Zoledronic acid-initiated fiber internal and external mineralized collagen and preparation method thereof

Abstract

The invention discloses zoledronic acid initiated fiber inside and outside mineralized collagen gel and a preparation method thereof, and belongs to the technical field of biological materials and tissue engineering. The technical problems that in the prior art, due to blending of phosphate and medicine, the combination stability and uniformity of mineralized crystals and collagen are insufficient, and medicine loading is limited are solved. The preparation method comprises the following steps: blending a type I collagen solution, a buffer solution, a sodium hydroxide solution and deionized water, controlling the pH value to be 7.0-7.5, and incubating to obtain unmineralized type I collagen gel; then blending the zoledronic acid solution and the calcium chloride-polyacrylic acid mixed solution to obtain a mineralized solution; and finally, uniformly mixing the unmineralized type I collagen gel with the mineralizing liquid, incubating, and discarding the liquid after the incubation is completed, thereby obtaining the zoledronic acid initiated inside and outside mineralized collagen of the fiber. The mineralized collagen not only has a more bionic three-dimensional mineralized structure, but also has good calcium ion deposition attraction capability and osteoclast proliferation inhibition capability.

Description

Technical Field

[0001] The present invention relates to a mineralized collagen inside and outside fibers induced by zoledronic acid (ZOL) and a preparation method thereof, belonging to the technical field of biomaterials and tissue engineering. Background Art

[0002] With the acceleration of the aging process of the population in China, osteoporosis (OP) has become a severe public health problem. Arthroplasty is an effective means for treating end-stage joint diseases, but OP patients face a higher risk of prosthesis-related complications after surgery. Some studies have shown that after 5 years, the risk of revision due to periprosthetic fracture (PPF) in OP patients is 1.8 times that of patients without osteoporosis, and the cumulative incidence of PPF is 0.8%, which is significantly higher than 0.4% of non-OP patients. In addition, the revision risks of aseptic loosening and periprosthetic joint infection (PJI) in OP patients both increase by 20%, and the 5-year cumulative incidences are 1.0% and 1.3% respectively, higher than 1.1% and 1.5% of the control group. The precise matching of the prosthesis shape and the interface bone integration effect are the core factors determining the long-term survival rate after surgery. Although the mechanism of long-term stability achieved by bio-fixed prostheses through bone integration has been widely applied, there are still three major technical bottlenecks in OP patients: ① the stress shielding effect caused by the mismatch of elastic modulus between the prosthesis and bone tissue; ② insufficient prosthesis conformity at the cancellous bone defect site; ③ lack of anti-osteoporosis function to actively regulate bone metabolism balance. Therefore, developing a new type of prosthesis interface material with both biomechanical compatibility and bone metabolism regulation ability has become a key technical problem to be solved urgently in the field of orthopedic implants.

[0003] In response to the above problems, the modification strategy of filling mineralized collagen materials into the interface of porous titanium alloy prostheses shows good application prospects. As the core component of natural bone matrix, biomimetic mineralized collagen has significant advantages of composition biomimesis (the composite of type I collagen and nano-hydroxyapatite) and structure biomimesis (fibrous ordered assembly). Its unique properties include: no immunogenicity, excellent biocompatibility, injectable bonding fluidity and controllable degradability, and it can effectively fill the irregular pores of the prosthesis microporous interface. More importantly, the mineralized crystal structure can induce the directional deposition of calcium ions in body fluid through surface hydroxyl and carboxyl groups to form a precursor of interface bone bonding; the collagen polypeptides and mineral ions released during its degradation process can directly participate in the regulation of osteogenic signaling pathways, and the nano-scale spatial structure formed by the gaps between collagen fibers provides an ideal carrier for drug or growth factor loading, which can synergistically promote the adhesion, proliferation and differentiation of osteoblasts into a mineralized phenotype.

