A multiphasic biomimetic mineralized collagen scaffold and methods of making and using same

Abstract

The present application relates to the technical field of biomimetic materials, in particular to a multiphase biomimetic mineralized collagen scaffold, a preparation method and application thereof.The preparation method is as follows: type I collagen solution is self-assembled with solid copper sulfide nanoparticles, and through the technology of liquid phase mineralization, intracellular mineralization of collagen fibrils occurs, forming a multiphase biomimetic mineralized collagen scaffold similar to the nanostructure and Young's modulus of natural bone.The scaffold can improve the energy metabolism of cells by regulating the oxidative phosphorylation of bone marrow mesenchymal stem cells, thereby promoting the osteogenic differentiation of the cells; in addition, the scaffold releases copper ions to simulate enzyme activity, catalyzing the generation of a large amount of active oxygen in bacteria, leading to the rupture of the bacterial membrane and the overflow of the contents, thereby achieving the effect of antibacterial.The scaffold can treat simple bone defects or infected bone defects by regulating the energy metabolism of cells and simulating enzyme activity, thereby providing more new methods for the preparation of drugs for realizing bone regeneration and anti-infection in clinic.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomimetic materials, in particular to a multiphase biomimetic mineralized collagen scaffold, a preparation method and an application in the preparation of a medicine for regulating energy metabolism and repairing an infectious bone defect. BACKGROUND

[0002] Infection of bone defects often occurs after fracture repair, bone-related surgery or trauma, which has a significant impact on the daily life and physical function recovery of patients. With the aging of the population and the frequent occurrence of sports-related injuries, the prevalence of infectious bone defects is rising. The main pathogenic bacteria include Staphylococcus aureus and Escherichia coli, which can spread through the blood or directly infect the bone, leading to complications such as osteomyelitis.

[0003] Traditional methods for treating infectious bone defects include the use of antibiotics, surgical removal of infected tissue, and the use of various bone graft materials. Although these methods can control infection and promote bone healing to some extent, they each have limitations. In particular, antibiotic resistance is becoming increasingly prominent, and the emergence of drug-resistant strains reduces the effectiveness of traditional antibiotic therapy and prolongs the treatment period. In addition, excessive use of antibiotics can lead to imbalance of microorganisms in the human body, thereby exacerbating infection. Surgical procedures, while able to remove necrotic tissue, are invasive in nature and may not completely remove the infecting bacteria, increasing the risk of recurrent infection. At the same time, traditional bone graft materials, such as autologous bone, allogeneic bone and artificial bone materials, while having some effect in promoting bone healing, have limitations such as difficulty in obtaining, donor site complications, immune rejection and lack of antibacterial properties, limiting their range of application.

[0004] Developing new bone repair materials to improve the treatment of infectious bone defects has become an important direction in bone tissue engineering research. In the field of bone tissue engineering, strategies to promote bone formation by adjusting cell energy metabolism show great potential. The energy metabolism state of cells is crucial for key biological processes such as survival, proliferation and differentiation. The energy metabolism state of bone marrow mesenchymal stem cells is directly related to their osteogenic differentiation ability. When bone marrow mesenchymal stem cells are in a high energy metabolism state, they can more effectively perform aerobic respiration, producing enough energy to support cell growth and function. In a suitable metabolic environment, these cells not only proliferate rapidly, but also synthesize bone matrix efficiently, improving the efficiency of osteogenesis. Therefore, optimizing the energy metabolism of bone marrow mesenchymal stem cells not only accelerates the osteogenic process, but also improves the quality and strength of the newly formed bone, providing a more stable foundation for the repair of bone defects. SUMMARY

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

[0006] Specifically, the application provides the following technical solutions:

[0007] A preparation method of a multi-phase biomimetic mineralized collagen scaffold: synthesizing copper sulfide nanoparticles, self-assembling type I collagen solution and solid-phase copper sulfide nanoparticles in a bottom-up manner, and combining liquid-phase mineralization technology to cause intracellular mineralization of collagen fibrils to form a multi-phase biomimetic mineralized collagen scaffold.

[0008] The specific method for synthesizing copper sulfide nanoparticles is to mix a copper-containing compound and sodium citrate, dropwise add an aqueous sodium sulfide solution under heating conditions, obtain copper sulfide nanoparticles after ultracentrifugation, and fully disperse the copper sulfide nanoparticles into an aqueous copper sulfide nanoparticle solution using ultrapure water.

