Collagen scaffold with fixed-axis orientation structure as well as preparation method and application of collagen scaffold

Abstract

The invention relates to a collagen scaffold with a fixed-axis orientation structure and a preparation method and application thereof, and the preparation method comprises the following steps: (1) dropwise adding a soluble phosphate solution into a collagen-soluble calcium salt mixed solution, and adjusting the pH value with ammonia water; (2) injecting the obtained mixed solution into a mold of which the bottom is provided with a heat-conducting bottom plate; vacuumizing is conducted, heat insulation treatment is conducted on the part, except for the heat conduction bottom plate, of the mold, and then the mold is frozen and formed at the temperature ranging from-20 DEG C to-180 DEG C; and (3) freeze drying. According to the preparation method, heat insulation treatment is conducted on the part, except for a heat conduction bottom plate, of a mold, only the heat conduction bottom plate is left for heat exchange with the external environment, during freezing, low temperature is gradually transmitted upwards from the heat conduction bottom plate to form a temperature gradient, ice crystals of an oriented structure are formed, and then the ice crystals are removed through freeze drying. The interior of the scaffold can be provided with a structure which is similar to natural bones and is arranged in a Harvard system, so that osteoblast proliferation is facilitated, and good conditions are created for growth of cells and blood vessels.

Description

Technical Field

[0001] The present invention relates to the technical field of medical biomaterials, and in particular to a collagen scaffold with a fixed-axis orientation structure, and a preparation method and application thereof. Background Art

[0002] Bone is a hard tissue organ in the body. Its main components include type I collagen and multi-level ordered structures of hydroxyapatite crystals. The most important part of bone tissue is nano-hydroxyapatite, which accounts for 69%. The other part is composed of 20% collagen and 9% water. There are also a small amount of organic substances, such as proteins, lipids, etc. Bone tissue has inherent self-repair ability, but in most cases it cannot complete bone regeneration or needs to be stimulated to complete bone regeneration. Every year, millions of patients with bone defects need bone transplants or bone substitute implants. Therefore, the use of biological scaffold materials to simulate the microenvironment and stimulate bone tissue regeneration to achieve bone defect repair has become a hot topic of concern.

[0003] The hard tissues of natural organisms have a hierarchical structure from micro to macro. How to achieve bionic manufacturing from micro-nano structures based on component simulation is a major challenge currently faced in the design and manufacture of tissue engineering scaffold materials. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a method for preparing a collagen scaffold with a simple process, good repeatability and an axially oriented structure of natural bionic bone, so as to obtain a composite material scaffold with good mechanical properties, cell adhesion and osteogenic activity.

[0005] One aspect of the present invention provides a method for preparing a collagen scaffold with an axially oriented structure, comprising the following steps:

[0006] (1) adding a soluble phosphate solution dropwise to a collagen-soluble calcium salt mixed solution under stirring at 37° C.; then adjusting the pH to 9 to 12 with aqueous ammonia, and continuing stirring at 37° C. for 6 to 24 hours;

[0007] (2) injecting the mixed solution obtained in step (1) into a mold, wherein a heat-conducting bottom plate is provided at the bottom of the mold; evacuating the mold, performing heat insulation treatment on the mold except for the heat-conducting bottom plate, and then placing the mold in a cold environment of -20°C to -180°C until the mold is completely frozen and formed;

[0008] (3) Taking out the sample frozen in step (2), freeze-drying it, and obtaining the collagen scaffold with a fixed-axis oriented structure.

[0009] By constructing a solution system similar to bone components and then freezing it in a mold that conducts heat only through a heat-conducting base plate, a directional heat-conducting freezing can be obtained, which is more conducive to cell proliferation than the collagen / collagen composite scaffold prepared by conventional freeze-drying.

[0010] Furthermore, the soluble calcium solution is calcium chloride or calcium nitrate tetrahydrate solution.

