Multiphase biomimetic mineralized collagen scaffold as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biomimetic materials, in particular to a multiphase biomimetic mineralized collagen scaffold as well as a preparation method and application thereof. The preparation method comprises the following steps: self-assembling an I-type collagen solution and solid-phase copper sulphide nanoparticles, and carrying out intra-fiber mineralization on collagen fibrils by virtue of a liquid-phase mineralization technology, so as to form the multiphase biomimetic mineralized collagen scaffold which is similar to a natural bone nanostructure and Young modulus. The scaffold can improve energy metabolism of cells by regulating oxidative phosphorylation of bone marrow mesenchymal stem cells, so as to promote osteogenic differentiation of the bone marrow mesenchymal stem cells; in addition, copper ions are released through the stent to play the activity of mimic enzyme, and a large amount of active oxygen is generated in bacteria through catalysis, so that membrane rupture and content overflow of the bacteria are caused, and the antibacterial effect is achieved. The scaffold can be used for treating simple bone defects or infectious bone defects by regulating energy metabolism of cells and exerting mimic enzyme activity, and more novel methods for realizing bone regeneration and preparing anti-infection drugs are provided for clinic.

Description

Technical Field

[0001] The present invention relates to the technical field of biomimetic materials, and particularly to a multiphase biomimetic mineralized collagen scaffold, a preparation method thereof, and uses thereof in the preparation of drugs for regulating energy metabolism and repairing infectious bone defects. Background Art

[0002] Infectious bone defects often occur after fracture repair, bone-related surgeries, or trauma when the bone is invaded by bacteria, which has a significant impact on the patient's daily life and recovery of physical function. With the aging of the population and the frequent occurrence of sports-related injuries, the prevalence of infectious bone defects is on the rise. The main pathogenic bacteria include Staphylococcus aureus and Escherichia coli, etc., which may spread through the blood or directly infect the bone, leading to complications such as osteomyelitis.

[0003] Traditional methods for treating infectious bone defects include using antibiotics, performing surgical removal of infected tissues, and using various bone graft materials. Although these methods can control infection and promote bone healing to a certain extent, they each have limitations. In particular, the problem of antibiotic resistance is becoming increasingly prominent. The emergence of drug-resistant strains reduces the effectiveness of traditional antibiotic treatments and prolongs the treatment cycle. In addition, the overuse of antibiotics may lead to a disorder of the microbial balance in the human body, thereby exacerbating the infection. Although surgery can remove necrotic tissues, the surgery itself is invasive and may not completely remove the infected bacteria, increasing the risk of re-infection. At the same time, traditional bone graft materials, such as autologous bone, allogeneic bone, and artificial bone materials, although having certain effects in promoting bone healing, have problems such as difficult acquisition, donor site complications, immune rejection, and lack of antibacterial properties, which limit their scope of application.

[0004] Developing new bone repair materials to improve the treatment effect of infectious bone defects has become an important direction in bone tissue engineering research. In the field of bone tissue engineering, the strategy of promoting bone formation by adjusting cell energy metabolism shows great potential. The energy metabolism state of cells is crucial for key biological processes such as their survival, proliferation, and differentiation. There is a direct connection between the energy metabolism state of bone marrow mesenchymal stem cells and their osteogenic differentiation ability. When bone marrow mesenchymal stem cells are in a high energy metabolism state, they can carry out aerobic respiration more effectively, producing sufficient energy to support cell growth and function. In a suitable metabolic environment, these cells can not only proliferate rapidly but also efficiently synthesize bone matrix, improving the efficiency of osteogenesis. Therefore, optimizing the energy metabolism of bone marrow mesenchymal stem cells can not only accelerate the osteogenic process but also improve the quality and strength of the newly formed bone, providing a more solid foundation for the repair of bone defects. Summary of the Invention

[0005] In view of the deficiencies of existing treatment methods and for the purpose of improving cell energy metabolism to promote bone regeneration, the present invention provides a multiphase biomimetic mineralized collagen scaffold, a preparation method thereof, and an application thereof.

[0006] Specifically, the present application provides the following technical solutions: A preparation method of a multiphase biomimetic mineralized collagen scaffold: Synthesize copper sulfide nanoparticles, self-assemble a type I collagen solution and solid-phase copper sulfide nanoparticles in a bottom-up manner, and combine a liquid-phase mineralization technique to cause intrafibrillar mineralization of collagen fibrils to form a multiphase biomimetic mineralized collagen scaffold.

