Multifunctional mineralized collagen with uniform layered structure as well as preparation method and application of multifunctional mineralized collagen

Abstract

The invention discloses multifunctional mineralized collagen with a uniform layered structure as well as a preparation method and application of the multifunctional mineralized collagen, and particularly relates to the technical field of biomedical materials. The preparation method comprises the following steps: adding soluble calcium salt into a collagen stock solution, adjusting the pH value of a reaction system to 7-9, and reacting; after the reaction is finished, adding low-valence phosphate and deionized water, and reacting; after the reaction is finished, adding a silver ion solution, adjusting the pH value to 7.4-8.0, and carrying out mineralization reaction; after the mineralization is finished, cleaning, centrifuging, freezing and forming, and freezing and drying to obtain the multifunctional mineralized collagen with a uniform layered structure. According to the preparation method, the mineralized collagen which is provided with a mineralized structure which is assembled by apatite and nano-silver particles in a layered manner and is uniformly distributed along collagenous fibers and has good antibacterial performance and osteogenic performance is prepared on the micron scale and the nano-scale by utilizing the reaction of collagen groups and phosphate and introducing silver ions with oxidation and antibacterial performance.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and particularly relates to a multifunctional mineralized collagen with a uniform layered structure, a preparation method thereof, and an application thereof. Background Art

[0002] Bone is an important part of the human locomotor system, playing a role in supporting and protecting the body. However, bone diseases are one of the important diseases affecting people's physical health in modern society. Bone defect is a common bone disease at present, and bone repair by bone transplantation surgery is one of the important ways to treat bone defects.

[0003] Bone repair materials are an important means for bone transplantation and bone defect repair. Traditional bone transplantation mostly uses autologous bone or allogeneic bone for transplantation. Although autologous bone has always been an ideal material for bone repair and transplantation, it has disadvantages such as insufficient donors, secondary injury to patients, high blood loss, and easy occurrence of complications at the bone donation site. If allogeneic bone is used to replace autologous bone transplantation, there are risks such as rejection reactions. Therefore, the research on using artificial bone to replace natural bone for bone transplantation has attracted more and more attention from scientific research personnel.

[0004] The research on bone repair substitute materials is the main research direction for solving the problem of bone defect repair at present. Among them, since mineralized collagen is the basic structural unit of bone matrix and the key to the mechanical adaptability of bone, the research on biomimetic mineralized collagen is of great significance for preparing ideal bone repair substitute materials. At present, although mineralized collagen with different structural forms and mineral contents prepared through research has different mechanical properties, the mechanical properties of biomimetic mineralized collagen materials still have a certain distance from being used as substitute materials for load-bearing parts. At the same time, since other problems such as infection and inflammatory reactions often occur during the bone repair process, it is necessary to improve the mechanical properties of mineralized collagen materials so that the bone repair materials have multiple functions of high strength, anti-infection, and bone formation promotion. Summary of the Invention

[0005] Therefore, the present invention provides a multifunctional mineralized collagen with a uniform layered structure, a preparation method thereof, and an application thereof to solve the above-mentioned existing problems.

[0006] The control of the reaction between collagen groups and phosphite by the present invention is the key to forming more nucleation sites of apatite on collagen fibers and inducing the uniform distribution of apatite along collagen fibers. The oxidation reaction of silver ions and phosphorous acid enables phosphorous acid to participate in the formation of apatite, and at the same time, during the redox process, nano-silver is formed, making it have a layered assembly structure. This is the key to improving mechanical properties and making the material have antibacterial properties at the same time.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] A preparation method of a multifunctional mineralized collagen with a uniform hierarchical structure provided by the first aspect of the present invention includes:

[0009] Step 1: Add a soluble calcium salt to the collagen stock solution, and adjust the pH of the reaction system to 7 - 9, and react;

[0010] Step 2: After the reaction ends, add a low-valent phosphate and deionized water, and react;

[0011] Step 3: After the reaction ends, add a silver ion solution, adjust the pH to 7.4 - 8.0, and carry out a mineralization reaction;

[0012] Step 4: After the mineralization ends, after washing and centrifuging, carry out freeze molding and then freeze drying to obtain the multifunctional mineralized collagen with a uniform hierarchical structure.