[0011] Furthermore, the concentration of collagen in the collagen-soluble calcium salt mixed solution is 30-100 mg / ml, and the mass concentration of calcium ions: the mass concentration of collagen = (1-0.8): 1. Further preferably, the concentration of collagen in the collagen-soluble calcium salt mixed solution is 60-100 mg / ml.

[0012] Furthermore, the soluble phosphate is one or more of disodium hydrogen phosphate, sodium dihydrogen phosphate, diammonium hydrogen phosphate, ammonium dihydrogen phosphate, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate.

[0013] Furthermore, the soluble phosphate solution and the collagen-soluble calcium salt mixed solution have the same volume, and the molar concentration of calcium ions in the collagen-soluble calcium salt mixed solution: the molar concentration of phosphate ions in the soluble phosphate solution = 1.67.

[0014] Furthermore, the thermally conductive base plate material is metal copper, aluminum or iron.

[0015] Furthermore, the cold environment temperature may be -20°C, -40°C, -60°C, -80°C, -100°C, -120°C, -140°C, -160°C, -180°C, etc., and may be provided by a refrigerator, dry ice or liquid nitrogen, etc. Preferably, it is -150 to -180°C.

[0016] Furthermore, the freeze-drying time is 12 to 24 hours.

[0017] Another aspect of the present invention also provides a collagen scaffold prepared by the preparation method of the collagen scaffold with a fixed-axis oriented structure.

[0018] Another aspect of the present invention also provides the use of the collagen scaffold in preparing bone regeneration products.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The preparation method of the present invention performs heat insulation treatment on the part of the mold except the heat-conducting bottom plate, leaving only the heat-conducting bottom plate to exchange heat with the external environment, and the low temperature is gradually transferred upward from the heat-conducting bottom plate to form a temperature gradient. When the temperature is lowered below the freezing point of the system, the solvent water in the solution in the mold first crystallizes and nucleates on the frozen surface, and then grows directionally along the direction of the temperature gradient to form ice crystals with an oriented structure. After that, the ice crystals are removed by freeze-drying to obtain a porous skeleton with an oriented structure. The interior of the skeleton has a structure arranged in a Haversian system similar to natural bones, which is beneficial to the proliferation of osteoblasts and also creates good conditions for the growth of cells and blood vessels. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the preparation method of an embodiment of the present invention;

[0022] Figure 2 Schematic diagram of the traditional freeze-drying preparation method;

[0023] Figure 3 The absorbance test results of the collagen scaffolds of the embodiment and the comparative example at different time points. DETAILED DESCRIPTION

[0024] Reference Figure 1 The present invention provides a method for preparing a collagen scaffold with a fixed-axis orientation structure, comprising the following steps:

[0025] (1) adding a soluble phosphate solution dropwise to a collagen-soluble calcium salt mixed solution under stirring at 37° C.; then adjusting the pH of the mixed solution to 9 to 12 with aqueous ammonia, and continuing stirring at 37° C. for 6 to 24 hours;

[0026] (2) injecting the mixed solution obtained in step (1) into a mold, wherein a heat-conducting bottom plate is provided at the bottom of the mold; vacuuming the mold to remove air from the mold, performing heat insulation treatment on the mold except for the heat-conducting bottom plate, and then placing the mold in a cold environment of -20°C to -180°C until the mold is completely frozen and formed;

[0027] (3) Taking out the sample frozen in step (2), freeze-drying it, and obtaining the collagen scaffold with a fixed-axis oriented structure.

[0028] The preparation method of the present invention, referring to Figure 1, by insulating the part of the mold except the heat-conducting bottom plate, it can be wrapped with insulation materials or equipped with insulation devices, leaving only the heat-conducting bottom plate for heat exchange with the external environment. The low temperature is gradually transferred upward from the heat-conducting bottom plate to form a temperature gradient, which can conduct heat in a directional manner. When the temperature is lowered below the freezing point of the system, the solvent water in the solution in the mold first crystallizes and nucleates on the frozen surface, and then grows in a directional manner along the direction of the temperature gradient to form ice crystals with an oriented structure. After that, the ice crystals are removed by freeze-drying to obtain a porous material with an oriented structure, which can form a Haversian system arrangement structure with natural bone, which is beneficial to the proliferation of osteoblasts and also creates good conditions for the growth of cells and blood vessels.