[0007] Among them, the specific method for synthesizing copper sulfide nanoparticles is to mix a copper-containing compound and sodium citrate, and dropwise add an aqueous solution of sodium disulfide under heating conditions. After ultracentrifugation, copper sulfide nanoparticles are obtained and dispersed into an aqueous solution of copper sulfide nanoparticles with ultrapure water.

[0008] Among them, the copper-containing compound is copper chloride with a concentration of 10 mmol / L; the concentration of sodium citrate is 2 g / L; the heating condition is 90 °C; the concentration of the aqueous solution of sodium disulfide is 1 mol / L; the ultracentrifugation speed is 30000 r / min.

[0009] Among them, the type I collagen solution is prepared from type I rat tail collagen, which can be obtained commercially, and its concentration is adjusted to 1 mg / mL with a 0.1M acetic acid solution. The concentration of copper sulfide nanoparticles can be adjusted as needed.

[0010] Among them, the self-assembly is completed with the assistance of a potassium ion buffer solution. The potassium ion buffer solution contains 200 mM potassium chloride, 30 mM disodium hydrogen phosphate, and 10 mM potassium dihydrogen phosphate.

[0011] Among them, after the self-assembly of the type I collagen solution and the solid-phase copper sulfide nanoparticles, centrifuge, discard the supernatant and collect the precipitate, load it into a mold and freeze-dry and crosslink to form a solid scaffold material.

[0012] Among them, the centrifugation speed is 5000 r / min for 5 min; the crosslinking is to immerse the material in an 80% ethanol solution containing 1% 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide for crosslinking for 4 h.

[0013] Among them, the liquid-phase mineralization technique is to immerse the crosslinked material in an intrafibrillar mineralization solution of collagen fibrils at 37 °C and 40 r / min for 5-7 d; the intrafibrillar mineralization solution of collagen fibrils contains 4.2 mM potassium hydrogen phosphate, 9 mM calcium chloride dihydrate, and 0.2 mg / mL polyaspartic acid.

[0014] The multiphase biomimetic mineralized collagen scaffold prepared by the present invention can exert a mimetic enzyme effect to inhibit bacterial growth; it can also be used to prepare drugs for repairing infectious bone defects by regulating energy metabolism.

[0015] Compared with the prior art, the multiphase biomimetic mineralized collagen scaffold, preparation method and application of the present invention at least have the following beneficial effects: The multiphase biomimetic mineralized collagen scaffold prepared by the present invention self-assembles a type I collagen solution and solid-phase copper sulfide nanoparticles in a bottom-up manner, and through a liquid-phase mineralization technique, the collagen fibrils undergo intra-fibrillar mineralization, so that the topological structure and Young's modulus formed are similar to those of natural bone. Therefore, this scaffold can create a more favorable osteogenic microenvironment, exert mimetic enzyme activity antibacterial by slowly releasing copper ions, or assist the mitochondria of bone marrow mesenchymal stem cells to strengthen oxidative phosphorylation to promote new bone formation, which is of great significance for the treatment of infectious bone defects or the development of drugs related to infectious bone defects.

[0016] The following further illustrates the multiphase biomimetic mineralized collagen scaffold, preparation method and application of the present invention with reference to the accompanying drawings. Brief Description of the Drawings

[0017] Figure 1 It is a three-dimensional material entity diagram of the multiphase mineralized collagen scaffold (mp-MCS) prepared in Example 1.

[0018] Figure 2 It is the morphology diagram of copper sulfide nanoparticles and mp-MCS in Example 1 under a transmission electron microscope (TEM). Among them, A represents copper sulfide nanoparticles, and B represents mp-MCS. Arrow: copper sulfide nanoparticles.

[0019] Figure 3 It is the morphology diagram and energy spectrum diagram of mp-MCS in Example 1 under a scanning electron microscope (SEM).

[0020] Figure 4 It is the atomic force microscope (AFM) diagram and Young's modulus statistical chart of mp-MCS in Example 1. Among them, A represents AFM, and B represents Young's modulus.

[0021] Figure 5 It is the cell proliferation activity after co-culturing different concentrations of mp-MCS with bone marrow mesenchymal stem cells in Example 2.

