Method for observing mineralized collagen fibers for simulating in-vivo bone mineralization process and application

Abstract

The invention discloses a mineralized collagen fiber method for observing and simulating an in-vivo bone mineralization process and application, and the method comprises the following steps: obtaining mineralized collagen nanofibers with different mineralization degrees; carrying out pressure forming on the mineralized collagen nanofiber to obtain a block structure; wherein the pressure forming parameters are as follows: the pressure is increased at the room temperature by 500-900 MPa, and the constant pressure is kept for 10-15 min; and the block body structure is observed. The method relates to bionic preparation of mineralized collagen fibers and observation of the mineralization process, irradiation damage is avoided, and high-resolution morphology is obtained.

Description

Technical Field

[0001] The present invention relates to the technical field of organic chemistry, and in particular to a method and application for observing mineralized collagen fibers that simulate the bone mineralization process in vivo. Background Art

[0002] The biomimetic mineralization process of bone in vitro has been reported in many studies, evolving from the initial physical and mechanical mixing to the mineralized structure relying on chemical bonding methods. The biomimetic mineralization process is widely defined as a process in which an organic component is used as a template to induce mineralization to form a biomimetic structure with a special orientation relationship. The process of in vitro biomimetic mineralization using collagen as a template to simulate the bone structure has been successfully applied to the preparation of mineralized collagen fibers. However, for the microscopic structure observation of biomimetic mineralized collagen fibers, it has always remained in the traditional preparation technology, that is, the solution is dropped on the copper mesh sample stage. Since the sample to be observed is a composite material of organic / inorganic, there is radiation damage during direct observation. Even when the mineral content reaches 80%, it is still impossible to directly observe the growth process of mineralized collagen fiber crystals and information such as precise lattice fringes. Traditional preparation methods for collagen / hydroxyapatite mixtures have been reported, but more intuitive evidence regarding the inducing effect of collagen therein and a more intuitive observed morphology have not been reported. Therefore, the present invention aims to innovatively propose an observation means and preparation method for observing the mineralization process of in vitro simulated bone structure, to achieve low-cost and high-precision observation of the mineralization growth process of mineralized collagen fibers, the regulation effect of collagen fibers, and the special orientation relationship between collagen / hydroxyapatite, and to provide technical support for the direct observation of predicting other mineralization processes. Summary of the Invention

[0003] The purpose of the present invention is to provide a method and application for observing mineralized collagen fibers that simulate the bone mineralization process in vivo. This method involves the biomimetic preparation of mineralized collagen fibers and the realization of observing the mineralization process, avoiding radiation damage and obtaining high-resolution morphology. To achieve the above purpose, the present invention provides the following technical solutions:

[0004] The present invention provides a method for observing mineralized collagen fibers that simulate the bone mineralization process in vivo, and the method includes,

[0005] Obtaining mineralized collagen nanofibers with different mineralization degrees;

[0006] Performing pressure molding on the mineralized collagen nanofibers to obtain a bulk structure; wherein, the pressure molding parameters are pressurizing at room temperature at 500 - 900 MPa and maintaining a constant pressure for 10 - 15 min;

[0007] Observing the bulk structure.

[0008] Further, the mineralized collagen nanofibers with different mineralization degrees include:

[0009] Mineralized collagen fibers with a mineralization degree of 90% to 100%.

[0010] Furthermore, the average length of the mineralized collagen nanofibers with different mineralization degrees is 200 nm to 240 nm.

[0011] Furthermore, the average diameter of the mineralized collagen nanofibers with different mineralization degrees is 8 nm to 10 nm.

[0012] Furthermore, pressure forming the mineralized collagen nanofibers includes:

[0013] The pressure direction is perpendicular to the layered structure of the mineralized collagen nanofibers.

[0014] Furthermore, the preparation of the mineralized collagen nanofibers includes:

[0015] Mixing type I collagen sponge with a phosphoric acid solution to obtain a collagen template solution;

[0016] Dissolving type I collagen sponge in a solution containing CaCl 2 / HCl solution to obtain a calcium ion template solution;

[0017] Performing a mineralization reaction on the collagen template solution and the calcium ion template solution, and controlling the mineralization time to obtain mineralized collagen nanofibers with different mineralization degrees.