[0029] The traditional freeze-drying method (such as Figure 2 As shown in the figure, after freezing, the scaffold structure is uniform and cannot form an arrangement structure similar to the Haversian system of natural bone.

[0030] By further exploring the system concentration and freezing temperature, the scaffold structure was further optimized to be more conducive to osteoblast proliferation.

[0031] The present invention will be described more fully below in conjunction with specific embodiments. The following are only preferred embodiments of the present invention. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0032] In the following examples and comparative examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0033] Example 1

[0034] A method for preparing a collagen scaffold with a fixed-axis orientation structure comprises the following steps:

[0035] (1) fully dissolving collagen in a solution of calcium nitrate tetrahydrate to obtain a collagen-soluble calcium salt mixed solution, wherein the calcium ion concentration is 40 mg / mL and the collagen concentration is 40 mg / mL; and continuously stirring at 37° C.; while stirring, dropwise adding an equal volume of diammonium hydrogen phosphate solution having a phosphate ion concentration of 56.89 mg / mL; and then adjusting the pH of the obtained new mixed solution to 11 with ammonia water, and continuously stirring at 37° C. for 24 hours;

[0036] (2) according to the desired shape of the bracket, the mixed solution obtained in step (1) is injected into a mold with a heat-conducting base plate installed at the bottom, the air is removed by vacuum, and the mold is sealed with a heat-insulating material except for the heat-conducting base plate, and placed in a -80°C cold environment. The low temperature is gradually transferred upward from the heat-conducting base plate to form a temperature gradient until it is completely frozen and formed;

[0037] (3) The sample formed by freeze-forming in step (2) is taken out and freeze-dried for 24 hours until it is completely freeze-dried, thereby obtaining a collagen scaffold with a fixed axis orientation structure.

[0038] Example 2

[0039] A method for preparing a collagen scaffold with a fixed-axis orientation structure comprises the following steps:

[0040] (1) fully dissolving collagen in a solution of calcium nitrate tetrahydrate to obtain a collagen-soluble calcium salt mixed solution, wherein the calcium ion concentration is 40 mg / mL and the collagen concentration is 40 mg / mL; and continuously stirring at 37° C.; while stirring, dropwise adding an equal volume of diammonium hydrogen phosphate solution having a phosphate ion concentration of 56.89 mg / mL; and then adjusting the pH of the obtained new mixed solution to 11 with ammonia water, and continuously stirring at 37° C. for 24 hours;

[0041] (2) Injecting the mixed solution obtained in step (1) into a mold according to the desired shape of the bracket, installing a heat-conducting bottom plate on the bottom plate of the mold, removing air and sealing the mold except the heat-conducting bottom plate with a heat-insulating material or device, and placing the mold in a -180°C cold environment, so that the low temperature is gradually transferred upward from the heat-conducting bottom plate to form a temperature gradient until the mold is completely frozen and formed;

[0042] (3) The sample formed by freeze-forming in step (2) is taken out and freeze-dried for 24 hours until it is completely freeze-dried, thereby obtaining a collagen scaffold with a fixed axis orientation structure.

[0043] Example 3

[0044] A method for preparing a collagen scaffold with an axially oriented structure comprises the following steps:

[0045] (1) fully dissolving collagen in a solution of calcium nitrate tetrahydrate to obtain a collagen-soluble calcium salt mixed solution, wherein the calcium ion concentration is 60 mg / mL and the collagen concentration is 60 mg / mL; and continuously stirring at 37° C.; while stirring, dropwise adding an equal volume of diammonium hydrogen phosphate solution having a phosphate ion concentration of 85.33 mg / mL; and then adjusting the pH of the obtained new mixed solution to 11 with ammonia water, and continuously stirring at 37° C. for 24 hours;