[0022] Figure 6 It is the cell live / dead staining diagram and semi-quantification diagram after co-culturing 60 μg / mL of mp-MCS with bone marrow mesenchymal stem cells in Example 2.

[0023] Figure 7This shows the expression of osteogenesis-related factors after co-culturing mp-MCS and bone marrow mesenchymal stem cells for 7 days or 14 days in Example 3. Among them, A shows the protein expression of osteogenesis-related genes at 7 days, and B shows the protein expression of osteogenesis-related genes at 14 days.

[0024] Figure 8 This shows the expression of oxidative phosphorylation-related factors after co-culturing mp-MCS and bone marrow mesenchymal stem cells for 7 days in Example 3. Among them, A shows the relative gene expression level, and B shows the relative protein expression level.

[0025] Figure 9A - Figure 9B This is the evaluation of the effect of mp-MCS in repairing non-infected rat cranial defects in Example 4. Among them, Figure 9A shows the micro-CT images and quantitative analysis images of new bone. Figure 9B shows the tissue HE and Masson staining images. ST: soft tissue, S: scaffold, ECM: extracellular matrix, NB: new bone.

[0026] Figure 10 This is the performance of the mimetic enzyme activity of mp-MCS in Example 5. Among them, A shows the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) after mp-MCS reacts with hydrogen peroxide or water. B shows the ability of mp-MCS to produce reactive oxygen species detected by electron paramagnetic resonance (EPR).

[0027] Figure 11 This is the detection of the in vitro antibacterial situation of mp-MCS in Example 5. Among them, A shows the bacterial colony experiment, and B shows the bacterial live / dead staining experiment, SEM and TEM detection.

[0028] Figure 12A - Figure 12B This is the evaluation of the effect of mp-MCS in repairing infected rat cranial defects in Example 6. Among them, Figure 12A shows the micro-CT images and quantitative analysis images of new bone. Figure 12B shows the tissue HE and Masson staining images. ST: soft tissue, S: scaffold, ECM: extracellular matrix, NB: new bone. Specific implementation manners Example 1 Preparation method and morphological characterization of a multiphase biomimetic mineralized collagen scaffold

[0029] (1) Synthesis of copper sulfide nanoparticles: In a beaker containing 100 mL of aqueous solution, dissolve 1 mmol of CuCl 2 and 0.2 g of sodium citrate. Continuously stir the mixture for 5 min to ensure complete dissolution of sodium citrate. Add 1 mmol of Na 2 S 2Dissolve it in 1 mL of aqueous solution, and then gradually add the resulting solution dropwise to the above-mentioned 100 mL of mixed solution. Heat the mixture to 90 °C and stir for 15 min to ensure complete reaction. Subsequently, immerse the solution in an ice-water bath for cooling. After ultracentrifugation at 30000 r / min for 10 min, disperse the nanoparticles with ultrapure water, and store the obtained sample at 4 °C for a long time.

[0030] (2) Assembly of type I collagen and copper sulfide nanoparticles: Adjust the concentration of the type I collagen solution to 1 mg / mL using 0.1 M acetic acid solution, and then mix it with 0.1 mg / mL copper sulfide nanoparticles to make the final concentration 60 μg / mL. With the assistance of a potassium ion solution (containing 200 mM potassium chloride, 30 mM disodium hydrogen phosphate, and 10 mM potassium dihydrogen phosphate), assemble it for 24 h through a bottom-up self-assembly mode.

[0031] (3) Freeze-drying and cross-linking: Centrifuge the mixture in the dialysis bottle in step (2) at 5000 r / min for 5 min, collect the precipitate and put it into a mold, and freeze-dry it for 12 hours. After obtaining the three-dimensional material, immerse it in an 80% anhydrous ethanol solution containing 1% 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) for cross-linking for 4 hours.