[0018] Furthermore, the preparation of the mineralized collagen nanofibers further includes:

[0019] Freeze-drying the mineralized collagen fibers with different mineralization degrees, and the freeze-drying conditions are: -50°C, 50 Pa for 48 - 60 h.

[0020] Furthermore, the mixing of the type I collagen sponge and the phosphoric acid solution is carried out for 6 h to 12 h; the mixing temperature is 35°C to 38°C.

[0021] Furthermore, dissolving type I collagen sponge in a solution containing CaCl 2 / HCl solution for 6 h to 12 h, and the dissolution temperature is 35°C to 38°C.

[0022] The present invention also provides the application of a method for observing mineralized collagen fibers for simulating the in vivo bone mineralization process as described above in observing bionic materials for oil well equipment.

[0023] The technical effects and advantages of the present invention:

[0024] 1. Simulate the existence state of ions in simulated body fluid, including free calcium ions and phosphate ions, etc., and mainly carry out long-term mineralization in the reaction solution through two solutions to synthesize mineralized collagen fibers. Since the surface of collagen fibers has special binding sites for calcium ions and phosphate ions, taking advantage of this feature, first conjugate collagen fully with calcium ions and phosphate ions, and then carry out the mineralization reaction to make it fully approach the mineralization process in the animal body.

[0025] 2. The technical effects brought by the pressing process of this application; fusion can occur at room temperature, and it is an integral block structure. It is not physical polymerization, but a block structure with chemical interaction, which can withstand certain mechanical properties. It will not disintegrate immediately when immersed in water, and can maintain a certain shape even after long-term immersion. The structure after pressure fusion can stabilize the mineralized collagen structure and resist radiation damage.

[0026] This method can be extended to the research of the carbonate mineralization process, not limited to the mineralization process of phosphate biomimetic bone, such as shells, etc. However, in this invention, collagen fibers are selected as the inducer because collagen fibers have special binding sites, which are specific for the induction of the mineralization process. At present, only the difference between having and not having can be observed for the mineralized collagen fibers on the market, and the process cannot be observed and described. Taking advantage of the completely simulated body fluid mineralization process, this invention is more innovative and convenient for observation compared with the previous invention content. Currently, existing research on mineralized collagen, collagen / hydroxyapatite structure, etc. cannot provide direct evidence to support that this kind of substance is a polymer with chemical interaction rather than physical mixing. Here we provide technical and intuitive morphological support.

[0027] Other features and advantages of the present invention will be described in the subsequent specification, and, in part, will become obvious from the specification, or will be understood by implementing the present invention. The objectives and other advantages of the present invention can be achieved and obtained through the structures pointed out in the specification and the drawings. Description of the Drawings

[0028] Figure 1 It is a flow chart of the method for observing and simulating the in-vivo bone mineralization process of mineralized collagen fibers of the present invention;

[0029] Figure 2 It is the morphology after pressing of mineralized collagen fibers with two different mineralization degrees in the specific embodiment of the present invention;

[0030] Figure 3a It is the full spectrum of the Fourier transform infrared spectrum of collagen fibers and mineralized collagen fibers under different pressure conditions in the specific embodiment of the present invention in the range of 600 - 3700 cm -1 Full spectrum;

[0031] Figure 3bThis is the amide bond peak spectrum diagram of collagen fibers and mineralized collagen fibers under different pressure conditions in the specific embodiments of the present invention in Fourier transform infrared spectroscopy;

[0032] Figure 4 This is the Fourier spectrum diagram of the phosphate structural peak in the specific embodiments of the present invention;

[0033] Figure 5 This is the diagram showing the gradual increase in the ratio of stoichiometric hydroxyapatite to amide peak in the specific embodiments of the present invention;

[0034] Figure 6 This is the thermogravimetric analysis diagram of mineralized collagen fibers with two different degrees of mineralization in the specific embodiments of the present invention;

[0035] Figure 7 This is the TEM morphology of 90% mineralized collagen fibers in the specific embodiments of the present invention;

[0036] Figure 8 This is the TEM morphology of 100% mineralized collagen fibers in the specific embodiments of the present invention;

[0037] Figure 9 This is the morphology of the collagen fibers conjugated with calcium ions alone first in the specific embodiments of the present invention;