[0004] Bisphosphonates (BPs), as first-line drugs for anti-OP, the bisphosphonic acid group in their molecular structure can form a highly stable complex with calcium ions through multidentate chelation, and can directly participate in the biomimetic synthesis process of mineralized collagen; at the same time, the nitrogen-containing heterocyclic structure endows it with specific anti-osteoclast activity, which can reverse the bone metabolic imbalance in the OP state and reconstruct the dynamic balance of osteogenesis-osteoclast. As a third-generation nitrogen-containing bisphosphonate, zoledronic acid, in addition to the above pharmacological effects, its molecular anchoring effect can significantly improve the early interface stability of implants. For example, a Chinese patent application for a mineralized collagen hydrogel and its preparation method and application (CN 116785503A) uses a sodium hydrogen phosphate and zoledronic acid blend system to induce collagen mineralization. Although it can achieve the dual functions of osteoclast inhibition and osteogenesis promotion, this blend system has inherent defects: the phosphate ions generated by the ionization of sodium hydrogen phosphate compete with zoledronic acid molecules for the calcium binding sites on collagen fibers, and at the same time change the concentration gradient of free calcium ions in the solution, resulting in randomness in the nucleation sites and growth directions of hydroxyapatite crystals during the mineralization process, causing a significant decrease in the structural stability and compositional uniformity of the collagen mineralization layer, and ultimately affecting the biological properties of the material and the interface bone integration efficiency. Summary of the Invention

[0005] In order to solve the technical problems in the prior art that the blending of phosphates and drugs leads to insufficient stability and uniformity of mineralized crystallization and collagen binding, and limited drug loading, the present invention provides a zoledronic acid-induced mineralized collagen inside and outside the fiber and its preparation method. The zoledronic acid-induced mineralized collagen inside and outside the fiber of the present invention realizes the chelation of calcium ions and the regulation of calcium ion diffusion with the help of zoledronic acid and polyacrylic acid, and not only has a more biomimetic three-dimensional mineralized structure design, but also has good ability to attract calcium ion deposition and the ability to inhibit osteoclast proliferation.

[0006] To achieve the above object, the present invention adopts the following technical solutions.

[0007] The preparation method of the zoledronic acid-induced mineralized collagen inside and outside the fiber of the present invention includes the following steps:

[0008] (1) Blend a type I collagen solution, a buffer solution, a sodium hydroxide solution and deionized water according to a volume ratio of 1:0.5-1:0.01-0.1:0.3-0.5, and control the pH to be 7.0-7.5, and incubate to obtain an unmineralized type I collagen gel;

[0009] (2) Dissolve zoledronic acid in deionized water to obtain solution A;

[0010] Dissolve calcium chloride in deionized water, and mix the obtained calcium chloride solution with polyacrylic acid to obtain solution B;

[0011] Mix solution A and solution B to obtain a mineralization solution;

[0012] (3) Immerse the unmineralized type I collagen gel in the mineralization solution, incubate for more than 24 h, and replace the mineralization solution every 24 h. After incubation, discard the liquid to obtain the collagen with internal and external mineralization induced by zoledronic acid.

[0013] Preferably, in step (1), the volume ratio of the type I collagen solution, buffer solution, sodium hydroxide solution and deionized water is 1:0.5:0.023:0.5.

[0014] Preferably, in step (1), the concentration of the type I collagen solution is 3 - 5 mg / ml, and the concentration of the sodium hydroxide solution is 0.1 - 1 mol / L.

[0015] Preferably, in step (1), the buffer solution is triethanolamine buffered saline solution (TBS buffer solution),

[0016] More preferably, the buffer solution is 1×TBS buffer solution or 10×TBS buffer solution (200 mM Tris, 1.37 M NaCl, pH 7.6 ± 0.1 (25 °C)).

[0017] Preferably, in step (1), the incubation temperature is 37 - 40 °C, and the incubation time is 1.5 - 2 h.

[0018] Preferably, in step (2), in the mineralization solution, the concentration of zoledronic acid is 4×10 -6 - 10×10 -4 mol / L, the concentration of calcium chloride is 6.68×10 -6 - 16.7×10 -4 mol / L, the concentration of polyacrylic acid is 40 - 50 mg / L, and the molar ratio of Ca to P is 1.67 - 2:1.

[0019] Preferably, in step (2), in solution A, the concentration of zoledronic acid is 8×10 -6 - 20×10 -4 mol / L.

[0020] Preferably, in step (2), the concentration of the calcium chloride solution is 13.36×10 -6 - 33.4×10 -4 mol / L.

[0021] Preferably, in step (2), the molecular weight of the polyacrylic acid is 2 kDa - 50 kDa.