[0032] (4) Liquid-phase mineralization: Wash the cross-linked scaffold material in step (3) with ultrapure water, and then immerse it in a mixed solution prepared with TBS containing 4.2 mM dipotassium hydrogen phosphate, 9 mM calcium chloride dihydrate, and 0.2 mg / mL polyaspartic acid, and carry out mineralization at 37 °C and 40 r / min for 5 - 7 d to obtain the mp-MCS three-dimensional material ( Figure 1 )

[0033] (5) TEM ( Figure 2 ) : Place the copper sulfide nanoparticle solution and the prepared mp-MCS sample on nickel grids respectively, stain them negatively with 3% sodium phosphotungstate for 5 min, wash and dry them, and observe the ultrastructure of copper sulfide nanoparticles and mp-MCS through TEM. The results show that the diameter of copper sulfide nanoparticles is less than 10 nm, and its lattice spacing is about 0.268 nm. After assembling with collagen fibrils, it can adhere to the surface or enter the interior of the fibrils.

[0034] (6) SEM ( Figure 3 ) : Take about 1 mm 3 of mp-MCS and stick it on the conductive adhesive and perform platinum spraying treatment. Observe the surface morphology of each material through SEM, and use an energy spectrometer to make an elemental surface distribution map of mp-MCS to understand the distribution of Cu, Ca, P, O, and N. The results show that the large areas of each element coincide, indicating the successful assembly of the scaffold material.

[0035] (7) AFM ( Figure 4 ): The nickel mesh sample prepared in step (6) was scanned under a microscope with an amplitude of 250 mV, a rate of 1 Hz, and a spring constant of 25.89 N / cm, and the Young's modulus of the sample was measured using a TAP525A silicon cantilever beam. The results showed that copper sulfide nanoparticles were distributed inside or on the surface of collagen fibrils, and its Young's modulus was about 10 GPa, similar to that of natural bone.

[0036] Comparative example Preparation of mineralized collagen scaffold (MCS): The concentration of type I collagen solution was adjusted to 1 mg / mL using 0.1 M acetic acid solution, and a potassium ion solution (containing 200 mM potassium chloride, 30 mM disodium hydrogen phosphate, and 10 mM potassium dihydrogen phosphate) was added. Freeze-drying crosslinking and liquid-phase mineralization were carried out with reference to steps (3) and (4) in Example 1 to obtain a three-dimensional MCS material.

[0037] Example 2 Determination of biocompatibility of multiphase biomimetic mineralized collagen scaffold (1) MCS and mp-MCS containing different concentrations of copper sulfide nanoparticles (10 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL) were prepared according to the methods of the comparative example and Example 1.

[0038] (2) Bone marrow mesenchymal stem cells (BMSCs) were seeded in a 96-well plate, and 100 μL of α-MEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (100×) was added to each well. After 24 h, the materials in step (1) were placed. After continued culturing for 1 d, 3 d, and 7 d, the α-MEM medium containing CCK-8 was replaced and incubated at 37 °C for 1 h. The absorbance was detected and recorded using a microplate reader at a wavelength of 450 nm ( Figure 5 ). The results showed that mp-MCS had good biocompatibility, and the cell survival rate was greater than 90% when the concentration ≤ 60 μg / mL.

[0039] (3) BMSCs were seeded in a 6-well plate, and α-MEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (100×) was added. After 24 h, 60 μg / mL mp-MCS in step (1) was placed. After continued culturing for 24 h, the cells were stained with calcein and propidium iodide. After culturing in an incubator for 30 min, observation was carried out under a fluorescence microscope ( Figure 6 ). The results showed that the proportion of live cells in the mp-MCS group was greater than 90%, which was consistent with the results of the CCK-8 experiment. Example 3 Detection of osteogenic properties and mechanisms of multiphase biomimetic mineralized collagen scaffold

[0040] (1)Prepare MCS and 60 μg / mL mp-MCS according to the methods of the comparative example and Example 1.

[0041] (2)Inoculate BMSCs into 6-well plates, and add α-MEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (100×). After 24 h, place MCS and mp-MCS in the material groups. Set up a group without adding scaffold materials as the blank control group, and change the medium to osteogenic induction medium. After culturing for 7 d and 14 d, extract cell RNA and proteins, and detect the expression of osteogenesis-related factors by qRT-PCR and Western blot ( Figure 7 ). The results show that mp-MCS can up-regulate the expression of osteogenic genes and proteins, and its expression level is higher than that of MCS and the blank control group, proving that mp-MCS can promote the osteogenic differentiation of BMSCs and has good osteogenic performance.

