Composite drug-loaded biological material as well as preparation method and application thereof

Abstract

The invention discloses a composite drug-loaded biological material as well as a preparation method and application thereof, and particularly relates to the technical field of biological materials. The invention discloses a composite drug-loaded biological material. The composite drug-loaded biological material is formed by mixing and solidifying vancomycin, mineralized collagen and bone cement. The preparation method of the composite drug-loaded biological material comprises the following steps: S1, mixing and shaping mineralized collagen and vancomycin in a beaker; s2, adding the bone cement, and uniformly mixing and stirring; and S3, the mixture is poured into a mold with the length, width and height being 12 mm, and solidification is conducted. The composite drug-loaded biological material and the preparation method thereof provided by the invention can be applied to clinical treatment of bone defect infection.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and particularly to a composite drug-loaded biomaterial, a preparation method thereof, and an application thereof. Background Art

[0002] In clinical work, the incidence of bone defects caused by trauma, limb deformity, tumors, and infectious diseases is increasing day by day. Large-area bone defects will cause great inconvenience to patients, seriously affect the postoperative recovery of patients, and endanger the physical and mental health of patients. Bone defects are extremely prone to bacterial infection. Post-fracture infection has become one of the most common forms of bone infection. Staphylococcus aureus is the most common pathogenic bacterium. The incidence of infection after internal fixation of fractures can reach 0.4% - 16.1%, and the average incidence is about 5%. If bone defects with infection are not treated in time or improperly, it will cause serious limb deformity and dysfunction. Preventing and treating post-fracture infection is extremely urgent.

[0003] At present, the treatment of bone defect infection mainly adopts repeated thorough debridement, negative pressure drainage, and antibiotic treatment. However, its treatment cycle is long, the cost is high, the recurrence rate is high, and it may cause antibiotic resistance and organ toxicity damage. The treatment is difficult and extremely intractable to handle. At present, polymethylmethacrylate (PMMA) bone cement plus antibiotics is a simple and effective technique for repairing segmental bone defects. By loading antibiotics, a scaffold material with antibacterial properties is prepared and filled into the infected bone defect site. However, some patients using PMMA bone cement plus antibiotics to treat bone defects with infection do not achieve ideal treatment effects, and sometimes the infection even worsens. The possible reasons are problems such as poor biocompatibility of the system, low antibiotic release rate, short effective antibiotic release time, and non-biodegradable characteristics.

[0004] In view of the problems of low antibiotic release rate and short effective antibiotic release time of existing PMMA-loaded antibiotics, it is proposed to use a mineralized collagen material (MC) plus bone cement (PMMA) plus vancomycin to construct a novel MC-PMMA-vancomycin composite material to solve the problems of low antibiotic release rate and short effective antibiotic release time. Summary of the Invention

[0005] In view of the above situation, in order to overcome the defects of the prior art, the present invention provides a composite drug-loaded biomaterial and a preparation method thereof, which can be applied to the clinical treatment of bone defect infection.

[0006] To achieve the above object, the technical scheme adopted by the present invention is as follows:

[0007] A composite drug-loaded biomaterial, which is formed by mixing and solidifying vancomycin, mineralized collagen, and bone cement.

[0008] Preferably, the content ratio of vancomycin, mineralized collagen and bone cement is 1:1 - 2:13.

[0009] A preparation method of a composite drug-loaded biomaterial, comprising the following steps:

[0010] S1: Mix and shape mineralized collagen and vancomycin in a beaker;

[0011] S2: Add bone cement and mix and stir evenly;

[0012] S3: Pour the mixture into a mold with a length, width and height of 12 mm and solidify.

[0013] Preferably, all instruments in the preparation process are pre-sterilized, should be dry during use, and have the same temperature as the overall operating room environment.

[0014] Application of the composite drug-loaded biomaterial as a bone tissue repair material in the treatment of bone defects.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] (1) The composite drug-loaded biomaterial has good sustained-release effect and antibacterial performance. After adding vancomycin and bone cement to the mineralized collagen material, the drug release efficiency is more excellent than that of simple bone cement plus vancomycin.

[0017] (2) The in vitro antibacterial test of the composite drug-loaded biomaterial shows that the composite material has excellent antibacterial effect on Staphylococcus aureus.