[0038] Figure 10 This is the TEM morphology of 90% mineralized collagen fibers in Comparative Example 1 in the specific embodiments of the present invention;

[0039] Figure 11 This is the partial TEM morphology of 100% mineralized collagen fibers in Comparative Example 1 in the specific embodiments of the present invention;

[0040] Figure 12 This is the TEM morphology of 100% mineralized collagen fibers in Comparative Example 1 in the specific embodiments of the present invention;

[0041] Figure 13 This is the partial TEM morphology of 90% mineralized collagen fibers in Comparative Example 1 in the specific embodiments of the present invention. Detailed implementation manners

[0042] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0043] To solve the deficiencies of the prior art, the present invention discloses a method for observing mineralized collagen fibers simulating the in vivo bone mineralization process, asFigure 1 As shown, the method includes obtaining mineralized collagen nanofibers with different mineralization degrees; performing pressure molding on the mineralized collagen nanofibers to obtain a bulk structure; wherein the pressure molding parameters are pressurizing at room temperature of 350 - 900 MPa for 10 - 15 minutes under constant pressure; and observing the bulk structure.

[0044] In a specific embodiment of the present invention, the biomimetic mineralized collagen fibers simulate the composition and structure of bone structure at the nanoscale, which are mainly composed of collagen and hydroxyapatite. And there is a special orientation relationship between collagen and hydroxyapatite. To observe the important role of collagen in mineralization, biomimetic mineralized collagen fibers with different mineralization times are prepared for detection. Simulate the existence state of ions in simulated body fluid, including free calcium ions and phosphate ions, etc., and mainly perform long-term mineralization in the reaction solution through two solutions to synthesize mineralized collagen fibers. Since the surface of collagen fibers has special binding sites for calcium ions and phosphate ions, using this feature, first fully conjugate collagen with calcium ions and phosphate ions, and then perform the mineralization reaction to make it fully close to the mineralization process in animals.

[0045] In a specific embodiment of the present invention, the mineralized collagen nanofibers with different mineralization degrees include: the mineralization time is 12 - 16 h and 28 - 36 h; control the mineralization degree to reach 90% to 100% mineralized collagen fibers. The preparation process of 90% mineralized collagen fibers requires strict control of the mineralization time within the time range. Since it is a collagen-mediated mineralization process, this process must be slow, so it is a controllable process. In currently known methods, most are explosive growth that is difficult to control, and it is very difficult to track and observe the growth process of mineralized collagen fibers. For 100% mineralized collagen fibers, it is necessary to meet the requirement that the mineralization time reaches more than 28 - 36 hours, but it will not continue to mineralize with the increase of time and reaches a stable state.

[0046] In a specific embodiment of the present invention, the average length of the mineralized collagen nanofibers with different mineralization degrees is 240 nm to 300 nm.

[0047] In a specific embodiment of the present invention, the average diameter of the mineralized collagen nanofibers with different mineralization degrees is 8 nm to 10 nm.

[0048] The observation process of the mineralized collagen fibers is different from the traditional sample preparation method. First, a dense bulk structure is prepared. In an implementation manner provided by the present application, during the pressure molding process of the mineralized collagen fibers with different mineralization degrees, performing pressure molding on the mineralized collagen nanofibers includes: the pressure direction is perpendicular to the structure of the mineralized collagen nanofibers.

[0049] In the pressure operation process, the pressure direction is applied vertically, but it must be supported by a mold. The pressure magnitude is between 350 - 1000 MPa, and the constant pressure is maintained for 10 - 15 min. This process does not require heating. Compared with the traditional polymer compression method, pressurization at room temperature will not affect the quality of collagen and will not damage the crystal structure. And there is a process of fusion and transformation of crystals between fibers. It realizes the protective effect on the electron beam during the long process of observing mineralized collagen fibers.

[0050] Then a dense block structure is formed. And FIB is used for the preparation of TEM specimens, and specimens with a thickness of 100 - 150 nm are cut for observation. Usually, the crystal structure can stably observe the crystal morphology, but for materials containing minerals, it is often impossible to achieve long-term irradiation observation with high-intensity electron beams. Therefore, in order to observe the growth process of mineralized collagen fibers, a pressurization process is adopted, which is particularly important compared with the traditional method.