[0042] (3)Inoculate BMSCs into 6-well plates, and add α-MEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (100×). After 24 h, place MCS and mp-MCS in the material groups. Set up a group without adding scaffold materials as the blank control group, and change the medium to osteogenic induction medium. After culturing for 7 d, extract cell RNA and proteins, and detect the expression of oxidative phosphorylation-related factors by qRT-PCR and Western blot ( Figure 8 ). The results show that mp-MCS can up-regulate the expression of key genes and proteins in the oxidative phosphorylation electron transport chain, and its expression level is higher than that of MCS and the blank control group, proving that mp-MCS can promote the osteogenic differentiation of BMSCs by improving oxidative phosphorylation. Example 4 Evaluation of the repair of non-infected rat cranial defects by a multiphase biomimetic mineralized collagen scaffold

[0043] (1)Prepare MCS and 60 μg / mL mp-MCS according to the methods of the comparative example and Example 1.

[0044] (2)The experiment is divided into three groups: the blank control group and the MCS and mp-MCS groups implanted with materials, with 9 rats in each group. Anesthetize the rats with 2% sodium pentobarbital, shave the hair on the top of the skull, incise the full-thickness skin along the middle of the skull, and expose the bone surface. Use a dental implant machine to remove the full-thickness bone on the top of the skull at a speed of 800 rpm / min with a ring drill with an outer diameter of 5 mm and expose the dura mater. After implanting MCS and mp-MCS in the material groups respectively, suture the wound.

[0045] (3)After constructing the model for 4 w, 8 w, and 12 w, randomly select 3 rats from each group, and use CO 2Rats were sacrificed by asphyxiation. The parietal bones of the rats were isolated and fixed with 4% paraformaldehyde. The bone tissues were scanned using Micro-CT and three-dimensional reconstruction was performed on the original files. The CT-An analysis software was used to analyze the bone defect area to evaluate the ability of mp-MCS to repair bone defects. The scanned tissues were decalcified, then dehydrated, embedded, made into wax blocks and sections. The situation of new bone was observed by HE and Masson staining ( Figure 9A - Figure 9B ).

[0046] The Micro-CT results showed that after 4 weeks of implanting the material, obvious new bone could be seen in the defect area in the mp-MCS group. According to the semi-quantitative results, the volume and percentage of new bone in the mp-MCS group were higher than those in the MCS group and the blank control group. After 8 weeks of implantation, bone regeneration was achieved in most of the defect areas in the mp-MCS group, and its osteogenic ability was better than that of MCS. Moreover, only a small amount of new bone was formed in the blank control group. When implanted for 12 weeks, a large amount of new bone could be seen in the bone defect area of the mp-MCS group, almost covering all the defect areas. According to the semi-quantitative results, although MCS had good osteogenic ability, it was still inferior to mp-MCS. At the same time, there were still a large number of defect areas not repaired in the blank control group. The HE and Masson results showed that after 4 weeks of implanting the material, obvious osteoid extracellular matrix was generated in the mp-MCS group, a large amount of scaffold material and a small amount of extracellular matrix were seen in the MCS group, while only soft tissue filling was seen in the blank control group. At 8 weeks, a small amount of new bone was formed in the mp-MCS group, the extracellular matrix increased in the MCS group, while only soft tissue was still seen in the blank control group. After 12 weeks of implantation, almost all of the bone defect area in the mp-MCS group was occupied by new bone, there was partial new bone formation and a large amount of extracellular matrix in the MCS group, and no obvious new bone was seen in the blank control group. From the above experimental results, it can be seen that mp-MCS has good osteogenic performance and can repair rat skull defects in vivo. Example 5 Detection of the mimetic enzyme activity and antibacterial properties of mp-MCS

[0047] (1) Prepare MCS and 60 μg / mL mp-MCS according to the methods described in the comparative example and Example 1.

[0048] (2) Mimetic enzyme activity: An oxidation experiment was carried out using 41.6 mM TMB (and 1040 μM hydrogen peroxide). MCS and mp-MCS were respectively put in, and a blank control was set up. According to the time dependence, the absorbance was measured at 652 nm to detect the ability to generate hydroxyl radicals (·OH) and superoxide anions (O 2 · - ). DMPO was used as ·OH and O 2 · -The spin trap was used to mix mp-MCS with 50 mM hydrogen peroxide and 100 mM DMPO solution (50 μL of methanol was added). The mixture was thoroughly mixed and the reaction was carried out for 5 min. Then the sample was placed in a sample tube and sealed, and data was collected using EPR ( Figure 10 ).