[0018] (3) Co-culture of osteoblasts, osteoclasts and mesenchymal stem cells with the composite drug-loaded biomaterial shows that the cell morphology on the surface of the composite material is complete, the growth state is good, and it has excellent bone tissue repair ability;

[0019] (4) Adding mineralized collagen material to the composite drug-loaded biomaterial can increase the porosity of the bone cement surface, improve the release efficiency and release time of vancomycin in the composite drug-loaded biomaterial, and can be applied to the clinical treatment of bone defect infections. Description of the Drawings

[0020] Figure 1 It is a scanning electron microscope experimental result diagram of the composite drug-loaded biomaterial provided by the present invention;

[0021] Figures 2-6 It is a drug sustained-release result diagram of the composite drug-loaded biomaterial;

[0022] Figure 7 : A is the experimental result of the composite drug-loaded biomaterial against Pseudomonas aeruginosa, and B is the experimental result of the composite drug-loaded biomaterial against Staphylococcus aureus;

[0023] Figure 8 : A shows the growth of mouse osteoblast cell line (MC3T3-E1) on the surface of the composite drug-loaded biomaterial; B shows the growth of rabbit primary mesenchymal stem cells (BMSC) on the surface of the composite drug-loaded biomaterial; C shows the growth of mouse RAW264.7 cells on the surface of the composite drug-loaded biomaterial. Detailed implementation manners

[0024] The present invention will be further described below in conjunction with the accompanying drawings and embodiments. The implementation manners of the present invention include but are not limited to the following embodiments.

[0025] The treatment of infectious bone defects has always been a thorny problem for orthopedic surgeons. Anti-infective tissue-engineered bone is currently the most promising transplantation material for repairing infectious bone defects. One of the very crucial technologies is the application of a local antibiotic release system. PMMA bone cement, as the most commonly used antibiotic carrier at present, has been widely used in clinical treatments at home and abroad.

[0026] Mineralized collagen material is a biomimetic composite material mainly composed of collagen and hydroxyapatite, which simulates the chemical composition and microstructure of natural bone matrix. Mineralized collagen has characteristics such as good bone induction and angiogenesis potential, biocompatibility, biodegradability, controllable shape and size, long shelf life, and low cost.

[0027] In the present invention, mineralized collagen and vancomycin are mixed and shaped in a beaker; then bone cement is added and stirred evenly; the mixture is poured into a mold with a length, width, and height of 12 mm, and solidified to obtain a composite drug-loaded biomaterial, that is, a mineralized collagen material-bone cement-vancomycin (MC-PMMA-vancomycin) biomaterial.

[0028] All instruments during the preparation process are pre-sterilized, should be dry when used, and have the same temperature as the overall operating room environment (23°C ± 1°C).

[0029] The following experiments are used to verify the antibiotic release rate and effective antibiotic release time of the composite drug-loaded biomaterial.

[0030] Samples with different mineralized collagen contents are prepared in 3 groups (Example 1, Example 2, and Example 3 respectively) using the preparation method provided by the present invention. The ratios of vancomycin, mineralized collagen material, and bone cement in each group are shown in Table 1.

[0031] Table 1: Ratio table of mineralized collagen material-bone cement-vancomycin (MC-PMMA-vancomycin).

[0032]

[0033] Scanning electron microscopy analysis and results of the composite drug-loaded biomaterial:

[0034] Scanning electron microscope (JEOL, JSM-IT700HR) and ion sputtering instrument (JEOL, Smart Coater) were used to perform scanning electron microscopy experiments on samples. First, PBS was used to clean and remove surface attachments, mucus, etc., and 3% glutaraldehyde was added for fixation. Then, ultrapure water was used to wash 3 times, each time for 10 minutes, and 1% osmium acid was used for post-fixation for 1h-2h, and then ultrapure water was used to wash 3 times, each time for 10 minutes. Alcohol was dehydrated step by step, and the concentration gradient of the dehydrating agent was 30%→50%→70%→90%→100% (100% concentration was changed 3 times), each time for 15 minutes; the sample was placed in a critical point dryer for drying, and then adhered to the sample stage with conductive glue, and placed in an ion sputtering instrument for gold spraying. Finally, the JSM-IT700HR scanning electron microscope produced by JEOL was used to collect images of the sample. Each sample was first observed at low magnification in its entirety, and then the area to be observed was selected to collect pictures.

[0035] Scanning electron microscopy experiments were performed on all four sides of the cubic samples obtained in Examples 1-3. The results showed that in the samples with added mineralized collagen material, obvious voids could be observed on the surface of the bone cement, while no voids were observed on the surface of the materials in the bone cement group (without adding mineralized collagen material). Figure 1 shown. Figure 1 A: Scanning electron microscopy experimental results of Example 1, there are many voids on the surface of the material; B: Scanning electron microscopy experimental results of Example 2, no obvious voids are found on the surface of the material; C: Scanning electron microscopy experimental results of Example 3, no obvious voids are found on the surface of the material.