[0051] The mineralized collagen fibers are pressure-molded at 350 - 1000 MPa and room temperature for 10 min, and finally a block structure is obtained. To avoid the influence of temperature on collagen fibers, compression is carried out at a temperature close to room temperature of 20 - 30 °C to form a dense block structure. For the mineralized collagen fibers with a mineralization degree of 90%, the pressure parameters are set at 500 MPa - 1000 MPa, and for the mineralized collagen fibers with a mineralization degree of 100%, the pressure parameters are set at 350 MPa - 1000 MPa.

[0052] The preparation process of the mineralized collagen fibers that fully simulates the in vitro bone mineralization process involved in the present invention is as follows: First, prepare a mineralized collagen fiber that fully simulates the in vivo bone mineralization process. In the preparation process of the mineralized collagen fiber, the forms of collagen and calcium and phosphate ions in the simulated body fluid are simulated, and mineralized collagen fibers with special orientation relationships are formed. This special orientation relationship is similar to the bone formation process and nano-scale structural morphology in animals.

[0053] In a specific embodiment of the present invention, the ratio of Ca ions to phosphate ions in hydroxyapatite is approximately equal to 1.52. This is less than the theoretical ratio of 1.67, because a small amount of carbon dioxide is introduced during the mineralization process. This is consistent with the situation in natural bone.

[0054] The preparation of the mineralized collagen nanofibers includes: mixing a type I collagen sponge with a phosphoric acid solution to obtain a collagen template solution; dissolving the type I collagen sponge in a CaCl 2 / HCl solution to obtain a calcium ion template solution; carrying out a mineralization reaction on the collagen template solution and the calcium ion template solution, and controlling the mineralization time to obtain mineralized collagen nanofibers with different mineralization degrees.

[0055] Mix type I collagen sponge with diluted phosphoric acid solution at a mixing temperature of 35°C to 38°C for 6 to 12 hours; dissolve the type I collagen sponge completely to obtain a collagen template solution for use; the collagen content is 0.3-0.5 g / L, and the phosphoric acid concentration is 6 mM.

[0056] Dissolve type I collagen sponge in 10 mM CaCl 2 / HCl solution for 6h to 12h, dissolving temperature at 35℃ to 38℃; hydrochloric acid concentration is about 0.5M, slowly add dropwise to adjust pH=2-3. Until collagen is completely dissolved and the solution is clear and transparent. Then add sodium hydroxide solution containing 20mmol, and the solution is slightly turbid. The ratio of calcium ion to phosphate ion is 5:3.

[0057] Prepare a buffer solution with a concentration of 10-30 mol / L, that is, dissolve 1-3 mol of the buffer solution in 100 mL of deionized water. The buffer solution is selected from any one or more of Tris-HCl and phosphate buffer solution.

[0058] The collagen template solution and the calcium ion template solution are titrated in a buffer solution, maintaining a stable titration rate of 400-500 mL / h to obtain a mixed solution; during the titration process, the pH value of the mixed solution is maintained between 8 and 10; the mixed solution is sealed and stirred for more than 24 hours, preferably, stirred for 36 hours, the concentration of the calcium ion solution is 10 mM, and after the titration is completed, the reaction temperature is controlled at 37°C.

[0059] After a period of incubation, the mixed solution is centrifuged to retain the bottom precipitate. The mineralization time can be selected as 12-16 hours and 28-36 hours, and a mineralized collagen fiber solution containing 90% mineralization and a mineralized collagen fiber solution containing 100% mineralization are obtained, and the mineralized products are extracted respectively.

[0060] The extraction of mineralized products includes centrifuging and washing a mineralized collagen fiber solution containing 90% mineralization and a mineralized collagen fiber solution containing 100% mineralization at 3000 rpm respectively, and then centrifuging at 16500 rpm to obtain mineralized collagen fibers with different mineralization degrees, namely, mineralized collagen fibers with 90% mineralization and mineralized collagen fibers with 100% mineralization.

[0061] The mineralized collagen fibers with different mineralization degrees were freeze-dried in a cold well environment of -50 degrees Celsius and 50 Pa for 48-60 hours to obtain mineralized collagen fibers with a mineralization degree of 90% and mineralized collagen fibers with a mineralization degree of 100%.