[0022] Preferably, in step (2), dissolve zoledronic acid in deionized water by ultrasonic treatment to obtain solution A;

[0023] Calcium chloride was ultrasonically dissolved in deionized water, and the obtained calcium chloride solution was mixed with polyacrylic acid and ultrasonically homogenized to obtain Solution B;

[0024] Solution A and Solution B were mixed evenly by oscillation at an oscillation speed of 15 - 120 r / min.

[0025] Preferably, in step (3), the incubation temperature is 37 - 40 °C and the incubation time is 3 - 7 days.

[0026] The present invention also provides the intra - and extra - fibrillar mineralized collagen initiated by zoledronic acid prepared by the above - mentioned preparation method.

[0027] The principle of the present invention is as follows: For the intra - and extra - fibrillar mineralized collagen initiated by zoledronic acid of the present invention, zoledronic acid is used as a single phosphate donor. The bisphosphonic acid group in its molecular structure specifically binds to calcium ions through multidentate chelation, forming active sites with uniform charge distribution on the surface of collagen fibers, guiding the mineral phase to attach to the surface of collagen fibers in a highly ordered manner. On this basis, the carboxyl groups on the polyacrylic acid molecular chain bind to calcium ions through dynamic complexation and enrich phosphate ions through electrostatic adsorption, constructing a locally supersaturated calcium - phosphorus solution in the collagen fiber microenvironment. This promotes the mineralization precursor to enter the interior of collagen fibers in the form of nanoparticles, realizing the attachment of mineralized substances inside the fibers.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] For the intra - and extra - fibrillar mineralized collagen initiated by zoledronic acid of the present invention, with zoledronic acid as a single phosphate donor, this single - donor design avoids the competitive interference of phosphate ions in the traditional blending system, making the mineralization process exhibit excellent uniformity and controllability, and endowing the mineralized collagen with the unique function of inhibiting the activation of pathological osteoclasts. Moreover, through the synergistic effect of the single phosphate donor and polyacrylic acid, the present invention effectively inhibits the rapid and disordered precipitation of calcium and phosphorus ions, promoting the penetration of mineralization precursors such as amorphous calcium phosphate into the internal pores of collagen fibers in the form of nanoparticles. It realizes the uniform attachment and directional deposition of mineral phases inside and outside the collagen fibers, forming a mineralized collagen material with bionic structure and synergistic function. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for description in the embodiments. Obviously, the following - described drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0031] Figure 1Transmission electron microscopy (TEM) images of the collagen fibers with internal and external mineralization induced by zoledronic acid prepared in Example 1 of the present invention, the unmineralized type I collagen gel prepared in Comparative Example 1, and the collagen fibers with internal and external mineralization induced by disodium hydrogen phosphate-zoledronic acid prepared in Comparative Example 2. Among them, A is the unmineralized type I collagen gel, B is the collagen fibers with internal and external mineralization induced by disodium hydrogen phosphate-zoledronic acid, and C is the collagen fibers with internal and external mineralization induced by zoledronic acid. The scales in A, B, and C are the same, all being 200 nm.

[0032] Figure 2 Scanning electron microscopy (SEM) images of the collagen fibers with internal and external mineralization induced by zoledronic acid prepared in Example 1 of the present invention and the collagen fibers with internal and external mineralization induced by disodium hydrogen phosphate-zoledronic acid prepared in Comparative Example 2. Among them, A is the SEM image of the collagen fibers with internal and external mineralization induced by disodium hydrogen phosphate-zoledronic acid, and B is the SEM image of the collagen fibers with internal and external mineralization induced by zoledronic acid. The scales in A and B are the same, all being 1 µm.

[0033] Figure 3 Fourier transform infrared spectroscopy (FTIR) spectra of the collagen fibers with internal and external mineralization induced by zoledronic acid prepared in Example 1 of the present invention, the unmineralized type I collagen gel prepared in Comparative Example 1, and the collagen fibers with internal and external mineralization induced by disodium hydrogen phosphate-zoledronic acid prepared in Comparative Example 2. In the figure, the curves from bottom to top are the collagen fibers with internal and external mineralization induced by zoledronic acid, the collagen fibers with internal and external mineralization induced by disodium hydrogen phosphate-zoledronic acid, and the unmineralized type I collagen gel, respectively.

