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Back to Journal »International Journal of Nanomedicine» Volume 16
Gold nanoparticles and PLGA/L-lysine-g-graphene oxide composite scaffolds inspired by mussels are used for bone defect repair
Authors: Fu C, Jiang Y, Yang X, Wang Y, Ji Wei, Jia Geng
Published on September 30, 2021, the 2021 volume: 16 pages 6693-6718
Single anonymous peer review
Editor approved for publication: Professor Dongwoo Khang
Fu Chuan,1 Jiang Yikun,1 Yang Xiaoyu,1 Wang Yu,2 Wei Ji,3 Jia Guoliang1 Department of Orthopedics, Second Hospital of Jilin University, Changchun, 130041; 2Key Laboratory of Polymer Ecological Materials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022 ; 3 College of Life Science and Technology, Changchun University of Science and Technology, Changchun 130022, Address: Jia Guoliang, Department of Orthopedics, Second Hospital of Jilin University, Changchun 130041, Tel 86 157 54306089 Email [email protected] Purpose: Insufficient biological activity severely restricts the application of biodegradable bone implants And development. The functional modification of bone implants is essential to improve osseointegration and bone regeneration. Methods: In this study, L-lysine functionalized graphene oxide (Lys-g-GO) nanoparticles and polydopamine-assisted gold nanoparticles (AuNPs-PDA) coating were used to improve the biological functions of PLGA scaffold materials. The effects of Lys-g-GO nanoparticles and AuNPs-PDA functional coating on the physical and chemical properties of PLGA stents were detected by scanning electron microscope (SEM), contact angle measurement and mechanical testing equipment. In vitro, the effects of the composite scaffold on the proliferation, adhesion and osteogenic differentiation of MC3T3-E1 cells were studied. Finally, the radial defect model was used to evaluate the effect of composite scaffolds on bone defect healing. Results: The prepared [email protection]/Lys-g-GO composite scaffold exhibited excellent mechanical strength, hydrophilicity and antibacterial properties. In vitro, this composite scaffold can significantly improve osteoblast adhesion, proliferation, osteogenic differentiation, calcium deposition and other cell behaviors. In vivo, this composite scaffold can significantly promote the formation of new bone and the deposition of collagen at the defect site of the radius, and exhibits good biocompatibility. Conclusion: The combination of bioactive nanoparticles and surface coatings shows great potential for enhancing osseointegration of bone implants. Keywords: gold nanoparticles, graphene oxide, bone defect, poly(dopamine), PLGA, L-lysine
With the development of tissue engineering technology, people are paying more and more attention to the use of biodegradable polymer scaffolds for bone defect treatment. At present, a large number of natural materials and synthetic polymers have been prepared as bone implants and used in the treatment of bone defects, such as gelatin, chitosan, polypeptides, polylactic acid and PLGA. 1-5 Among many degradable polymer materials, PLGA is the most widely used because of its good biocompatibility, degradability and adjustable mechanical properties. 6 Many studies have used PLGA to prepare bone tissue engineering scaffolds and applied them to treat bone defects. 7,8 However, PLGA materials have some disadvantages, such as hydrophobicity9,10 Therefore, many strategies have been used to improve the bone repair ability of PLGA through functional modification. 11 A common modification is to mix some biologically active nanoparticles with PLGA to prepare composite materials, such as hydroxyapatite, graphene oxide, and carbon nanotubes. 12-14 These nanoparticles can significantly improve the mechanical strength of the scaffold, regulate cell function, and promote the growth of bone cells and the fusion of the material with the surrounding bone tissue. This strategy can effectively improve the bone repair ability of PLGA as a solution to overcome the defects of a single material.