[0013] Further, in the above step 1, the collagen stock solution is a rat tail type I collagen stock solution; the concentration range is 3 - 5 mg / mL.

[0014] Further, in the above step 1, the concentration of the soluble calcium salt is 0.1 M / L of calcium chloride.

[0015] Further, in the above step 2, the concentration of the low-valent phosphate is 0.1 M / L.

[0016] Further, in the above step 2, the reaction time is not less than 3 h.

[0017] Further, in the above step 3, the concentration of the silver ion solution is 0.1 M / L.

[0018] A multifunctional mineralized collagen with a uniform hierarchical structure provided by the second aspect of the present invention has an armor-like multi-layer mineral structure formed by two mineral components.

[0019] Further, taking the mineralized collagen as 5 mL, the addition amount of each component is that the final concentration of collagen is 1 mg / mL, the final concentration of calcium ions is 10 mM, the final concentration of mass fraction of phosphite is 10 mM, and the final concentration of silver ions is 10 mM - 20 mM.

[0020] An application of a multifunctional mineralized collagen with a uniform hierarchical structure provided by the third aspect of the present invention in the preparation of bone defect repair products.

[0021] The present invention has the following advantages:

[0022] The present invention utilizes the reaction between collagen groups and phosphates and introduces silver ions with oxidation and antibacterial properties. At the micro and nano scales, a mineralized structure with hierarchical assembly of apatite and nano silver particles and uniformly distributed along collagen fibers is prepared, and the mineralized collagen has good antibacterial and osteogenic properties. The present invention can provide technical support for the preparation of multifunctional bionic bone repair materials with a uniform mineralized structure.

[0023] Compared with traditional bone matrix mineralization methods, the present invention fully considers the effects of ion sources, chemical reaction equilibrium, reaction sequence, etc. in mineralization, constructs a multi-factor synergistic regulation system, and better endows the material with more functions through hierarchical regulation, while improving the key properties of the material, that is, mechanical properties. In the present invention, phosphite combines with collagen, changing the surface properties of collagen and endowing it with more nucleation sites for apatite. Under the action of silver ions, the phosphite combined with collagen undergoes an oxidation reaction, forming a large amount of orthophosphate radicals on the collagen fibers. The orthophosphate radicals combine with calcium ions to directly form a layer of minerals uniformly distributed along the collagen fibers. While oxidizing phosphite, silver ions are reduced to nano silver particles. Therefore, the whole process shows that while forming a layer of apatite, a uniformly distributed nano silver coating of another layer is formed. This novel mineralized bone matrix material has both good osteogenic and antibacterial properties, and also has mechanical properties matching the bone matrix in human bone tissue, and has a broad application space. Brief Description of the Drawings

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only exemplary, and for those of ordinary skill in the art, without creative efforts, other implementation drawings can be obtained according to the provided drawings.

[0025] The structures, ratios, sizes, etc. depicted in this specification are only used to cooperate with the content disclosed in the specification for those familiar with this technology to understand and read, and are not used to limit the limited conditions under which the present invention can be implemented. Therefore, they do not have technical substantive significance. Any modification of the structure, change of the proportional relationship, or adjustment of the size, without affecting the effects that the present invention can produce and the purposes that can be achieved, should still fall within the scope covered by the technical content disclosed in the present invention.

[0026] Figure 1 It is a flow chart for preparing multifunctional mineralized collagen with a uniform hierarchical structure provided by Embodiment 1 of the present invention;

[0027] Figure 2Differences between the TEM / EDS nanoscale control group and experimental group provided in Experimental Example 1 of the present invention; among them, 1 - experimental group; 2 - control group 1; 3 - control group 2; 4 - control group 3; the symbol C on the figure represents the collagen skeleton, Ca and P respectively represent calcium and phosphorus elements, and Ag represents silver element;

[0028] Figure 3 Differences between the SEM micron-scale control group and experimental group provided in Experimental Example 1 of the present invention; among them, 1 - experimental group; 2 - control group.