[0009] The copper-containing compound is copper chloride, and the concentration is 10 mmol / L; the concentration of the sodium citrate is 2 g / L; the heating condition is 90 DEG C; the concentration of the aqueous sodium sulfide solution is 1 mol / L; and the ultracentrifugation speed is 30000 r / min.

[0010] The type I collagen solution is prepared from type I mouse tail collagen, which can be obtained from the market, and the concentration of the type I mouse tail collagen is adjusted to 1 mg / mL using 0.1M acetic acid solution. The concentration of the copper sulfide nanoparticles can be adjusted as needed.

[0011] The self-assembly is completed with the assistance of a potassium ion buffer solution containing 200 mM potassium chloride, 30 mM sodium phosphate dibasic and 10 mM potassium dihydrogen phosphate.

[0012] After the self-assembly of the type I collagen solution and the solid-phase copper sulfide nanoparticles, the solution is centrifuged, the supernatant is discarded, the precipitate is collected, the material is loaded into a mold, freeze-dried and crosslinked to form a solid scaffold material.

[0013] The centrifugation speed is 5000 r / min for 5 min, and the crosslinking is to immerse the material in an 80% ethanol solution containing 1% 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide for 4 h.

[0014] The liquid-phase mineralization technology is to immerse the crosslinked material in a collagen fibril intracellular mineralization solution at 37 DEG C and 40 r / min for 5-7 days; the collagen fibril intracellular mineralization solution contains 4.2 mM potassium phosphate dibasic, 9 mM calcium chloride dihydrate and 0.2 mg / mL polyaspartic acid.

[0015] The multiphase biomimetic mineralized collagen scaffold prepared by the application can play a simulation enzyme effect and inhibit bacterial growth; and can also be used for preparing drugs for repairing infectious bone defects by regulating energy metabolism.

[0016] Compared with the prior art, the multiphase biomimetic mineralized collagen scaffold, the preparation method and the application have at least the following beneficial effects:

[0017] The multiphase biomimetic mineralized collagen scaffold prepared by the application is self-assembled by a bottom-up method with a type I collagen solution and solid copper sulfide nanoparticles, and the collagen fibrils are mineralized in fibers by a liquid phase mineralization technology, so that the topological structure and Young's modulus of the collagen fibrils are similar to those of natural bone. Thus, the scaffold can create a more favorable osteogenic microenvironment, play a simulation enzyme activity antibacterial effect by slowly releasing copper ions, or assist mitochondria of bone marrow mesenchymal stem cells to strengthen oxidative phosphorylation to promote new bone formation, which has important significance for treating infectious bone defects or developing drugs related to infectious bone defects.

[0018] The multiphase biomimetic mineralized collagen scaffold, the preparation method and the application of the application will be further described below with reference to the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is a three-dimensional material entity diagram of the multiphase mineralized collagen scaffold (mp-MCS) prepared in Example 1.

[0020] Figure 2 is a morphology diagram of copper sulfide nanoparticles and mp-MCS in Example 1 under a transmission electron microscope (TEM). Wherein, A represents copper sulfide nanoparticles, and B represents mp-MCS. Arrow: copper sulfide nanoparticles.

[0021] Figure 3 is a scanning electron microscope (SEM) morphology diagram and an energy spectrum diagram of mp-MCS in Example 1.

[0022] Figure 4 is an atomic force microscope (AFM) diagram and a Young's modulus statistical diagram of mp-MCS in Example 1. Wherein A represents AFM, and B represents Young's modulus.

[0023] Figure 5 is cell proliferation activity of bone marrow mesenchymal stem cells after being co-cultured with different concentrations of mp-MCS in Example 2.

[0024] Figure 6 is cell live / dead staining diagram and semi-quantitative diagram of bone marrow mesenchymal stem cells after being co-cultured with 60 μg / mL of mp-MCS in Example 2.

[0025] Figure 7The expression of osteogenesis-related factors after 7 or 14 days of co-culture of mp-MCS and bone marrow mesenchymal stem cells in Example 3. A represents the protein expression of osteogenesis-related genes at 7 days, and B represents the protein expression of osteogenesis-related genes at 14 days.

[0026] Figure 8 The expression of oxidative phosphorylation-related factors after 7 days of co-culture of mp-MCS and bone marrow mesenchymal stem cells in Example 3. Wherein, A represents the relative gene expression level, and B represents the relative protein expression level.

[0027] Figures 9A-9B This is the evaluation of the effect of mp-MCS in repairing non-infectious rat skull defects in Example 4. Figure 9A Represents micro-CT images and quantitative analysis of new bone, Figure 9B Figure 5. Tissue HE and Masson staining images. ST: soft tissue, S: scaffold, ECM: extracellular matrix, NB: new bone.