In recent years, graphene oxide (GO) has been widely used in functional modification of polymer materials due to its excellent physical and chemical properties. The special surface structure and abundant functional groups provide GO with many excellent properties, such as good hydrophilicity, antibacterial properties, dispersibility and mechanical strength. 15-18 In addition, many studies have shown that GO has a strong osteoinductive ability. 19,20 Some studies have found that GO can promote osteogenic differentiation by increasing the tension of the cytoskeleton. 21 In addition, GO's strong protein adsorption capacity can attract nutrients needed for cell growth to the surface of the material, creating a better living environment. 22 More importantly, the abundant oxygen contains active groups such as carboxyl, hydroxyl and epoxy groups, which is beneficial to modify the surface of GO with biologically active factors to improve biological properties. Many studies have used various active substances such as amino acids, growth factors, and drugs to further modify the surface of GO to improve the bone repair ability of composite materials. 23-25 Although the addition of bioactive nanoparticles can effectively enhance the repair ability of bone polymer materials, this method still has some shortcomings, such as uneven distribution of nanoparticles on the surface. In order to maximize the bone repair ability of bone implants, many studies have used other modification methods combined with bioactive nanoparticles to enhance the biological properties of bone implants. For example, Gao et al. used both hydroxyapatite nanoparticles and gelatin coating to improve the biological activity of PLGA materials. The results showed that the combination of these two modification methods can effectively enhance cell proliferation and osteogenic differentiation. 6 Yang et al. modified PLGA to prepare nanofibers simultaneously through cellulose and polydopamine coatings. The results show that the surface of composite nanofibers has good applicability for cell adhesion, proliferation and growth. 26 In addition, the introduction of cellulose can effectively accelerate the deposition rate of polydopamine coatings. These studies show that different modification methods can simultaneously improve the function of scaffold materials and achieve complementary advantages. This multi-component composite material has broad application prospects in the field of bone repair. In addition to mixing with bioactive nanoparticles, surface modification is another classic strategy to improve the performance of PLGA materials. Transplanting or coating the surface of the stent with bioactive substances such as gelatin, metal nanoparticles, and hydroxyapatite can effectively regulate cell functions. 27-29 Many studies combine different material modification methods to maximize the bone repair ability of scaffold materials.
Gold nanoparticles (AuNPs) have attracted considerable attention in the field of bone repair due to their superior properties in biomedical applications, such as good biocompatibility, osteoinduction and easy functionalization. 30-32 Recently, some studies have found that AuNPs can effectively promote the differentiation and mineralization of osteoblasts, ALP activity, and the expression of osteoblast-related genes (Runx-2, OPN, OCN) after the contact between osteoblasts and AuNPs. 33-35 Other studies have shown that AuNPs can induce cell osteogenesis by regulating (MPAK)-p38 and ERK/MAPK pathways. 36,37 A recent clinical trial showed that surface modification of titanium implants with AuNPs can promote bone interface formation and bone regeneration. 38 In addition, AuNPs can effectively regulate bone formation. The behavior of osteoblasts and changes the way in which osteoclasts are formed. Therefore, these characteristics make AuNPs very suitable for the functional modification of bone repair materials. In the application process of gold nanoparticles, their concentration, properties, morphology and synthesis method have a very important influence on their biological applications. 39,40 However, the traditional mixing method of AuNPs has some limitations in enhancing material properties. Effective mixing requires high concentrations of gold nanoparticles, which can cause biological toxicity and reduce the biological activity of bone repair materials. Therefore, many studies tend to use coating technology to introduce AuNPs into bone repair materials, which can not only maintain effective contact between cells and AuNPs, but also avoid toxicity to a large extent. At present, the methods of applying AuNPs coating on the surface of materials mainly include sputtering coating and physical/chemical vapor deposition; however, these methods usually require complex equipment. In recent years, polydopamine reduction has become a popular method for preparing metal nanoparticle coatings. Dopamine can oxidize and self-polymerize in oxygen and alkaline environments, forming a polydopamine (PDA) coating on the surface of most solid materials. 41 The adhesion and reducibility of polydopamine can reduce gold ions to AuNP and promote their adhesion to the surface of the material. In addition, the PDA coating can increase the wettability of the material surface, accelerate the formation of hydroxyapatite, and further improve the bone repair ability of the material. Therefore, the combination of polydopamine-assisted AuNPs functional coating and bioactive nanoparticles is an excellent choice to improve the performance of bone implants, and it is worthy of further study.
In this study, we modified GO with L-lysine (Lys) by chemical grafting, and mixed Lys-g-GO with PLGA to prepare a composite porous scaffold (PLGA/Lys-g-GO). Then, we applied the AuNPs-PDA functional coating to the surface of the stent using polydopamine as a reducing agent. The physical and chemical properties of the stent were characterized by scanning electron microscopy, contact angle measurement, mechanical test and antibacterial experiment. We also studied the effects of [email protection]/Lys-g-GO composite scaffold on cell proliferation, adhesion, osteogenic differentiation and new bone regeneration. This study aims to improve the biological activity of PLGA through the above-mentioned material modification methods, and to prepare composite materials with excellent bone repair capabilities for bone defect repair.