[0046] (2) Injecting the mixed solution obtained in step (1) into a mold according to the desired shape of the bracket, installing a heat-conducting bottom plate on the bottom plate of the mold, vacuuming to remove air, and sealing the mold except the heat-conducting bottom plate with a heat-insulating device, placing the mold in a -180°C cold environment, so that the low temperature is gradually transferred upward from the heat-conducting bottom plate to form a temperature gradient until the mold is completely frozen and formed;

[0047] (3) The sample formed by freeze-forming in step (2) is taken out and freeze-dried for 24 hours until it is completely freeze-dried, thereby obtaining a collagen scaffold with a fixed axis orientation structure.

[0048] Example 4

[0049] A method for preparing a collagen scaffold with an axially oriented structure comprises the following steps:

[0050] (1) fully dissolving collagen in a solution of calcium nitrate tetrahydrate to obtain a collagen-soluble calcium salt mixed solution, wherein the calcium ion concentration is 100 mg / mL and the collagen concentration is 100 mg / mL; and continuously stirring at 37° C.; while stirring, dropwise adding an equal volume of diammonium hydrogen phosphate solution having a phosphate ion concentration of 142.21 mg / mL; and then adjusting the pH of the obtained new mixed solution to 11 with ammonia water, and continuously stirring at 37° C. for 24 hours;

[0051] (2) Injecting the mixed solution obtained in step (1) into a mold according to the desired shape of the bracket, installing a heat-conducting bottom plate on the bottom plate of the mold, vacuuming to remove air, and sealing the mold except the heat-conducting bottom plate with a heat-insulating device, placing the mold in a -180°C cold environment, so that the low temperature is gradually transferred upward from the heat-conducting bottom plate to form a temperature gradient until the mold is completely frozen and formed;

[0052] (3) The sample formed by freeze-forming in step (2) is taken out and freeze-dried for 24 hours until it is completely freeze-dried, thereby obtaining a collagen scaffold with a fixed axis orientation structure.

[0053] Comparative Example 1

[0054] A method for preparing a collagen scaffold with an axially oriented structure comprises the following steps:

[0055] (1) fully dissolving collagen in a solution of calcium nitrate tetrahydrate to obtain a collagen-soluble calcium salt mixed solution, wherein the calcium ion concentration is 100 mg / mL and the collagen concentration is 100 mg / mL; and continuously stirring at 37° C.; while stirring, dropwise adding an equal volume of diammonium hydrogen phosphate solution having a phosphate ion concentration of 142.21 mg / mL; and then adjusting the pH of the obtained new mixed solution to 11 with ammonia water, and continuously stirring at 37° C. for 24 hours;

[0056] (2) injecting the mixed solution obtained in step (1) into a mold according to the desired shape of the scaffold, vacuuming to remove air, and placing in a -80°C cold environment until it is completely frozen into shape;

[0057] (3) The sample formed by freeze-forming in step (2) is taken out and freeze-dried for 24 hours until it is completely freeze-dried, thereby obtaining a collagen scaffold.

[0058] Comparative Example 2

[0059] A method for preparing a collagen scaffold with an axially oriented structure comprises the following steps:

[0060] (1) fully dissolving collagen in a solution of calcium nitrate tetrahydrate to obtain a collagen-soluble calcium salt mixed solution, wherein the calcium ion concentration is 100 mg / mL and the collagen concentration is 100 mg / mL; and continuously stirring at 37° C.; while stirring, dropwise adding an equal volume of diammonium hydrogen phosphate solution having a phosphate ion concentration of 142.21 mg / mL; and then adjusting the pH of the obtained new mixed solution to 11 with ammonia water, and continuously stirring at 37° C. for 24 hours;

[0061] (2) injecting the mixed solution obtained in step (1) into a mold according to the desired shape of the stent, vacuuming to remove air, and placing in a -180° C. cold environment until it is completely frozen into shape;

[0062] (3) The sample formed by freeze-forming in step (2) is taken out and freeze-dried for 24 hours until it is completely freeze-dried, thereby obtaining a collagen scaffold with a fixed axis orientation structure.

