite bone scaffolds of collagen and hydroxyapatite prepared in Examples 13-22 and Example 26 were placed in centrifuge tubes containing water, and then subjected to shaking at 37°C and 120 rpm for 8 h, and the results showed that the composite bone scaffold of Example 18 collapsed in a large area, and there was turbidity in the water; the composite bone scaffold of Example 26 had a slight edge scattering; while the composite bone scaffolds of Examples 13-17 and Examples 19-22 all maintained good integrity and did not have a collapse and scattering phenomenon. The composite bone scaffolds of Examples 13-17 and Examples 19-22 were continued to be shaken for 4 h, and they all maintained good integrity.

[0071] It is illustrated that when the initial product is crosslinked by heat crosslinking, if the temperature is too low and the crosslinking time is too short, the composite bone scaffold structure will be unstable, and it will be easy to scatter and drop slag after rehydration shock. In addition, when the initial product is crosslinked by heat crosslinking, the step of soaking the initial product in an ethanol solution can help maintain the integrity of the composite bone scaffold.

[0072] Test Example 3 The composite bone scaffolds of collagen and hydroxyapatite prepared in Example 1, Example 5, Example 23 and Example 24 were rehydrated, and the results are shown in Figures 5-8 As shown in the table, the composite bone scaffolds of Example 1 and Example 5 did not crumble and drop slag after rehydration; while the composite bone scaffolds of Example 23 and Example 24 all had different degrees of slag drop, and the slag drop of Example 24 was more serious than that of Example 23.

[0073] It is illustrated that the mass ratio of collagen and hydroxyapatite will affect the structural stability of the composite bone scaffold, and the application further limits the mass ratio of collagen and hydroxyapatite in the composite to be 0.5-3:1; when the mass ratio of collagen and hydroxyapatite is not within the limited range, especially when the proportion of hydroxyapatite exceeds the limited range, the composite bone scaffold will drop more slag after rehydration.

[0074] Test Example 4 The leaching solution of the composite bone scaffold of Example 1 was obtained by leaching method, and the pH values of the leaching solution at 0h, 24h, 48h and 72h were tested, and the results are shown in Table 2.

[0075]

[0076] The pH value of the leaching solution of the composite bone scaffold of Example 1 was stable at neutral within 72h, and the pH change was not more than 0.3. It is illustrated that the preparation method of Example 1 can basically remove the residual acetic acid, so as to keep the pH value of the leaching solution stable at neutral.

[0077] Test Example 5 The composite bone scaffolds prepared in Example 1 and Example 13 were observed under an electronic scanning fiber microscope, and the SEM images thereof are shown in Figure 2 and Figure 4 .

[0078] From Figure 2 and Figure 4 , it can be seen that the composite bone scaffolds of Example 1 and Example 13 both have a large number of irregular pore structures, and most of the pore diameters are micron-sized small pores.

[0079] The above descriptions 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 can have various modifications and changes. Any modifications, equivalent replacements, improvements, 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 preparing a composite bone scaffold of collagen and hydroxyapatite, characterized in that, Includes the following steps: Collagen was dissolved in an acidic solution to obtain a collagen solution with a concentration of 10-80 mg / L. The collagen solution was then mixed with hydroxyapatite and stirred to obtain a complex. The composite was freeze-dried and then cross-linked to obtain a preliminary product. The initial product was washed with water to remove acid residue, and then freeze-dried to obtain a composite bone scaffold of collagen and hydroxyapatite.

2. The method for preparing the composite bone scaffold of collagen and hydroxyapatite according to claim 1, characterized in that, The mass ratio of collagen to hydroxyapatite in the composite is 0.5~3:

1.

3. The method for preparing the composite bone scaffold of collagen and hydroxyapatite according to claim 1, characterized in that, The acid solution includes any one or both of acetic acid solution and hydrochloric acid solution; optionally, the concentration of the acetic acid solution is 0.1~2 mol / L.

4. The method for preparing the composite bone scaffold of collagen and hydroxyapatite according to claim 1, characterized in that, The hydroxyapatite has a particle size in the micrometer range, and optionally, the particle size of the hydroxyapatite is 10~100μm.

5. The method for preparing the composite bone scaffold of collagen and hydroxyapatite according to any one of claims 1 to 4, characterized in that, Crosslinking methods include either crosslinking using a crosslinking agent solution or thermal crosslinking.

6. The method for preparing the composite bone scaffold of collagen and hydroxyapatite according to claim 5, characterized in that, The crosslinking agent solution contains 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and the solvent of the crosslinking agent solution is an ethanol solution; optionally, the concentration of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 1~8 mg / mL; optionally, the concentration of the ethanol solution is 50%~80%.

7. The method for preparing the composite bone scaffold of collagen and hydroxyapatite according to claim 5, characterized in that, The crosslinking method is thermal crosslinking, and the step of soaking the initial product in an ethanol solution is included between the thermal crosslinking and the water washing step; optionally, the temperature of the thermal crosslinking is 110~150℃, and the treatment time of the thermal crosslinking is 20~48h; optionally, the concentration of the ethanol solution is 30%~60%.

8. The method for preparing the composite bone scaffold of collagen and hydroxyapatite according to any one of claims 1 to 4, characterized in that, The collagen is provided in the form of collagen sponge or collagen liquid to dissolve in an acidic solution.

9. A composite bone scaffold of collagen and hydroxyapatite, prepared by the preparation method according to any one of claims 1 to 8, wherein the porosity of the composite bone scaffold is ≥90%.

10. The application of a composite bone scaffold of collagen and hydroxyapatite as described in claim 9 in the preparation of bone tissue repair materials.

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