The detailed schematic diagram of scaffold preparation is shown in Figure 1. Figure 1 A brief schematic diagram of the preparation of the [email protection]/Lys-g-GO composite scaffold.
Figure 1 A brief schematic diagram of the preparation of the [email protection]/Lys-g-GO composite scaffold.
PLGA (molecular weight=100,000, LA/GA=75/25) was purchased from Changchun Zhonghe Biomaterials Co., Ltd. GO was purchased from China Chengdu Organic Chemical Co., Ltd. (thickness: 0.55-1.2nm, diameter: 0.5-3μm) (MFG code 021519). L-Lysine was purchased from China Beijing Chemical Reagent Co., Ltd. (Cat. No. 62016734). HAuCl4·3H2O (CAS number 16961-25-4), cetylpyridinium chloride (CPC) (CAS number 6004-24-6) and dopamine hydrochloride (CAS number 62-31-7) were purchased from China Latin Reagent Co., Ltd. N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) (CAS No. 25952-53-8), N-methylpyrrolidone (NMP ) (CAS No. 872-50-4), 3-(4,5-dimethyl)-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) (CAS No. 298-93-1), 4,6-Diamino-2-phenylindole (DAPI) (CAS No. 28718-90-3) and Rhodamine-Phalloidin (Product No. P1951) were purchased from Sigma- Aldrich (United States). Cell test reagents were purchased from Sigma-Aldrich (USA). Hematoxylin and Eosin (H&E) (product code G1120), Masson's tricolor stain (product code G1340) and Sirius Red kit (product code S8060) were purchased from China Solar Biotechnology Co., Ltd.
L-lysine was grafted onto the surface of GO by chemical grafting. In short, first disperse 100 mg GO in 50 mL deionized water, and then continuously sonicate for 2 hours. Subsequently, 0.25 g of EDC was added to the above solution to activate the carboxyl group of GO. Then, 0.3 g of L-lysine powder was added to the solution system and stirred at room temperature for 24 h. After the reaction is completed, the mixed solution is centrifuged in a centrifuge (8000 rpm) (eppendorf, Centrifuge 5427 R, GER), and the resulting precipitate is repeatedly washed with deionized water. Finally, Lys-g-GO is obtained by vacuum drying. Fourier transform infrared spectroscopy (FTIR, Perkin Elmer, 1730, USA) was used to identify the crosslinks between L-lysine and GO. The surface structure of Lys-g-GO was observed by transmission electron microscope (TEM, FEI, Tecnai G2 F20 S-TWIN, USA).
In order to compare the effects of GO and Lys-g-GO on the physical and chemical properties of PLGA materials, we added GO and Lys-g-GO to PLGA, and made the composite material into a thin film (the ratio of PLGA to Lys-g-GO) nano The particles are 99:1). Then, the surface hydrophilicity of the composite sample was studied by a water contact angle tester (Kruss, DSA 10, GER). Analyze the protein adsorption characteristics of PLGA, PLGA/GO and PLGA/Lys-g-GO by using BSA as a model protein for bicinchoninic acid (BCA) protein determination (Abcam, ab207003). In short, add the sample to 4mL BSA solution (2mg/mL), and take a liquid sample to analyze the BSA concentration of the solution after 1 hour. The amount of BSA loaded on the sample is determined by the decrease in BSA concentration.
The PLGA/Lys-g-GO composite scaffold was prepared by a preparation process combining phase inversion and particle leaching. In short, dissolve 2 g of PLGA in 10 mL of NMP, and then add 20 mg of Lys-g-GO. After the above mixture is fully stirred, add foaming agent (sodium chloride particles, 220~450 μm), pour it into a cylindrical mold and freeze. After the mixture is completely frozen, the cylindrical mold is immersed in deionized water for 3 days, and the deionized water is replaced every 8 hours. After the NMP solvent and sodium chloride particles in the mixed solution are completely replaced by water, the solidified stent material is freeze-dried and stored in a drying box.