[0063] Experimental results analysis

[0064] The collagen scaffolds prepared by conventional freeze-drying in Comparative Examples 1 and 2 and Examples 1, 2, 3, and 4 were co-cultured with MC3T3-E1 cells, and the CCK-8 method was used to detect the effects of the collagen scaffolds prepared under different conditions on cell proliferation. The absorbance values ​​at different time points are as follows: Figure 3 shown.

[0065] It can be seen from the results that the collagen scaffold with fixed axis orientation prepared by the method proposed in the present invention is more conducive to cell proliferation than the collagen / collagen composite scaffold prepared by conventional freeze-drying. Moreover, as the concentration of the prepared solution increases and the freezing temperature decreases, the performance of the scaffold improves. This may be because as the solid content in the solution system increases and the efficiency of freezing increases, the growth rate of ice crystals increases, and along the direction of the temperature gradient, the solutes and particles have no time to be expelled to form small holes, and then the transverse holes in the scaffold gradually become regular circles and the pore size decreases, and the longitudinal layered structure has a good layered structure and the pore wall becomes thinner, which makes it easier to form a Haversian system arrangement structure with natural bone, which is more conducive to osteoblast proliferation and creates good conditions for cell and blood vessel growth.

[0066] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0067] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing a collagen scaffold with a fixed axis orientation structure, which is characterized by the following steps: (1) adding a soluble phosphate solution dropwise to a collagen-soluble calcium salt mixed solution under stirring at 37°C; Then adjust the pH to 9-12 with aqueous ammonia and continue stirring at 37°C for 6-24 hours; (2) injecting the mixed solution obtained in step (1) into a mold, wherein a heat-conducting bottom plate is provided at the bottom of the mold; and evacuating the mold. The mold is subjected to heat insulation treatment except for the heat-conducting bottom plate, and then placed in a cold environment of -20°C to -180°C for freezing molding; (3) Taking out the sample frozen in step (2), freeze-drying it, and obtaining the collagen scaffold with a fixed-axis oriented structure.

2. The method for preparing the collagen scaffold with a fixed-axis oriented structure according to claim 1, characterized in that: The soluble calcium solution is calcium chloride or calcium nitrate tetrahydrate solution.

3. The method for preparing the collagen scaffold with a fixed-axis oriented structure according to claim 1, characterized in that: The concentration of collagen in the collagen-soluble calcium salt mixed solution is 30-100 mg / ml, and the mass concentration of calcium ions: the mass concentration of collagen = (1-0.8):

1.

4. The method for preparing the collagen scaffold with a fixed-axis oriented structure according to claim 1, characterized in that: The soluble phosphate is one or more of disodium hydrogen phosphate, sodium dihydrogen phosphate, diammonium hydrogen phosphate, ammonium dihydrogen phosphate, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate.

5. The method for preparing the collagen scaffold with a fixed-axis oriented structure according to claim 1, characterized in that: The soluble phosphate solution and the collagen-soluble calcium salt mixed solution have the same volume, and the molar concentration of calcium ions in the collagen-soluble calcium salt mixed solution: the molar concentration of phosphate ions in the soluble phosphate solution = 1.

67.

6. The method for preparing the collagen scaffold with a fixed-axis oriented structure according to claim 1, characterized in that: The heat-conducting base plate is made of copper, aluminum or iron.

7. The method for preparing the collagen scaffold with a fixed-axis oriented structure according to claim 1, characterized in that: The temperature of the cold environment is -150 to -180°C.

8. The method for preparing the collagen scaffold with a fixed-axis oriented structure according to claim 1, characterized in that: The freeze-drying time is 12 to 24 hours. 9 . The collagen scaffold prepared by the method for preparing a collagen scaffold with a fixed-axis oriented structure according to any one of claims 1 to 8 .

10. Use of the collagen scaffold according to claim 9 in preparing bone regeneration products.

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