[0036] The composite drug-loaded biomaterial drug sustained-release test and results:

[0037] The samples were subjected to drug sustained release test using a high performance liquid chromatography (HPLC) analysis system. First, the chromatographic conditions of vancomycin were determined; chromatographic column: Hypersil ODS2 (250 mm × 4.6 mm, 5 μm); mobile phase: acetonitrile-50 mmol potassium dihydrogen phosphate solution (phosphoric acid adjusted pH = 3.2) = 10:90 (see Figure 2) Flow rate: 1.0 mL·min-1; Column temperature: 30 °C; Detection wavelength: 236 nm; Injection volume: 20 μL; Under these chromatographic conditions, the retention time of vancomycin is around 6 - 7 min. A linear regression was performed with peak area (A) against mass concentration (C), and the standard curve equation was obtained as A = y = 16087*C + 2786.8, r = 1 (n = 6). Vancomycin showed a good linear relationship within the range of 1 - 100 μg·mL-1. Finally, a phosphate buffer solution with pH 7.4 simulating the human physiological environment was used as the release medium to detect the in vitro release of the sample. The sample was transferred to a conical flask containing 40 mL of the release medium and shaken at 37 °C and 100 r / min. 2 mL of the sample was taken at the set time points, and an equal volume of the release medium was replenished simultaneously. After the sample solution was filtered through a microporous membrane (0.45 μm), its content was detected by HPLC, the cumulative release amount of vancomycin was calculated, and the release curve was plotted.

[0038] The High Performance Liquid Chromatography (HPLC)

[0039] The analysis system was used to conduct drug sustained-release tests on the samples of Examples 1 - 3. The results showed that the group of Example 1 (GROUP1) had good release efficiency and release time. 26.3% of the drug was released on the first day, and the drug release reached 45.6% on the 14th day and tended to be stable. In Example 2 (GROUP2), 16% of the drug was released on the first day, and the drug release reached 46.5% on the 38th day. In Example 3 (GROUP3), the drug release efficiency was the worst, with 8.5% of the drug released on the first day and only reaching 19.1% on the 38th day. The results are shown in Figures 2-6 Table 2 and

[0040]

[0041]

[0042] Figures 2-6 represent the drug sustained-release results of the composite drug-loaded biomaterial, where Figure 2 : A: Standard curve of vancomycin release, Figure 3 : B: Release curves of the drug in Example 1 on the first day and the 14th day, Figure 4 : C: Release curves of the drug in Example 2 on the first day and the 14th day, Figure 5 : D: Release curves of the drug in Example 3 on the first day and the 14th day, Figure 6 : E: Comparison of the release curves of vancomycin in the samples of Examples 1 - 3.

[0043] In vitro antibacterial experiment and results of the composite drug-loaded biomaterial:

[0044] The composite drug-loaded biomaterial was subjected to in vitro antibacterial experiments against Staphylococcus aureus and Pseudomonas aeruginosa. First, 100 μL of glycerol bacteria was taken and spread on LB agar medium, and cultured at 37 °C for 24 h. A single colony was picked and inoculated into LB broth, and cultured at 37 °C with shaking at 150 rpm for 16 h for bacterial culture. Secondly, the McFarland turbidity method was used to determine the bacterial concentration, and the bacterial suspension concentration was adjusted to 1×108 CFU / mL using sterile liquid medium, and the bacterial suspension was reserved. Finally, 200 μL of 1×108 CFU / mL bacterial suspension was pipetted onto LB agar culture, and a gypsum block containing the drug at the bottom (the medicine was melted with physiological saline and applied to the bottom of the gypsum block) was placed on each plate, and the antibacterial effect was observed at 37 °C, and the antibacterial effect was detected.

[0045] The results showed that the samples of Examples 1-3 all had excellent antibacterial effects against Staphylococcus aureus, but had no antibacterial effect against Pseudomonas aeruginosa. Since vancomycin itself has no antibacterial effect against Pseudomonas aeruginosa, the antibacterial experiment of the samples against Pseudomonas aeruginosa can be used as a control group for the antibacterial experiment. The results of the antibacterial experiment are shown in Tables 3, 4 and Figure 7 . Among them Figure 7 : A. Results of the anti-Pseudomonas aeruginosa experiment of the composite drug-loaded biomaterial. The samples of Examples 1-3 all had no antibacterial effect against Pseudomonas aeruginosa. B. Results of the anti-Staphylococcus aureus experiment of the composite drug-loaded biomaterial. The samples of Examples 1-3 all had antibacterial effects against Staphylococcus aureus.