[0029] Figure 4 Antibacterial performance comparison chart provided in Experimental Example 2 of the present invention;

[0030] Figure 5 Osteogenic performance evaluation chart provided in Experimental Example 2 of the present invention; among them, A - histological staining using HE / Masson; B - statistical analysis chart of trabecular bone separation amount (Tb.Sp) and new bone increment (BV / TV). Detailed implementation manners

[0031] The following specific embodiments illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of them. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0032] Reaction principle of the present invention:

[0033] Firstly, controlling the reaction between collagen groups and phosphite is the key to forming more nucleation sites of apatite on collagen fibers and inducing the uniform distribution of apatite along collagen fibers. Secondly, the oxidation reaction of silver ions and phosphorous acid enables phosphorous acid to participate in the formation of apatite. At the same time, during the redox process, nano-silver is formed, making it have a hierarchical assembly structure. This is the key to improving mechanical properties and endowing the material with antibacterial properties at the same time.

[0034] Example 1

[0035] This example provides a multifunctional mineralized collagen with a uniform hierarchical structure:

[0036] Step 1, preparation of the reaction stock solution

[0037] Calcium chloride: 0.1 M / L;

[0038] Sodium phosphite: 0.1 M / L;

[0039] Silver nitrate solution: 0.1 M / L;

[0040] Sodium hydroxide solution: 1M / L and 0.1M / L;

[0041] Acetic acid: 2%

[0042] Collagen stock solution: Rat tail type I collagen purchased from BD biocoat is the collagen reaction stock solution with a concentration range of 3-5 mg / mL.

[0043] Step 2: Limit the dosage

[0044] The total amount of solution in the reaction system was 5 mL. Figure 1 Add the components in the reaction system in the order shown. Calculate the amount of stock solution required for each component according to the final concentration of each component in the reaction system. The final concentration of collagen is 1 mg / mL, the final concentration of calcium ions is 10 mM, the final concentration of phosphite is 10 mM, and the final concentration of silver ions is 10 mM-20 mM.

[0045] Step 3: Specific operations

[0046] Add the substances in order:

[0047] First, add soluble calcium salt to the collagen stock solution, adjust the pH of the reaction system to 7-9, and react for 30 minutes;

[0048] Secondly, after the reaction is completed, add phosphite, add deionized water to make the reaction system reach 5mL, and maintain the reaction for no less than 3h;

[0049] Next, add silver ion solution, adjust the pH to 7.4-8.0, and mineralize for more than 24 hours;

[0050] Finally, after the reaction is completed, the mixture is washed with deionized water and centrifuged three times. A certain amount of deionized water is added to concentrate the mixture into a high-concentration bone matrix mineralization liquid, which is placed in a refrigerator at -20°C for freezing overnight, and then transferred to a vacuum freeze dryer for freeze drying to obtain multifunctional mineralized collagen with a uniform layered structure.

[0051] The material preparation process of multifunctional mineralized collagen with uniform distribution of minerals in micro-nano scale and good antibacterial and osteogenic properties. Different reaction regulating factors and the order of adding these regulating factors have different effects on the structure, morphology, etc. of the mineralized product. Therefore, the present invention fully considers the influence of ion source, redox reaction balance and reaction order in mineralization.

[0052] Example 2

[0053] This embodiment provides a multifunctional mineralized collagen with a uniform layered structure:

[0054] Step 1, Preparation of the reaction stock solution

[0055] Calcium nitrate: 0.1 M / L;

[0056] Sodium phosphite: 0.1 M / L;

[0057] Silver nitrate solution: 0.1 M / L;

[0058] Sodium hydroxide solution: 1 M / L and 0.1 M / L;

[0059] Acetic acid: 2%;

[0060] Collagen stock solution: The murine tail type I collagen purchased from BD biocoat is used as the reaction stock solution of collagen, and the concentration range is 3 - 5 mg / mL.