[0062] The freeze-dried mineralized collagen fibers are subjected to pressure molding. Different from the traditional sample preparation method, observations are made after molding. The molding parameters are pressurization at room temperature of 350 - 1000 MPa, with constant pressure maintained for 10 - 15 min. A bulk structure is obtained, as Figure 2 shown. Then, FIB is used for TEM sample preparation.

[0063] The bulk structure can be processed by FIB to obtain a thin slice for TEM observation with a thickness of 100 - 150 nm, which is relatively easy to process. It will not be damaged due to excessive ion beam energy. The reason is that during the compression process, the crystal structures between the mineralized collagen fibers undergo a certain degree of fusion, so a complete bulk structure can be formed. This process is not a physical packing process, but a certain phase change occurs. In addition, the pressure acts to fix the collagen fiber structure and reduce the thermal motion of the collagen fibers. Therefore, when observing with a high-energy electron beam, irradiation damage can be effectively avoided.

[0064] Figure 3a and 3b show the Fourier transform infrared spectra of collagen fibers and mineralized collagen fibers under different pressure conditions. Figure 3a shows the full spectrum from 600 - 3700 cm -1 ; Figure 3b shows the amide bond peak spectrum. Compared with the original collagen fibers, the phosphate structure peaks of the mineralized collagen fibers appear at 632 cm -1 , 963 cm -1 , 1029 cm -1 , 1099 cm -1 . It is proved that an obvious mineralized substance hydroxyapatite structure is formed. And during the process of increasing pressure, a certain degree of fusion occurs between the mineralized collagen fibers, resulting in an increase in density (which can be seen from the gradual decrease in spectral intensity). Figure 3b is the amide bond. P1 represents pressing at 1 MPa; P70 represents pressing at 70 MPa, P250 represents pressing at 250 MPa; P400 represents pressing at 400 MPa; P750 represents pressing at 750 MPa; P900 represents pressing at 900 MPa and P1000 represents pressing at 1000 MPa.

[0065] Figure 4 shows the Fourier spectrum of the phosphate structure peak. The pressing pressures of the curves in the figure are represented by P1, P70, P250, P400, P750, P900, and P1000 from top to bottom in sequence. The phosphate structure peak can reflect the crystal maturity of the mineralized collagen fibers, mainly including stoichiometric hydroxyapatite structure (mature) and non-stoichiometric hydroxyapatite structure (immature); Figure 5It shows that the ratio of stoichiometric hydroxyapatite to amide peaks gradually increases, indicating that 350 MPa - 1000 MPa can stimulate the phase transition process, i.e., the proportion of mature hydroxyapatite gradually increases. This shows that during the pressure action process, the crystal structure of low crystallinity transforms into high crystallinity. The above results indicate that the pressure action promotes the transformation of crystals from low crystallinity to high crystallinity, promotes the fusion between crystals, enables the mineralized collagen fibers to fuse together and become more stable, making it more resistant to irradiation during the observation process.

[0066] The mineralized collagen nanofibers with different mineralization degrees in this application have good bone compatibility, osteoconductivity, and certain osteogenic induction ability. This process fully simulates the mineralization process of the bone structure in vivo. On the basis of ensuring that the observed sample has radiation resistance, the morphologies of two kinds of mineralized collagen fibers with different mineralization degrees are observed. Furthermore, the mineralization process of collagen-induced hydroxyapatite is analyzed, providing a good theoretical basis for the mineralization of natural bone structures.

[0067] The present invention also provides an application of a method for observing mineralized collagen fibers that simulate the in vivo bone mineralization process in observing bionic materials for oil well equipment.

[0068] The method of the present invention can be extended to the research of the carbonate mineralization process, not limited to the mineralization process of phosphate biomimetic bone, such as shells, etc. However, the reason for selecting collagen fibers as the induction in the present invention is that collagen fibers have special binding sites, which are specific for the induction of the mineralization process. And currently, the commercially available mineralized collagen fibers can only observe the difference between having and not having, and cannot observe and describe this process. The present invention takes advantage of fully simulating the mineralization process of body fluid, which is more innovative and convenient for observation compared with the previous invention content. Currently, existing research on mineralized collagen, collagen / hydroxyapatite structures, etc. cannot provide direct evidence to support that this kind of substance is a polymer with chemical interactions rather than a physical mixture. Here we provide technical and intuitive morphological support.