[0028] Figure 10 The following are the enzyme activity profiles of mp-MCS in Example 5. A represents the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by mp-MCS after reaction with hydrogen peroxide or water. B represents the ability of mp-MCS to generate reactive oxygen species as determined by electron paramagnetic resonance (EPR).

[0029] Figure 11 This is the in vitro antibacterial test of mp-MCS in Example 5. Wherein, A represents the bacterial cloning test, and B represents the bacterial live / dead staining test, SEM and TEM detection.

[0030] Figures 12A-12B This is the evaluation of the effect of mp-MCS on repairing infected rat skull defects in Example 6. Figure 12A Represents micro-CT images and quantitative analysis of new bone, Figure 12B Figure 5. Tissue HE and Masson staining images. ST: soft tissue, S: scaffold, ECM: extracellular matrix, NB: new bone. DETAILED DESCRIPTION

[0031] Example 1 Preparation method and morphology characterization of multiphase biomimetic mineralized collagen scaffold

[0032] (1) Synthesis of copper sulfide nanoparticles: In a beaker containing 100 mL of aqueous solution, 1 mmol of CuCl2and 0.2 g of sodium citrate were dissolved. The mixture was continuously stirred for 5 min to ensure complete dissolution of sodium citrate. 1 mmol of Na2S2was dissolved in 1 mL of aqueous solution, and the resulting solution was gradually added dropwise to the above 100 mL mixed solution. The mixture was heated to 90°C and stirred for 15 min to ensure complete reaction. Subsequently, the solution was cooled in an ice water bath, and the nanoparticles were dispersed with ultrapure water after ultracentrifugation at 30000 r / min for 10 min, and the obtained sample was stored at a temperature of 4°C for a long time.

[0033] (2) Assembly of type I collagen and copper sulfide nanoparticles: The concentration of type I collagen solution was adjusted to 1 mg / mL using 0.1 M acetic acid solution, and then mixed with 0.1 mg / mL copper sulfide nanoparticles to a final concentration of 60 μg / mL. With the assistance of potassium ion solution (containing 200 mM potassium chloride, 30 mM sodium phosphate dibasic, and 10 mM potassium dihydrogen phosphate), the assembly was carried out by bottom-up self-assembly mode for 24 h.

[0034] (3) Freeze-drying crosslinking: The mixture in the dialysis bottle of step (2) was centrifuged at 5000 r / min for 5 min, the precipitate was collected and placed in a mold, and freeze-dried for 12 hours. After obtaining the three-dimensional material, it was immersed in a 1% 1-ethyl (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) solution in 80% anhydrous ethanol for 4 hours.

[0035] (4) Liquid phase mineralization: The scaffold material after crosslinking in step (3) was washed with ultrapure water, and then immersed in a mixed solution prepared using TBS, containing 4.2 mM potassium phosphate dibasic, 9 mM calcium chloride dihydrate, and 0.2 mg / mL polyaspartic acid, and mineralized at 37°C and 40 r / min for 5-7 d to obtain mp-MCS three-dimensional material. Figure 1 ).

[0036] (5) TEM ( Figure 2 ): The copper sulfide nanoparticle solution and the prepared mp-MCS sample were placed on a nickel mesh, and 3% sodium tungstate was used for negative staining for 5 min. After washing and drying, the ultra-micro morphology of copper sulfide nanoparticles and mp-MCS was observed by TEM. The results showed that the diameter of copper sulfide nanoparticles was less than 10 nm, and the lattice spacing was about 0.268 nm. After assembly with collagen fibrils, it could adhere to the surface or enter the interior of the fibrils.

[0037] (6) SEM ( Figure 3 ): About 1 mm 3The mp-MCS was bonded to the conductive adhesive and then platinum-sprayed. The surface morphology of each material was observed using a SEM, and an energy dispersive spectrometer was used to generate an elemental map of the mp-MCS to investigate the distribution of Cu, Ca, P, O, and N. The results showed a large overlap of elements, indicating successful assembly of the scaffold.

[0038] (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 an elastic modulus of 25.89 N / cm. The Young's modulus of the sample was measured using a TAP525A silicon cantilever. The results showed that copper sulfide nanoparticles were distributed inside or on the surface of the collagen fibrils, and their Young's modulus was approximately 10 GPa, similar to that of natural bone.