[0034] Figure 4 Water contact angle measurement images of the collagen fibers with internal and external mineralization induced by zoledronic acid prepared in Example 1 of the present invention, the unmineralized type I collagen gel prepared in Comparative Example 1, and the collagen fibers with internal and external mineralization induced by disodium hydrogen phosphate-zoledronic acid prepared in Comparative Example 2. Among them, A is the collagen fibers with internal and external mineralization induced by zoledronic acid, B is the unmineralized type I collagen gel, and C is the collagen fibers with internal and external mineralization induced by disodium hydrogen phosphate-zoledronic acid.

[0035] Figure 5 Qualitative alkaline phosphatase staining images of the collagen fibers with internal and external mineralization induced by zoledronic acid prepared in Example 1 of the present invention, the unmineralized type I collagen gel prepared in Comparative Example 1, and the collagen fibers with internal and external mineralization induced by disodium hydrogen phosphate-zoledronic acid prepared in Comparative Example 2. Among them, A is the unmineralized type I collagen gel, B is the collagen fibers with internal and external mineralization induced by disodium hydrogen phosphate-zoledronic acid, and C is the collagen fibers with internal and external mineralization induced by zoledronic acid.

[0036] Figure 6Alizarin red staining diagrams of the collagen fibrils mineralized inside and outside by zoledronic acid prepared in Example 1 of the present invention, the unmineralized type I collagen gel prepared in Comparative Example 1, and the collagen fibrils mineralized inside and outside by disodium hydrogen phosphate-zoledronic acid prepared in Comparative Example 2, where A is the unmineralized type I collagen gel, B is the collagen fibrils mineralized inside and outside by disodium hydrogen phosphate-zoledronic acid, and C is the collagen fibrils mineralized inside and outside by zoledronic acid.

[0037] Figure 7 Trap staining diagrams of the collagen fibrils mineralized inside and outside by zoledronic acid prepared in Example 1 of the present invention, the unmineralized type I collagen gel prepared in Comparative Example 1, and the collagen fibrils mineralized inside and outside by disodium hydrogen phosphate-zoledronic acid prepared in Comparative Example 2, where A is the unmineralized type I collagen gel, B is the collagen fibrils mineralized inside and outside by disodium hydrogen phosphate-zoledronic acid, and C is the collagen fibrils mineralized inside and outside by zoledronic acid. The scales in A, B, and C are the same, all being 360 µm.

[0038] Figure 8 F-actin diagrams of the collagen fibrils mineralized inside and outside by zoledronic acid prepared in Example 1 of the present invention, the unmineralized type I collagen gel prepared in Comparative Example 1, and the collagen fibrils mineralized inside and outside by disodium hydrogen phosphate-zoledronic acid prepared in Comparative Example 2, where A is the unmineralized type I collagen gel, B is the collagen fibrils mineralized inside and outside by disodium hydrogen phosphate-zoledronic acid, and C is the collagen fibrils mineralized inside and outside by zoledronic acid. The scales in A, B, and C are the same, all being 90 nm. Detailed implementation manners

[0039] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below in conjunction with embodiments.

[0040] In the following embodiments, various processes and methods not described in detail are conventional methods well known in the art. The materials, reagents, devices, instruments, equipment, etc. used in the following embodiments can be obtained from commercial channels without special instructions.

[0041] Example 1

[0042] A preparation method of collagen fibrils mineralized inside and outside by zoledronic acid, the steps are as follows:

[0043] (1) Mix a type I collagen solution, a TBS buffer solution, a sodium hydroxide solution, and deionized water according to a volume ratio of 1:0.5:0.023:0.5, and incubate at 37 °C for 2 h to obtain an unmineralized type I collagen gel; wherein, the concentration of the type I collagen solution is 3 mg / ml, the buffer solution is 10×TBS (model ST667-1L), and the concentration of the sodium hydroxide solution is 1 mol / L;

[0044] (2) Add 5.44 mg of zoledronic acid to 10 ml of deionized water. After ultrasonic dissolution, solution A is obtained.

[0045] Add 3.7 mg of calcium chloride to 10 ml of deionized water. After ultrasonic dissolution, add 0.8 mg of polyacrylic acid (2 kDa) to the obtained calcium chloride solution and mix to obtain solution B.