[0069] The technical solution of the present invention will be further described below in conjunction with specific embodiments.

[0070] To make the purpose, technical solution, and advantages of this application clearer, the embodiments of this application are described in detail below. It should be noted that, without conflict, the embodiments in this application and the features in the embodiments can be combined arbitrarily with each other.

[0071] In the embodiments provided in this application, the raw material sources are as follows: anhydrous calcium chloride (CaCl 2 , analytical pure), sodium hydroxide (NaOH, analytical pure), and ammonia water (NH 4OH (analytical reagent grade) was purchased from Sinopharm Chemical Reagent Co., Ltd.; type I collagen sponge (telopeptide - removed collagen from bovine tail skin, MW = 300,000, bovine tail, Hebei Kailisen Co., Ltd.); phosphoric acid (H 3 PO 4 , analytical reagent grade, Shanghai Titan Scientific Co., Ltd.); Tris - HCl (C 4 H 11 NO 3 ·HCl, analytical reagent grade, Beijing Baidi Biotechnology Co., Ltd.).

[0072] Example 1

[0073] (1) Dissolve the type I collagen sponge in an aqueous phosphoric acid solution. Add 0.5 g of the collagen sponge to a phosphoric acid solution with a concentration of 6 mM. Stir for 12 h, maintaining the stirring temperature at 37 °C until the final solution is clear and transparent.

[0074] (2) Dissolve the type I collagen sponge in an aqueous calcium chloride / hydrochloric acid solution. Add 0.5 g of the collagen sponge to a calcium chloride / hydrochloric acid solution with a concentration of 10 mM. Stir for 12 h, maintaining the stirring temperature at 37 °C until the final solution is clear and transparent. Then add a 20 mM sodium hydroxide solution and stir evenly.

[0075] (3) Prepare a Tris - HCl buffer solution. Dissolve 1 mM of Tris - HCL in 100 mL of water, and adjust the pH value of the reaction pool between 8 and 10 using 0.5 mol / L ammonia water and HCl solution.

[0076] (4) Slowly drip the phosphoric acid / collagen solution and the calcium chloride / collagen solution into the reaction pool solution at a dripping rate of 500 mL / h, and maintain the pH between 8 - 10. The reaction temperature is 37 °C. Seal the tube mouth and stir thoroughly for mineralization for 12 - 16 h and 28 - 36 h, and finally end the mineralization reaction. Mineralized collagen fibers with mineralization degrees of 90% and 100% are obtained respectively.

[0077] (5) Wash and centrifuge the reacted liquid for standby. The washing process is achieved through multiple low - speed centrifugation processes. First, centrifuge at 2500 rpm, then measure the pH of the upper clear liquid; then add deionized water and pipette evenly. Repeat the same centrifugation speed and operation 3 - 5 times until the pH of the surface liquid is 7. Finally, centrifuge at 16500 rpm and remove the upper layer of water.

[0078] (6) Finally, obtain centrifugation products with two mineralization degrees and perform freeze - drying. Place them in a freeze - dryer with a cold well environment of - 50 °C and 50 Pa for freeze - drying. The freeze - drying conditions are: - 50 °C, 50 Pa for 48 h to obtain powdered mineralized collagen fibers.

[0079] The steps of this embodiment can prepare mineralized collagen fibers doped with elements, only other cations or anions need to be incorporated, but it is necessary to satisfy that the ion introduction amount is less than 7%. The total molar ratio of cations to anions is 5:3. And by controlling the mineralization time, mineralized collagen fibers with different low mineral contents can be obtained. However, it is very difficult to distinguish the degree of mineralization by ordinary observation methods, and we have obtained and successfully observed mineralized collagen fibers with a mineralization degree of about 90% and 100% through this process.

[0080] Figure 6 Thermogravimetric analysis tests were performed on mineralized collagen fibers with two different degrees of mineralization. The mineral content of the mineralized collagen fibers with 100% mineralization degree is 81.3%, while the mineral content of the mineralized collagen fibers with 90% mineralization degree is calculated according to about 90% complete mineralization degree, and the final mineral content is about 70.9%. Such mineralized collagen fibers with this mineral content are denoted as 90% mineralization degree.