[0039] Comparative Example

[0040] 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 potassium ion solution (containing 200 mM potassium chloride, 30 mM disodium hydrogen phosphate, and 10 mM potassium dihydrogen phosphate) was added. Lyophilization cross-linking and liquid-phase mineralization were performed according to steps (3) and (4) in Example 1 to obtain the MCS three-dimensional material.

[0041] Example 2 Determination of biocompatibility of multiphase biomimetic mineralized collagen scaffold

[0042] (1) MCS and mp-MCS containing copper sulfide nanoparticles at different concentrations (10 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, and 80 μg / mL) were prepared according to the methods of Comparative Example and Example 1.

[0043] (2) Bone marrow mesenchymal stem cells (BMSCs) were seeded into a 96-well plate. 100 μL of α-MEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (100×) was added to each well. After 24 hours, the materials in step (1) were added. After culturing for 1 day, 3 days, and 7 days, the α-MEM medium containing CCK-8 was replaced and incubated at 37°C for 1 hour. 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 was ≤60 μg / mL.

[0044] (3) BMSCs were seeded in 6-well plates with a-MEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (100x), and 60 pg / mL of mp-MCS prepared in step (1) was added after 24 h. After 24 h of continuous culture, cells were stained with calcein and propidium iodide. After incubation in the incubator for 30 min, the cells were observed under a fluorescence microscope. Figure 6 The results showed that the proportion of living cells in the mp-MCS group was greater than 90%, which was consistent with the results of the CCK-8 experiment.

[0045] Example 3 Osteogenic performance and mechanism detection of the multiphase biomimetic mineralized collagen scaffold

[0046] (1) MCS and 60 pg / mL of mp-MCS were prepared according to the method of the comparative example and example 1.

[0047] (2) BMSCs were seeded in 6-well plates with a-MEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (100x), and MCS and mp-MCS were added to the material groups after 24 h. A group without the addition of scaffold material was set as a blank control group, and the culture medium was replaced with osteogenic induction medium. After 7 d and 14 d of culture, cell RNA and protein were extracted, and the expression of osteogenic related factors was detected by qRT-PCR and Western blot (Fig. 2). Figure 7 The results showed that mp-MCS could up-regulate the expression of osteogenic genes and proteins, and the expression level was higher than that of MCS and the blank control group, proving that mp-MCS could promote the osteogenic differentiation of BMSCs and had good osteogenic performance.

[0048] (3) BMSCs were seeded in 6-well plates with a-MEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (100x), and MCS and mp-MCS were added to the material groups after 24 h. A group without the addition of scaffold material was set as a blank control group, and the culture medium was replaced with osteogenic induction medium. After 7 d of culture, cell RNA and protein were extracted, and the expression of oxidative phosphorylation related factors was detected by qRT-PCR and Western blot (Fig. 3). Figure 8 The results showed that mp-MCS could up-regulate the expression of key genes and proteins in the oxidative phosphorylation electron transport chain, and the expression level was higher than that of MCS and the blank control group, proving that mp-MCS could promote the osteogenic differentiation of BMSCs by improving oxidative phosphorylation.

[0049] Example 4 Evaluation of the multiphase biomimetic mineralized collagen scaffold for repairing non-infected rat skull defects

[0050] (1) MCS and 60 pg / mL of mp-MCS were prepared according to the method of the comparative example and example 1.

[0051] (2) The experiment was divided into three groups: blank control group and material implanted MCS and mp-MCS groups, 9 rats in each group. The rats were anesthetized with 2% sodium pentobarbital, the scalp hair was shaved, the skin was cut along the middle of the skull, and the bone surface was exposed. A drill with an outer diameter of 5 mm was used at a speed of 800 rpm / min to remove the full layer of bone on the skull and expose the dura mater. The materials were implanted into the MCS and mp-MCS groups, respectively, and the wound was sutured.

[0052] (3) After the model was constructed for 4 w, 8 w and 12 w, 3 rats were randomly taken from each group and sacrificed using CO2 asphyxiation. The rat skull bone was isolated and fixed with 4% paraformaldehyde. The bone tissue was scanned using Micro-CT, and the original file was reconstructed in three dimensions. The CT-An analysis software was used to analyze the defect bone region and evaluate the ability of mp-MCS to repair bone defects. The tissues after scanning were decalcified, then dehydrated, embedded, wax blocks were made and sections were cut, and the new bone was observed by HE and Masson staining. Figures 9A-9B