[0046] Mix solution A and solution B and shake for 15 minutes to obtain 20 ml of mineralization solution. Among them, the final concentration of zoledronic acid is 10×10 -4 M, the final concentration of calcium chloride is 16.7×10 -4 M, and the final concentration of polyacrylic acid is 40 mg / L.

[0047] (3) Immerse the unmineralized type I collagen gel in the mineralization solution and incubate at 37 °C for 72 h, and change the mineralization solution every 24 h. After the incubation is completed, discard the liquid to obtain the collagen mineralized inside and outside the fibers induced by zoledronic acid, denoted as sample 1.

[0048] Comparative Example 1

[0049] Mix the type I collagen solution, TBS buffer, sodium hydroxide solution and deionized water according to a volume ratio of 1:0.5:0.023:0.5 and incubate at 37 °C for 2 h to obtain an unmineralized type I collagen gel, denoted as sample 2; among them, the concentration of the type I collagen solution is 3 mg / ml, the buffer is 10×TBS (model ST667-1L), and the concentration of the sodium hydroxide solution is 1 mol / L.

[0050] Comparative Example 2

[0051] (1) Mix the type I collagen solution, TBS buffer, sodium hydroxide solution and deionized water according to a volume ratio of 1:0.5:0.023:0.5 and incubate at 37 °C for 2 h to obtain an unmineralized type I collagen gel; among them, the concentration of the type I collagen solution is 3 mg / ml, the buffer is 10×TBS (model ST667-1L), and the concentration of the sodium hydroxide solution is 1 mol / L.

[0052] (2) Add 2.72 mg of zoledronic acid and 1.42 mg of disodium hydrogen phosphate to 10 ml of deionized water (the molar ratio of zoledronic acid to disodium hydrogen phosphate is 1:1). After ultrasonic dissolution, solution A is obtained.

[0053] Add 3.7 mg of calcium chloride to 10 ml of deionized water. After ultrasonic dissolution, add 0.8 mg of polyacrylic acid (2 kDa) to the calcium chloride solution and mix well to obtain solution B.

[0054] After mixing solution A and solution B and shaking for 15 minutes, 20 ml of zoledronic acid-disodium hydrogen phosphate-calcium mineralization solution was obtained, in which the concentration of zoledronic acid was 5×10 -4 M, the concentration of disodium hydrogen phosphate was 5×10 -4 M, the concentration of calcium chloride was 16.7×10 -4 M, and the concentration of polyacrylic acid was 40 mg / L;

[0055] (3) Immerse the unmineralized type I collagen gel in the mineralization solution, incubate at 37 °C for 72 h, and change the mineralization solution every 24 h. After incubation, discard the liquid to obtain the zoledronic acid-disodium hydrogen phosphate-induced intra- and extra-fibrillar mineralized collagen, denoted as sample 3.

[0056] The zoledronic acid-induced intra- and extra-fibrillar mineralized collagen (sample 1) prepared in Example 1, the unmineralized type I collagen gel (sample 2) prepared in Comparative Example 1, and the zoledronic acid-disodium hydrogen phosphate-induced intra- and extra-fibrillar mineralized collagen (sample 3) prepared in Comparative Example 2 were characterized. The transmission electron micrographs are as shown in Figure 1 As can be seen from Figure 1 , in the mineralized groups (sample 1 and sample 3), the deposition of minerals could be achieved, and the mineral deposition of the zoledronic acid-induced mineralized collagen was more adherent to the collagen fibers than that of the disodium hydrogen phosphate-zoledronic acid mineralized collagen. The scanning electron micrographs are as shown in Figure 2 As can be seen from Figure 2 , the mineral particles deposited on the surface of the zoledronic acid-induced intra- and extra-fibrillar mineralized collagen were larger in size and more in number, indicating that the zoledronic acid mineralization system had a more superior effect of attracting calcium ion deposition than the blending system due to its more uniform charge distribution. The Fourier transform infrared spectroscopy images are as shown in Figure 3 . As can be seen from the Fourier transform infrared spectroscopy diagram of the unmineralized type I collagen gel, the gel construction method of the present invention completely retained the core structure (amide I, II, III) of the collagen fibers. As can be seen from the Fourier transform infrared spectroscopy diagrams of the zoledronic acid-disodium hydrogen phosphate-induced intra- and extra-fibrillar mineralized collagen and the zoledronic acid-induced intra- and extra-fibrillar mineralized collagen, on the basis of not damaging the core structure of the collagen gel, the mineralization operation successfully loaded disodium hydrogen phosphate and zoledronic acid. The water contact angle diagrams are as shown in Figure 4 As can be seen from Figure 4 , as the water contact angles of the samples in the mineralized groups (sample 1 and sample 3) were both smaller than those of the pure collagen group (sample 2), and the water contact angle of the zoledronic acid-induced intra- and extra-fibrillar mineralized collagen was smaller than that of the zoledronic acid-disodium hydrogen phosphate-induced intra- and extra-fibrillar mineralized collagen. This was mainly because the number of mineral crystals on the surface of the zoledronic acid-induced intra- and extra-fibrillar mineralized collagen was more and the roughness was larger than that of the zoledronic acid-disodium hydrogen phosphate-induced intra- and extra-fibrillar mineralized collagen.