[0061] Step 2, Dosage limitation

[0062] The total volume of the solution in the reaction system is 5 mL. In the reaction system, add each component in the reaction system according to the addition sequence as shown. Calculate the addition amount of the stock solution required for each component according to the final concentration of each component in the reaction system. Among them, the final concentration of collagen is 1 mg / mL, the final concentration of calcium ions is 10 mM, the final concentration of phosphite is 10 mM in mass fraction, and the final concentration of silver ions is 10 mM - 20 mM. Figure 1

[0063] Step 3, Specific operation

[0064] Add each substance in sequence:

[0065] First, add soluble calcium salt to the collagen stock solution, and adjust the pH of the reaction system to 7 - 9, and react for 30 min;

[0066] Secondly, after the reaction is completed, add phosphite, and supplement deionized water to make the reaction system reach 5 mL, and maintain the reaction for no less than 12 h;

[0067] Then, add silver ion solution, adjust the pH to 7.4 - 8.0, and mineralize for more than 24 h;

[0068] Finally, after the reaction is completed, wash and centrifuge three times with deionized water. Add a certain amount of deionized water, concentrate it into a high-concentration bone matrix mineralization solution, place it in a refrigerator at -20 °C for overnight freeze molding, transfer it to a vacuum freeze dryer, and perform freeze drying to obtain the multifunctional mineralized collagen with a uniform layered structure.

[0069] Example 3

[0070] This example provides a multifunctional mineralized collagen with a uniform layered structure:

[0071] ​Step 1, Preparation of the reaction stock solution

[0072] Calcium chloride: 0.1 M / L;

[0073] Sodium hypophosphite: 0.1 M / L;

[0074] Silver nitrate solution: 0.1 M / L;

[0075] Sodium hydroxide solutions: 1 M / L and 0.1 M / L;

[0076] Acetic acid: 2%;

[0077] Collagen stock solution: The rat-tail type I collagen purchased from BD biocoat is used as the reaction stock solution of collagen, and the concentration range is 3 - 5 mg / mL.

[0078] Step 2, Dosage limitation

[0079] The total volume of the solution in the reaction system is 5 mL. In the reaction system, according to the addition sequence as shown, add each component in the reaction system. Calculate the addition amount of the stock solution of each component according to the final concentration of each component in the reaction system. Among them, the final concentration of collagen is 1 mg / mL, the final concentration of calcium ions is 10 mM, the final concentration of hypophosphates in mass fraction is 10 mM, and the final concentration of silver ions is 10 mM - 20 mM. Figure 1

[0080] Step 3, Specific operation

[0081] Add each substance in sequence:

[0082] First, add soluble calcium salt to the collagen stock solution, and adjust the pH of the reaction system to 7 - 9, and react for 30 min;

[0083] Second, after the reaction is completed, add hypophosphates, and supplement deionized water to make the reaction system reach 5 mL, and maintain the reaction for no less than 3 h;

[0084] Then, add silver ion solution, adjust the pH to 7.4 - 8.0, and mineralize for more than 24 h;

[0085] Finally, after the reaction is completed, wash and centrifuge three times with deionized water. Add a certain amount of deionized water, concentrate it into a high-concentration bone matrix mineralization solution, place it in a refrigerator at -20 °C for overnight freeze molding, and transfer it to a vacuum freeze dryer for freeze drying to obtain a multifunctional mineralized collagen with a uniform layered structure.

[0086] Comparative Example 1

[0087] Step 1, Preparation of the reaction stock solution

[0088] Calcium chloride: 0.1 M / L;

[0089] Sodium phosphite: 0.1 M / L;

[0090] Sodium hydroxide solutions: 1 M / L and 0.1 M / L;

[0091] Acetic acid: 2%;

[0092] Stock collagen solution: The type I collagen from rat tail purchased from BD biocoat was used as the stock solution for the reaction of collagen, with a concentration range of 3 - 5 mg / mL.