[0053] Micro-CT results showed that 4 w after implantation of materials, obvious new bone was observed in the defect area of 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. 8 w after implantation, most of the defect area in the mp-MCS group was regenerated, and its osteogenesis ability was better than that of MCS. The blank control group only had a small amount of new bone formation. 12 w after implantation, a large amount of new bone was observed in the bone defect area of the mp-MCS group, which almost covered all the defect area. According to the semi-quantitative results, although MCS had good osteogenesis ability, it was still inferior to mp-MCS, and the blank control group still had a large number of defect areas that were not repaired. HE and Masson results showed that 4 w after implantation of materials, obvious bone-like extracellular matrix was observed in the mp-MCS group, a large amount of scaffold material and a small amount of extracellular matrix were observed in the MCS group, and only soft tissue was observed in the blank control group. 8 w, a small amount of new bone was formed in the mp-MCS group, the extracellular matrix increased in the MCS group, and the blank control group still had only soft tissue. 12 w after implantation, the bone defect area of the mp-MCS group was almost entirely occupied by new bone, and a large amount of extracellular matrix was observed in the MCS group, and no obvious new bone was observed in the blank control group. Through the above experimental results, it can be known that mp-MCS has good osteogenesis performance and can restore the rat skull defect in vivo.

[0054] Example 5 Detection of simulated enzyme activity and antibacterial performance of mp-MCS

[0055] ​(1) MCS and mp-MCS were prepared according to the method described in Comparative Example and Example 1, and 60 μg / mL of mp-MCS.

[0056] (2) Mimic enzyme activity: Oxidation experiments were performed using 41.6 mM TMB (and 1040 μM hydrogen peroxide), and MCS and mp-MCS were placed in, respectively, and a blank control was set up. According to the time dependence, the absorbance was measured at 652 nm to detect the ability to produce hydroxyl radicals (·OH) and superoxide anion (O2· - ). DMPO was used as a spin trapping agent for ·OH and O2· - , and mp-MCS was mixed with 50 mM hydrogen peroxide and 100 mM DMPO solution (50 μL of methanol was added). The mixture was mixed well, and the reaction was performed for 5 min. Then the sample was placed in a sample tube and sealed, and data was collected using EPR. Figure 10

[0057] (3) Antimicrobial performance: The no-material group was set as a 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 final dilution of Staphylococcus aureus solution was added to 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, and was stained by the bacterial live / dead staining kit, and then observed under a fluorescence microscope. The samples that needed to be observed by SEM and TEM were first fixed with 2.5% glutaraldehyde, dried by gradient dehydration, and observed by SEM; after embedding in epoxy resin and negative staining, they were placed on a nickel mesh and observed by TEM. Figure 11

[0058] The results of the TMB oxidation experiment showed that the absorbance of the mp-MCS group increased significantly with time, while the absorbance of the MCS and blank control groups changed little, indicating that mp-MCS could produce ·OH and O2· - . The signals collected by EPR showed that mp-MCS could produce characteristic peaks of ·OH and O2· - , while the MCS and blank control groups showed no obvious change. These results suggest that mp-MCS has peroxidase-like and oxidase activity.

[0059] ​​The results of bacterial cloning experiments showed that mp-MCS could effectively inhibit the proliferation of S. aureus. The results of bacterial live / dead staining experiments showed that mp-MCS could not only inhibit bacterial growth but also kill bacteria. The results of SEM and TEM showed that mp-MCS could exert simulated enzyme activity to produce reactive oxygen species, destroy the cell membrane of bacteria and make the contents overflow, leading to the death of bacteria.

[0060] Example 6 Evaluation of mp-MCS for Repairing Infected Skull Defects in Rats

[0061] (1) MCS and mp-MCS at 60 μg / mL were prepared according to the method described in Comparative Example and Example 1.

[0062] (2) The experiment was divided into three groups: a blank control group and material-implanted MCS and mp-MCS groups, each group having 9 rats. The rats were anesthetized with 2% sodium pentobarbital, the scalp hair was shaved, the skin was incised along the middle of the skull to expose the bone surface. A trephine drill with an outer diameter of 5 mm was used at a speed of 800 rpm / min to remove the full-thickness bone on the skull and expose the dura mater, and 100 μL of S. aureus at 1 × 10 8 CFU / mL was applied, and the MCS and mp-MCS groups were implanted with the materials, respectively, and then the wound was sutured.