[0057] The ability of the fiber inner and outer mineralized collagen induced by zoledronic acid prepared in Example 1, the unmineralized type I collagen gel prepared in Comparative Example 1, and the fiber inner and outer mineralized collagen induced by zoledronic acid-disodium hydrogen phosphate prepared in Comparative Example 2 to regulate bone metabolism homeostasis was detected.

[0058] The experiments related to regulating the osteogenic process are as follows: In a 24-well plate, 2×10 4 bone marrow mesenchymal stem cells (derived from Sprague-Dawley Rats) were co-cultured with the samples in each well. Using osteogenic induction medium (450 ml DMEM-F12 medium, 10% fetal bovine serum, 1% penicillin-streptomycin double antibody, 12.8 mg / L vitamin C, 2.16 g / L β-glycerophosphate, 5 mmol / L dexamethasone), after culturing in a 37°C, 5% CO2 cell culture incubator for 14 days, an alkaline phosphatase (ALP) staining kit and an alizarin red staining kit were used for qualitative analysis of ALP and qualitative analysis of calcium nodules.

[0059] Figure 5 In A, B, and C in [reference], they are the alkaline phosphatase staining diagrams of Sample 2, Sample 3, and Sample 1 respectively. It can be seen from Figure 5 that the staining color of the fiber inner and outer mineralized collagen induced by zoledronic acid is deeper than that of the unmineralized type I collagen gel and the fiber inner and outer mineralized collagen induced by zoledronic acid-disodium hydrogen phosphate. Figure 6 In A, B, and C in [reference], they are the alizarin red staining diagrams of Sample 2, Sample 3, and Sample 1 respectively. It can be seen from Figure 6 that the number of calcium nodules of the fiber inner and outer mineralized collagen induced by zoledronic acid is more than that of the unmineralized type I collagen gel and the fiber inner and outer mineralized collagen induced by zoledronic acid-disodium hydrogen phosphate, and the calcium nodules are larger, indicating that the fiber inner and outer mineralized collagen induced by zoledronic acid has a more excellent osteogenic effect. Figure 5 and Figure 6 The results of [reference] show that the fiber inner and outer mineralized collagen induced by zoledronic acid of the present invention has a certain osteogenic effect, and the osteogenic effect is better than that of the unmineralized type I collagen gel and the fiber inner and outer mineralized collagen induced by zoledronic acid-disodium hydrogen phosphate.

[0060] The experiments related to regulating the osteoclastic process are as follows: In a 6-well plate, 2×10 4A number of RAW264.7 (mouse monocyte macrophage leukemia cells, derived from a tumor induced by Abelson murine leukemia virus in male mice, purchased from the Cell Bank of the Chinese Academy of Sciences, used as pre-osteoclast cells) were co-cultured with the samples. MEM-α medium containing 50 - 100 ng / ml osteoclast-inducing factor (RANKL) was used to induce the directional differentiation of RANW264.7 cells into osteoclasts. After culturing in a 37°C, 5% CO2 cell incubator for 5 days, a tartrate-resistant acid phosphatase (TRAP) staining kit was used to count the number of osteoclasts formed. At the same time, phalloidin and DAPI were used for fluorescence staining of the cells.