[0093] Step 2, Dosage limitation

[0094] The total volume of the solution in the reaction system is 5 mL. In the reaction system, according to the addition sequence as shown, add each component in the reaction system. Calculate the addition amount of the stock solution required for each component according to the final concentration of each component in the reaction system. Among them, the final concentration of collagen is 1 mg / mL, the final concentration of calcium ions is 10 mM, and the final concentration of phosphite is 10 mM in mass fraction. Figure 1

[0095] Step 3, Specific operation

[0096] Add each substance in sequence:

[0097] First, add soluble calcium salt to the stock collagen solution, and adjust the pH of the reaction system to 7 - 9, and react for 30 min;

[0098] Secondly, after the reaction is completed, add phosphite, supplement deionized water to make the reaction system reach 5 mL, adjust the pH to 7.4 - 8.0, and mineralize for more than 24 h;

[0099] Figure 2 Finally, after the reaction is completed, wash and centrifuge three times with deionized water. Add a certain amount of deionized water, concentrate it into a high - concentration bone matrix mineralization solution, place it in a refrigerator at - 20 °C for overnight freeze - forming, transfer it to a vacuum freeze - dryer, and freeze - dry to obtain the unmineralized collagen as Control group 2.

[0100] Comparative example 2

[0101] Step 1, Preparation of the stock reaction solution

[0102] Calcium chloride: 0.1 M / L;

[0103] Sodium phosphite: 0.1 M / L;

[0104] Silver nitrate solution: 0.1 M / L;

[0105] Sodium hydroxide solutions: 1 M / L and 0.1 M / L;

[0106] ​​

[0107] Collagen stock solution: Rat tail type I collagen purchased from BD biocoat is the collagen reaction stock solution with a concentration range of 3-5 mg / mL.

[0108] Step 2: Limit the amount of

[0109] The total amount of solution in the reaction system was 5 mL. Figure 1 Add the components in the reaction system in the order shown. Calculate the amount of stock solution required for each component according to the final concentration of each component in the reaction system. The final concentration of collagen is 1 mg / mL, the final concentration of calcium ions is 10 mM, the final concentration of phosphite is 10 mM, and the final concentration of silver ions is 10 mM-20 mM.

[0110] Step 3: Specific operations

[0111] Add the substances in order:

[0112] First, add soluble calcium salt to the collagen stock solution, adjust the pH of the reaction system to 7-9, and react for 30 minutes;

[0113] Secondly, after the reaction is finished, phosphite is added and deionized water is added to make the reaction system reach 5 mL;

[0114] At the same time as the previous step, add silver ion solution, adjust the pH to 7.4-8.0, and mineralize for more than 24 hours;

[0115] Finally, after the reaction, the mixture was washed with deionized water and centrifuged three times. A certain amount of deionized water was added to concentrate the mixture into a high-concentration bone matrix mineralization solution, which was placed in a refrigerator at -20°C to freeze overnight and then transferred to a vacuum freeze dryer for freeze drying. Due to the reaction sequence, the phosphite did not bind to the collagen and only aggregated mineral particles were obtained, i.e., the collagen and mineral particles were separated (e.g., Figure 2 Control group 3.

[0116] Experimental Example 1

[0117] The TEM / EDS nanometer scales of Example 1 (experimental group) and Comparative Example 1 (control group 1), Comparative Example 2 (control group 2), and Comparative Example 3 (control group 3) were compared. The results are as follows: Figure 2 shown.

[0118] Depend on Figure 2 It can be seen that in the preparation of mineralized collagen in which two phases of minerals are evenly distributed on the surface of collagen, the full reaction of phosphite with collagen is the key to the uniform distribution of minerals. The oxidation of silver ions is the key to the formation of minerals. Directly providing orthophosphate ions can only form one mineral, and the distribution is uneven, such as Figure 2As shown in Control Group 1. If only phosphite is provided and there is no participation of silver ions, just as Figure 2 shown in Control Group 2, no minerals are formed. With the participation of silver ions, but phosphite fails to bind to collagen, only two kinds of mineral particles can be formed, and the mineral particles cannot be distributed along the collagen fibers, as Figure 2 shown in Control Group 3.

[0119] Comparing the SEM micron scale of Example 1 (experimental group) and Comparative Example 1 (control group), the results are as Figure 3 shown.

[0120] It can be seen from Figure 3 that compared with ordinary mineralization, the mineralized collagen with a uniform distribution of two-phase minerals has a more uniform mineral distribution at the micron scale, and at the same time its pores are also more uniform.