[0081] For the two obtained mineralized collagen fiber powders, by using an appropriate pressing process, dense and complete bulk structures can be obtained. The pressing process is carried out at room temperature, and the bulk structure is formed by the fusion of crystals on the surface of the mineralized collagen fibers. At this time, under the action of a specific pressure, the crystal structure can be promoted to transform from a low crystalline state to a high crystalline state. The pressure range for the mineralized collagen fibers with 90% mineralization degree is set at 500 MPa - 1000 MPa, and the pressure range for the mineralized collagen fibers with 100% mineralization degree is set at 350 MPa - 1000 MPa, as Figure 2 .

[0082] For the bulk structures formed by mineralized collagen fibers with different degrees of mineralization under the same pressure conditions, sample preparation and observation were carried out. Under the observation conditions of a high-resolution and high-energy electron beam, irradiation damage will not be caused, and the crystal morphology can be stably observed for a long time, as Figure 7 , 8.

[0083] Figure 7 Shows the TEM morphology of the 90% mineralized collagen fibers prepared by the method of the present invention. By using the method of the present invention for TEM testing, the hydroxyapatite crystal structure in the fibers can be clearly observed. And under the condition of 90% mineralization, the crystal plane orientation has a special orientation relationship with the collagen fibers, and the hydroxyapatite crystals grow helically and orderly along the surface of the collagen fibers, and there are certain gaps between the crystals.

[0084] Figure 8 Shows the TEM morphology of the 100% mineralized collagen fibers. The degree of crystal mineralization is higher, and the crystal structure intersects diagram; from the above results, it shows that the crystals grow, the gaps between the crystals decrease, indicating a higher degree of mineralization, and it also shows that the growth process depends on special nucleation sites on the collagen surface and grows orderly. And a special orientation structure is formed.

[0085] The two types of mineralized collagen fibers only differ in mineral content, while other morphological features are similar. After compression molding, TEM was used for observation and analysis. The process of collagen-induced mineralization can be observed. Among them, hydroxyapatite minerals first grow along the y-axis direction of collagen fiber molecules. Then, mineralization forms a complete "shell" structure. In addition, the (002) crystal plane shows a special parallel relationship with the y-axis direction of collagen molecules. This method is surprisingly consistent with the natural bone structure, providing theoretical guidance for studying the bone mineralization process. For example Figure 7 , 8.

[0086] Comparative Example 1

[0087] Before mineralization, collagen fibers were separately dissolved in phosphoric acid or CaCl 2 solution. Other steps were the same as in Example 1. Although fiber morphology would also be formed, the fibers were prone to agglomeration and the yield was low. It was easy to form a mechanical mixture composed of collagen / hydroxyapatite, such as Figure 9 .

[0088] Figure 9 shows the morphology of mineralized collagen fibers. The morphology of mineralized collagen fibers obtained by first conjugating collagen fibers with calcium ions separately and then mineralizing for 24 hours. Agglomeration occurred between the fibers and the fiber morphology was uneven, making it difficult to distinguish the complete morphology of mineralized collagen fibers.

[0089] Comparative Example 2

[0090] The mineralized collagen fibers synthesized reported currently have certain differences from this method. The original method was described as collagen being only dissolved in calcium ion solution or dissolved in phosphate solution, with other methods remaining unchanged, but the morphology of the obtained mineralized collagen would not uniformly present a fibrous shape. The reason is that the interaction between collagen and Ca ions or phosphate ions is relatively strong. When another ion exists in the solution, its binding force may be higher than the interaction between collagen and the ion. Therefore, a large irregular mineralized collagen morphology will be formed.

[0091] By using this method, the free interaction between the two ions is fully avoided, and the template role of collagen is fully exerted. Furthermore, a mineralized collagen fiber morphology with uniform morphology is formed. Such as Figures 10 - 13 .

[0092] Such as Figure 10 and Figure 11 show the morphology of mineralized collagen fibers observed by the traditional sample preparation method, the TEM morphology of mineralized collagen fibers obtained at 90% mineralization degree (mineralized for 16 hours) and the locally selected diffraction pattern; such as Figure 12 and Figure 13Shown are the morphological features of mineralized collagen fibers observed by traditional sample preparation methods, the TEM morphological features of mineralized collagen fibers obtained at 100% mineralization degree (mineralization for more than 24 h), and the selected diffraction patterns locally. From the above data, it is difficult to visually observe and distinguish the mineralization degree. And in the case of higher resolution, due to irradiation damage, Figure 13 it is difficult to carefully observe the crystal structure in the fibers.