[0061] Figure 7 In A, B, and C are the TRAP staining diagrams of Sample 2, Sample 3, and Sample 1 respectively. Figure 8 In A, B, and C are the F-actin ring staining diagrams of Sample 2, Sample 3, and Sample 1 respectively. From Figure 7 and Figure 8 it can be seen that zoledronic acid induces fewer osteoclasts in the mineralized collagen inside and outside the fibers, and the F-actin ring staining of osteoclasts shows that its ability to inhibit osteoclasts is the most excellent among the three samples.

[0062] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the embodiments. For those of ordinary skill in the art, other different forms of changes or variations can be made based on the above description. It is not necessary and impossible to enumerate all embodiments here. And the obvious changes or variations derived therefrom are still within the protection scope of the present invention.

Claims

1. A method for preparing fiber-internal and fiber-external mineralized collagen induced by zoledronic acid, characterized in that, It includes the following steps: (1) Mix a type I collagen solution, a buffer solution, a sodium hydroxide solution, and deionized water in a volume ratio of 1:0.5 - 1:0.01 - 0.1:0.3 - 0.5, and control the pH to be 7.0 - 7.5, then incubate to obtain an unmineralized type I collagen gel; (2) Dissolve zoledronic acid in deionized water to obtain solution A; Dissolve calcium chloride in deionized water, and mix the obtained calcium chloride solution with polyacrylic acid to obtain solution B; Mix solution A and solution B to obtain a mineralization solution; (3) Immerse the unmineralized type I collagen gel in the mineralization solution, incubate for more than 24 h, and change the mineralization solution every 24 h. After the incubation is completed, discard the liquid to obtain zoledronic acid-induced collagen with internal and external mineralization of fibers.

2. The preparation method of the fiber-internal and fiber-external mineralized collagen induced by zoledronic acid according to claim 1, characterized in that, In step (1), the volume ratio of the type I collagen solution, the buffer solution, the sodium hydroxide solution, and the deionized water is 1:0.5:0.023:0.

5.

3. The preparation method of the in-fiber and out-fiber mineralized collagen induced by zoledronic acid according to claim 1, characterized in that, In step (1), the concentration of the type I collagen solution is 3 - 5 mg / ml, the concentration of the sodium hydroxide solution is 0.1 - 1 mol / L, and the buffer solution is a TBS buffer solution.

4. The preparation method of the in-fiber and out-fiber mineralized collagen induced by zoledronic acid according to claim 3, wherein, The buffer solution is 1×TBS buffer solution or 10×TBS buffer solution.

5. The preparation method of the intra- and extra-fibrillar mineralized collagen induced by zoledronic acid according to claim 1, wherein In step (1), the incubation temperature is 37 - 40 °C, and the incubation time is 1.5 - 2 h.

6. The preparation method of the intra- and extra-fibrillar mineralized collagen induced by zoledronic acid according to claim 1, characterized in that, In step (2), in the mineralization solution, the concentration of zoledronic acid is 4×10 -6 -10×10 -4 mol / L, the concentration of calcium chloride is 6.68×10 -6 -16.7×10 -4 mol / L, the concentration of polyacrylic acid is 40 - 50 mg / L, and the molar ratio of Ca to P is 1.67 - 2:

1.

7. The preparation method of the intra- and extra-fibrillar mineralized collagen induced by zoledronic acid according to claim 1, wherein, In step (2), in the solution A, the concentration of zoledronic acid is 8×10 -6 -20×10 -4 mol / L; The concentration of the calcium chloride solution is 13.36×10 -6 -33.4×10 -4 mol / L.

8. According to the method for preparing zoledronic acid-induced collagen with internal and external mineralization of fibers as described in claim 1, It is characterized in that In step (2), the molecular weight of the polyacrylic acid is 2 kDa - 50 kDa.

9. The preparation method of the zoledronic acid-induced mineralized collagen inside and outside fibers according to claim 1, characterized in that, In step (3), the incubation temperature is 37 - 40 °C, and the incubation time is 3 - 7 days.

10. Zoledronic acid-induced collagen with internal and external mineralization of fibers prepared by the preparation method according to any one of claims 1 - 9.

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