[0121] Experimental Example 2

[0122] Antibacterial and osteogenic evaluation experiments:

[0123] In order to verify the antibacterial performance and osteogenic performance evaluation of the obtained scaffold materials, the following operations were carried out:

[0124] Put the collagen scaffold material group at the bottom of the well plate, and irradiate the plate with a radiation dose of 15 kGy for 24 hours for sterilization. Place the agar medium in 6-well plates respectively, divided into a blank control group (without adding any material group), a control group (Comparative Example 1) and an experimental group (Example 1) (a mineralized collagen material group with a uniform hierarchical structure). Escherichia coli and Staphylococcus aureus were respectively planted on the surface of the agar, and then the materials were placed according to the grouping. The antibacterial properties of Example 1 (experimental group) and Comparative Example 1 (control group 1) and the blank group (control group 2) were compared, and the results are as Figure 4 shown. An obvious antibacterial circle was formed in the experimental group, indicating that the materials all had good antibacterial effects.

[0125] Similar to the above antibacterial experiment, the materials were first sterilized. The materials were respectively implanted into the infected bone defect sites of rats, divided into a control group (Comparative Example 1) and an experimental group (Example 1) (a mineralized collagen material group with a multi-level structure), and histological staining was carried out using HE / Masson, as Figure 5 shown in A. At the same time, osteogenic morphological imaging, as well as statistical analysis of bone mineral density (BMD), trabecular bone separation (Tb.Sp), and new bone increment (BV / TV)) were carried out using Micro-CT, as Figure 5 shown in B.

[0126] The antibacterial and osteogenic evaluation results show that the experimental group materials have good antibacterial properties, biosecurity and osteogenic ability, and can be used for the repair of infected bone defects.

[0127] The preparation process of the multifunctional mineralized collagen material with uniform distribution of minerals at the micro-nano scale, having good antibacterial performance and osteogenic performance. Different reaction regulation factors and the addition sequence of these regulation factors have different effects on the structure, morphology, etc. of the mineralized products. Therefore, the present invention fully considers the influence of ion sources, redox reaction balance and reaction sequence in mineralization.

[0128] Although the present invention has been described in detail with general descriptions and specific examples in the above text, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. A method for preparing multifunctional mineralized collagen with a uniform layered structure, characterized in that: include: Step 1, adding soluble calcium salt to the collagen stock solution, adjusting the pH of the reaction system to 7-9, and reacting; Step 2: After the reaction is completed, add low-valent phosphate and deionized water to react; Step 3: After the reaction is completed, add silver ion solution, adjust the pH to 7.4-8.0, and perform mineralization reaction; Step 4: After mineralization, the product is washed and centrifuged, freeze-formed, and then freeze-dried to obtain multifunctional mineralized collagen with a uniform layered structure.

2. The method for preparing a multifunctional mineralized collagen having a uniform layered structure according to claim 1, characterized in that: In the step 1, the collagen stock solution is rat tail type I collagen stock solution; the concentration range is 3-5 mg / mL.

3. The method for preparing a multifunctional mineralized collagen having a uniform layered structure according to claim 1, characterized in that: In the step 1, the concentration of soluble calcium salt is 0.1 M / L calcium chloride.

4. The method for preparing a multifunctional mineralized collagen having a uniform layered structure according to claim 1, characterized in that: In the step 2, the concentration of low-valent phosphate is 0.1 M / L.

5. The method for preparing a multifunctional mineralized collagen having a uniform layered structure according to claim 1, characterized in that: In the step 2, the reaction time is not less than 3 hours.

6. The method for preparing a multifunctional mineralized collagen having a uniform layered structure according to claim 1, characterized in that: In the step 3, the concentration of the silver ion solution is 0.1 M / L.

7. A multifunctional mineralized collagen having a uniform layered structure, characterized in that: The mineralized collagen has an armor-like multi-layered mineral structure formed of two mineral components.

8. The multifunctional mineralized collagen with a uniform layered structure according to claim 7, characterized in that: The mineralized collagen is calculated as 5 mL, and the amount of each component added is such that the final concentration of collagen is 1 mg / mL, the final concentration of calcium ions is 10 mM, the final concentration of phosphite is 10 mM by mass fraction, and the final concentration of silver ions is 10 mM-20 mM.

9. Application of a multifunctional mineralized collagen with a uniform layered structure in the preparation of bone defect repair products.

Images