[0046] Table 3: Results of the inhibition zone of Staphylococcus aureus of the composite drug-loaded biomaterial

[0047]

[0048] Table 4: Results of the inhibition zone of Pseudomonas aeruginosa of the composite drug-loaded biomaterial

[0049]

[0050] Co-culture of the composite drug-loaded biomaterial with mouse osteoblast cell line (MC3T3-E1), mouse RAW264.7 cells and rabbit primary mesenchymal stem cells in vitro and results:

[0051] In vitro, the composite drug-loaded biomaterial was co-cultured with mouse osteoblast cell line MC3T3-E1, mouse RAW264.7 cells and rabbit primary mesenchymal stem cells. First, after thoroughly washing the culture medium with PBS, each cell line was digested moderately with trypsin and then seeded at a density of 50,000 cells per well in a 6-well culture chamber for cultivation; the culture medium was changed every 3 days. After 5 days of cultivation, the composite drug-loaded biomaterial was added to the culture medium. After continuing the cultivation for one week, the materials for cultivation were taken out, fixed in an electron microscopy fixative, and then subjected to scanning electron microscopy detection. The remaining liquid in the culture medium was subjected to CCK-8 detection. The cell supernatant was removed, and serum-containing medium containing CCK-8 reagent was added, and then incubated in an incubator at 37 °C and 5% (volume fraction) CO2 for 2-4 h, and then taken out to measure the absorbance value. Each sample was measured 3 times and the average value was taken.

[0052] In vitro, the composite drug-loaded biomaterial was co-cultured with mouse osteoblast cell line MC3T3-E1, and the biocompatibility of the material was verified at the level of osteoblast cell line MC3T3-E1 by the CCK-8 method. The results showed that the composite drug-loaded biomaterial had good biocompatibility with osteoblast cell line MC3T3-E1. The scanning electron microscopy results showed that the cell states of each group of cells grew well on the surfaces of the samples in Examples 1-3. The results are shown in Figure 8 。

[0053] Figure 8 In A: the growth of mouse osteoblast cell line (MC3T3-E1) on the surface of the composite drug-loaded biomaterial, the cell morphology was complete and the state was good; in B: the growth of rabbit primary mesenchymal stem cells (BMSC) on the surface of the composite drug-loaded biomaterial, the cell morphology was complete and the state was good; in C: the growth of mouse RAW264.7 cells on the surface of the composite drug-loaded biomaterial, the cell morphology was complete and the state was good.

[0054] In this experiment, vancomycin, bone cement (PMMA) and mineralized collagen (MC) were mixed to obtain a new composite drug-loaded biomaterial. Scanning electron microscopy analysis of the surface of the new material found that obvious voids could be observed on the surface of the bone cement in the samples added with mineralized collagen material, and no voids were seen on the surface of the pure bone cement group (without adding mineralized collagen material), which was the reason for the better drug sustained-release efficiency of the composite drug-loaded biomaterial. In addition, an in vitro antibacterial experiment was also carried out on the composite drug-loaded biomaterial, which proved that the new material had excellent antibacterial performance against Staphylococcus aureus in vitro. The co-culture experiment of the composite drug-loaded biomaterial with mouse osteoblast cell line (MC3T3-E1), mouse RAW264.7 cells and rabbit primary mesenchymal stem cells (BMSC) also demonstrated its excellent cytocompatibility.

Claims

1. A composite drug-loaded biomaterial, characterized in that, The composite drug-loaded biomaterial is formed by mixing and solidifying vancomycin, mineralized collagen, and bone cement.

2. The composite drug-loaded biomaterial according to claim 1, characterized in that, The content ratio of vancomycin, mineralized collagen, and bone cement is 1:1 - 2:

13.

3. A method for preparing a composite drug-loaded biomaterial, which is used to prepare the composite drug-loaded biomaterial according to any one of claims 1-2, and is characterized in that, It includes the following steps: S1: Mix and shape mineralized collagen and vancomycin in a beaker. S2: Add bone cement and mix well by stirring. S3: Pour the mixture into a mold with a length, width, and height of 12 mm and let it solidify.

4. The preparation method of a composite drug-loaded biomaterial according to claim 1, wherein All instruments during the preparation process are pre-sterilized, should be dry when in use, and have the same temperature as the overall operating room environment.

5. Application of the composite drug-loaded biomaterial prepared by the preparation method of the composite drug-loaded biomaterial according to any one of claims 1 - 2 or the composite drug-loaded biomaterial according to any one of claims 3 - 4 as a bone tissue repair material in the treatment of bone defects.

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