[0093] Comparative Example 3

[0094] The method for preparing mineralized collagen fibers is the same as described above. However, in step (5) of Example 1, after centrifugal washing at 2500 rpm, the precipitate is dispersed in deionized water again. In the traditional sample preparation process, the suspension is dropped on a copper mesh and left to dry. The morphological features of the mineralized collagen fibers observed by this method are as Figures 10 - 13 shown. Due to the inconsistent high and low contrast of the sample and irradiation intolerance, it is difficult to observe the fine morphological features of the mineralized collagen fibers.

[0095] By extracting and observing the transmission electron microscope sample of the pressed block, the microscopic structural morphological features of the block are as Figure 7 shown, which can avoid such problems.

[0096] Comparative Example 4

[0097] Under high pressure, commercial hydroxyapatite powder does not fuse. The reason is that commercial hydroxyapatite has a mature crystal structure and no phase transformation occurs during the pressing process. Therefore, it is more likely to break. At the same time, the traditional preparation process can be used for observing commercial hydroxyapatite.

[0098] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for observing mineralized collagen fibers that simulate the in - vivo bone mineralization process, characterized in that, the method includes, obtaining mineralized collagen nanofibers with different mineralization degrees; performing pressure molding on the mineralized collagen nanofibers to obtain a bulk structure; wherein, the pressure molding parameters are pressurizing at room temperature to 500 - 900 MPa and maintaining a constant pressure for 10 - 15 min; observing the bulk structure.

2. The method for observing mineralized collagen fibers that simulate the in - vivo bone mineralization process according to claim 1, characterized in that, the mineralized collagen nanofibers with different mineralization degrees include: mineralized collagen fibers with a mineralization degree reaching 90% to 100%.

3. The method for observing mineralized collagen fibers that simulate the in - vivo bone mineralization process according to claim 1, characterized in that, the average length of the mineralized collagen nanofibers with different mineralization degrees is 200 nm to 240 nm.

4. The method for observing mineralized collagen fibers that simulate the in - vivo bone mineralization process according to claim 1, characterized in that, the average diameter of the mineralized collagen nanofibers with different mineralization degrees is 8 nm to 10 nm.

5. The method for observing mineralized collagen fibers that simulate the in - vivo bone mineralization process according to claim 1, characterized in that, performing pressure molding on the mineralized collagen nanofibers includes: the pressure direction is perpendicular to the layered structure of the mineralized collagen nanofibers.

6. The method for observing mineralized collagen fibers that simulate the in - vivo bone mineralization process according to any one of claims 1 - 5, characterized in that, the preparation of the mineralized collagen nanofibers includes: mixing type I collagen sponge with a phosphoric acid solution to obtain a collagen template solution; Dissolve type I collagen sponge in a solution containing CaCl 2 / HCl to obtain a calcium ion template solution; performing a mineralization reaction on the collagen template solution and the calcium ion template solution, and obtaining mineralized collagen nanofibers with different mineralization degrees by controlling the mineralization time.

7. The method for observing mineralized collagen fibers that simulate the in - vivo bone mineralization process according to claim 6, characterized in that, the preparation of the mineralized collagen nanofibers further includes: performing freeze - drying on the mineralized collagen fibers with different mineralization degrees, and the freeze - drying conditions are: - 50°C, 50 Pa for 48 - 60 h.

8. The method for observing mineralized collagen fibers that simulate the in - vivo bone mineralization process according to claim 6, characterized in that, the mixing time of the type I collagen sponge and the phosphoric acid solution is 6 h to 12 h; the mixing temperature is 35°C to 38°C.

9. The method for observing mineralized collagen fibers that simulate the in - vivo bone mineralization process according to claim 6, characterized in that, Dissolve type I collagen sponge in a solution containing CaCl 2 / HCl for 6 to 12 hours at a dissolution temperature of 35°C to 38°C.

10. The application of the method for observing mineralized collagen fibers that simulate the in - vivo bone mineralization process according to any one of claims 1 - 9 in observing bionic materials for oil well equipment.

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