[0049] (3)Antibacterial performance: The group without material was set as the blank control group. 100 μL of Staphylococcus aureus with a concentration of 1 × 10 7 CFU / mL was co-cultured with the material for 24 h, and then the supernatant was aspirated. Subsequently, 50 μL of the finally diluted Staphylococcus aureus solution was added to the solid LB medium for culture, and the colonies were counted after 24 h. In the bacterial live / dead staining, 1 × 10 7 CFU / mL of Staphylococcus aureus was co-cultured with the material for 24 h, stained with a bacterial live / dead staining kit, and then observed under a fluorescence microscope. For the samples to be observed by SEM and TEM, they were first fixed with 2.5% glutaraldehyde, observed by SEM after gradient dehydration and drying; after being embedded in epoxy resin and sectioned and negatively stained, they were placed on a nickel grid and observed by TEM ( Figure 11 ).

[0050] The results of the TMB oxidation experiment showed that with the increase of time, the absorbance of the mp-MCS group increased significantly, while the absorbance of the MCS and blank control groups changed little, indicating that mp-MCS could generate ·OH and O 2 · - . The signals collected by EPR showed that mp-MCS could generate characteristic peaks of ·OH and O 2 · - , while there were no obvious changes in the MCS and blank control groups. These results suggested that mp-MCS had peroxidase-like and oxidase activities.

[0051] The results of the bacterial cloning experiment showed that mp-MCS could effectively inhibit the proliferation of Staphylococcus aureus. The results of the bacterial live / dead staining experiment showed that mp-MCS could not only inhibit the growth of bacteria but also kill bacteria. From the results of SEM and TEM, it was known that mp-MCS could exert mimetic enzyme activity to generate reactive oxygen species, destroy the cell membrane of bacteria and cause the contents to overflow, resulting in the death of bacteria.

[0052] Example 6 Evaluation of the repair of infectious rat skull defects with a multiphase biomimetic mineralized collagen scaffold (1)MCS and 60 μg / mL of mp-MCS were prepared according to the methods described in the comparative example and Example 1.

[0053] (2)The experiment was divided into three groups: a blank control group and the MCS and mp-MCS groups with material implantation, with 9 rats in each group. The rats were anesthetized with 2% sodium pentobarbital, the hair on the top of the skull was shaved off, and the skin was incised along the middle of the skull to expose the bone surface. A dental implant machine was used to remove the full-thickness skull bone at a rotational speed of 800 rpm with a ring drill with an outer diameter of 5 mm to expose the dura mater, and 100 μL of Staphylococcus aureus at a concentration of 1 × 10 8 CFU / mL was applied, and the wounds were sutured after implanting MCS and mp-MCS in the material groups respectively.

[0054] (3)After 4 w, 8 w, and 12 w of model construction, 3 rats were randomly selected from each group, and the rats were sacrificed by the CO 2 asphyxiation method. The skull bones of the rats were isolated and fixed with 4% paraformaldehyde, and the bone tissues were scanned with Micro-CT and three-dimensional reconstruction was performed on the original files. The CT-An analysis software was used to analyze the bone defect area to evaluate the ability of mp-MCS to repair bone defects. The scanned tissues were decalcified, then dehydrated, embedded, made into wax blocks and sections, and the situation of new bone was observed by HE and Masson staining ( Figure 12A - Figure 12B ).

[0055] The Micro-CT results showed that after 4 w of implanting the materials, new bone formation was visible at the edge of the defect area in the mp-MCS group, very little new bone was visible in the MCS group, and almost no bone formation was seen in the blank control group. After 8 w of implantation, obvious new bone was visible at the edge and in the center of the defect area in the mp-MCS group, only a small amount of discontinuous bone was seen at the edge of the defect area in the MCS group, and only extremely little bone formation was seen in the blank control group. When implanted for 12 w, a large amount of new bone was seen at the edge and in the center of the bone defect area in the mp-MCS group, partial new bone was seen at the edge of the defect area in the MCS group, and only a small amount of new bone was seen in the blank control group. The semi-quantitative results showed that the osteogenic performance of MCS was weakened in the infected environment while mp-MCS was hardly affected. The HE and Masson results showed that after 4 w of implanting the materials, obvious osteoid extracellular matrix formation was visible in the mp-MCS group, the scaffold material in the MCS group was broken and infiltrated with a large number of inflammatory cells, and only soft tissues filled the blank control group. At 8 w, a small amount of new bone was formed in the mp-MCS group, a small amount of extracellular matrix was formed in the MCS group and inflammatory cells were still visible, and only soft tissues were still seen in the blank control group. After implanting for 12 w, a large amount of new bone was formed in the bone defect of the mp-MCS group, a large amount of soft tissues were formed in the MCS group and there was scaffold material that was not completely degraded, and no obvious new bone was seen in the blank control group. From the above experimental results, it can be seen that mp-MCS can resist infection and exert osteogenic performance, and restore the infectious skull defects of rats.