[0063] (3) After the model was constructed for 4 w, 8 w and 12 w, 3 rats were randomly taken from each group and sacrificed using CO2 asphyxiation. The rat skull was isolated and fixed with 4% paraformaldehyde, the bone tissue was scanned using Micro-CT, and the original file was three-dimensionally reconstructed. The CT-An analysis software was used to analyze the area of the defective bone, and the ability of mp-MCS to repair the bone defect was evaluated. The tissue after scanning was decalcified, then dehydrated, embedded, wax blocks were made and sections were cut, and the condition of the new bone was observed by HE and Masson staining Figures 12A-12B ).

[0064] Micro-CT results showed that after 4 weeks of implantation, new bone formation was observed at the edge of the defect area in the mp-MCS group, a small amount of new bone was observed in the MCS group, and almost no bone formation was observed in the blank control group. After 8 weeks of implantation, obvious new bone formation was observed at the edge and center of the defect area in the mp-MCS group, a small amount of discontinuous bone was observed at the edge of the defect area in the MCS group, and only a small amount of bone formation was observed in the blank control group. After 12 weeks of implantation, a large amount of new bone was observed at the edge and center of the defect area in the mp-MCS group, partial new bone was observed at the edge of the defect area in the MCS group, and only a small amount of new bone was observed in the blank control group. The semi-quantitative results showed that the osteogenic performance of MCS was weakened in the infected environment, while the mp-MCS was almost not affected. HE and Masson results showed that after 4 weeks of implantation, obvious bone-like extracellular matrix formation was observed 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 tissue filling was observed in the blank control group. At 8 weeks, a small amount of new bone formation was observed in the mp-MCS group, a small amount of extracellular matrix formation was observed in the MCS group, and inflammatory cells were still observed, while the blank control group still had only soft tissue. After 12 weeks of implantation, a large amount of new bone was observed in the bone defect of the mp-MCS group, a large amount of soft tissue was formed in the MCS group, and the scaffold material was not completely degraded, and no obvious new bone was observed in the blank control group. Through the above experimental results, it can be known that the mp-MCS can resist infection and exhibit osteogenic performance, and restore the infected skull defects of rats.

[0065] In summary, examples 1-6 can be obtained that the multiphase biomimetic mineralized collagen scaffold prepared by the present application can be used for inducing osteogenesis and antibiosis. The raw materials required by the preparation method of the present application are easy to obtain, the preparation process is simple and safe. 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 has enzyme activity simulation to inhibit microbial proliferation. Therefore, the multiphase biomimetic mineralized collagen scaffold has good prospects for application in clinical simple bone defects or infected bone defects.

[0066] The above-described examples only describe the preferred embodiments of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A method for preparing a multiphasic biomimetic mineralized collagen scaffold, characterized in that: The synthetic copper sulfide nanoparticles are self-assembled with a type I collagen solution and solid-phase copper sulfide nanoparticles in a bottom-up manner, and a liquid-phase mineralization technology is combined to cause intracellular mineralization of collagen fibrils to form a multiphase biomimetic mineralized collagen scaffold; the self-assembly is completed with the assistance of a potassium ion buffer solution containing 200 mM potassium chloride, 30 mM sodium phosphate dibasic and 10 mM potassium dihydrogen phosphate; The specific method for synthesizing the copper sulfide nanoparticles is to mix a copper-containing compound and sodium citrate, drop by drop add an aqueous sodium sulfide solution under heating conditions, obtain copper sulfide nanoparticles after ultracentrifugation, and disperse the copper sulfide nanoparticles into an aqueous solution of copper sulfide nanoparticles with ultrapure water; 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 DEG C; the concentration of the aqueous sodium sulfide solution is 1 mol / L; and the ultracentrifugation speed is 30000 r / min; The liquid sample after self-assembly of the type I collagen solution and the solid-phase copper sulfide nanoparticles is subjected to centrifugation, freeze-drying and cross-linking to form a solid scaffold material; The liquid-phase mineralization technology is to immerse the cross-linked material in a collagen intracellular mineralization solution at 37 DEG C and 40 r / min for 5-7 days; the collagen intracellular mineralization solution contains 4.2 mM potassium phosphate dibasic, 9 mM calcium chloride dihydrate and 0.2 mg / mL polyaspartic acid.

2. The method of claim 1, wherein: The centrifugation speed is 5000 r / min; and the cross-linking is to immerse the material in an 80% ethanol solution containing 1% 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide for cross-linking for 4 h.

3. The multiphase biomimetic mineralized collagen scaffold prepared by the preparation method of claim 1 or 2.

4. The use of the multiphase biomimetic mineralized collagen scaffold of claim 3 in the preparation of a drug for repairing infectious bone defects by regulating energy metabolism.

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