[0056] From Comprehensive Examples 1-6, it can be concluded that the multiphase biomimetic mineralized collagen scaffold prepared by the present invention can be used for osteogenesis induction and antibacterial purposes. The raw materials required for the preparation method of the present invention are easily available, the preparation process is simple and there is no safety risk. The obtained multiphase biomimetic mineralized collagen scaffold has low cytotoxicity, can activate the energy metabolism of bone marrow mesenchymal stem cells to promote osteogenesis, and at the same time has mimetic enzyme activity to inhibit microbial proliferation. Therefore, it has good prospects for its application in clinical simple bone defects or infectious bone defects.

[0057] The embodiments described above are only used to describe the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a multiphase biomimetic mineralized collagen scaffold, characterized in that: Copper sulfide nanoparticles were synthesized, and type I collagen solution and solid-phase copper sulfide nanoparticles were self-assembled in a bottom-up manner. The collagen fibrils were intrafiber mineralized using liquid-phase mineralization technology to form a multiphase biomimetic mineralized collagen scaffold.

2. The method for preparing the multiphase biomimetic mineralized collagen scaffold according to claim 1, characterized in that: The specific method for synthesizing copper sulfide nanoparticles is to mix a copper-containing compound and sodium citrate, add a sodium disulfide aqueous solution dropwise under heating conditions, obtain copper sulfide nanoparticles after ultracentrifugation, and use ultrapure water to fully disperse them into a copper sulfide nanoparticle aqueous solution.

3. The method for preparing the multiphase biomimetic mineralized collagen scaffold according to claim 2, characterized in that: The copper-containing compound is copper chloride with a concentration of 10 mmol / L; the concentration of sodium citrate is 2 g / L; the heating condition is 90° C.; the concentration of the sodium disulfide aqueous solution is 1 mol / L; and the ultracentrifugation speed is 30,000 r / min.

4. The method for preparing the multiphase biomimetic mineralized collagen scaffold according to claim 1, characterized in that: The self-assembly is completed with the assistance of potassium ion buffer.

5. The method for preparing the multiphase biomimetic mineralized collagen scaffold according to claim 4, characterized in that: The potassium ion buffer contains 200 mM potassium chloride, 30 mM disodium hydrogen phosphate and 10 mM potassium dihydrogen phosphate.

6. The method for preparing the multiphase biomimetic mineralized collagen scaffold according to claim 1, characterized in that: The liquid sample after self-assembly of the type I collagen solution and the solid phase copper sulfide nanoparticles is centrifuged, freeze-dried and cross-linked to form a solid scaffold material.

7. The method for preparing the multiphase biomimetic mineralized collagen scaffold according to claim 6, characterized in that: The centrifugal speed is 5000 r / min; the cross-linking is performed by immersing the material in an 80% ethanol solution containing 1% 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide for cross-linking for 4 hours.

8. The method for preparing the multiphase biomimetic mineralized collagen scaffold according to claim 7, characterized in that: The liquid phase mineralization technology is to immerse the cross-linked material in a collagen fibril mineralization solution at 37°C and 40 r / min for 5-7 days; the collagen fibril mineralization solution contains 4.2 mM dipotassium hydrogen phosphate, 9 mM calcium chloride dihydrate and 0.2 mg / mL polyaspartic acid.

9. The multiphase biomimetic mineralized collagen scaffold prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the multiphase biomimetic mineralized collagen scaffold according to claim 9 in the preparation of a drug for repairing infected bone defects by regulating energy metabolism.

Images