In this study, the surface of the scaffold material was functionally treated with polydopamine, and the gold nanoparticles were reduced and loaded on the surface of the scaffold through the reducibility of polydopamine. 42,43 The operation process is as follows. First, immerse the PLGA/Lys-g-GO composite scaffold in a dopamine solution (2 mg/mL, pH 8.5). After 24 hours, wash the PDA-modified stent ([email protection]/Lys-g-GO) with deionized water to remove unattached dopamine molecules. Then, the stent was immersed in a gold ion solution (2.0 mM) overnight at room temperature. Through the reducibility of PDA, AuNPs coating was constructed on the surface of the stent. Finally, rinse the [Email Protection]/Lys-g-GO holder with distilled water to remove the unconnected AuNP. Four sets of scaffold materials were prepared at the same time as the control group, including pure PLGA scaffold, PLGA/Lys-g-GO scaffold, PDA modified PLGA scaffold ([email protected]) and PDA assisted AuNPs coated PLGA scaffold ([email protection ]).
Cut off the composite stent to expose the middle section. Then, a scanning electron microscope (SEM, Zeiss, EVO 25, GRE) was used to observe the microstructure of the composite scaffold. The composite stent sample was prepared into a cylinder with a diameter of 8 mm and a height of 25 mm, and the mechanical properties of the composite stent were tested with a multifunctional mechanical tester (Instron, Instron 1121, UK). The water contact angle tester (Kruss DSA 10, GER) detects the hydrophilicity of the sample. X-ray photoelectron spectroscopy (XPS, Kratos, JPN) and X-ray diffraction (XRD, Bruker. D8 ADVANCE, GER) are used to detect the element composition and crystal structure of the composite scaffold.
Staphylococcus aureus (S. aureus, ATCC25923) and E. coli (ATCC25922) were used as strains to evaluate the antibacterial properties of the composite scaffold. All strains were provided by the Experimental Center of the Second Hospital of Jilin University. In short, the composite stent is first immersed in a 75% ethanol solution and sterilized with ultraviolet light. Then rinse the sterilized stent repeatedly with PBS to remove residual ethanol. Adjust the cell concentration to 4×104 CFU/mL, and incubate in a constant temperature incubator at 37°C for 2 h. Then, the bacteria are co-cultured with the sterilized composite scaffold. After 12 h, transfer 150 µL of the bacterial solution to a 96-well plate, and measure the absorbance at 600 nm with a multi-function microplate scanner (TECAN, Infinite M200, Swit). In order to further observe the antibacterial properties of the composite scaffold, 60 μL of bacterial solution was absorbed and diluted. Then, spread 30 µL of the diluted bacterial solution evenly on the LB agar plate and incubate overnight at 37°C. Finally, take pictures of the colonies in the LB agar plate. Previous studies have proved that PLGA materials have no obvious antibacterial properties, so PLGA samples were selected as the control group.
Mouse pre-osteoblastic MC3T3-E1 cells (Shanghai Institute of Cell Biology, China) were used as model cells to evaluate the biological activity of the composite scaffold. The cells were cultured in high-sugar DMEM medium containing 10% fetal bovine serum, 63mg/L penicillin and 100mg/L streptomycin at 37°C and 5% CO2, and the medium was changed every 2 days. For cell proliferation inspection, the composite scaffold is immersed in a 75% ethanol solution and sterilized by ultraviolet light. Then, the completely sterilized composite scaffold was repeatedly washed with PBS and soaked in the cell culture medium. Finally, the cells were seeded on the composite scaffold of a 24-well plate at a density of 2.5×104/well. On days 1, 3, and 7, MTT assay was used to determine cell proliferation on different composite scaffolds. At each time point, 80 µL of MTT solution (5 g L-1 in PBS) was added to the culture well and incubated for 4 h. After incubation, remove the medium and add 600 μL of acidified isopropanol to dissolve the converted dye. Finally, transfer 150 μL of the dissolving solution to a new 96-well plate, and measure the absorbance at 540 nm with a multi-function microplate scanner (TECAN, Infinite M200, Swit).
To evaluate cell adhesion, adherents cultured on various films (PLGA, [Email Protection], [Email Protection], PLGA/Lys-g-GO and [Email Protection]/Lys-g-GO) The cells were fixed with 4% paraformaldehyde and cultured for 3 days. Subsequently, the cells were washed with PBS to remove paraformaldehyde, and the cytoskeleton and nucleus were stained with phalloidin (red) and DIPI (blue), respectively. Finally, observe the adherent cells on the surface of different materials under a fluorescence microscope (Nikon, E80i, Japan).
Alizarin red (ARS) staining was used to detect calcium deposits in cells growing on composite scaffolds. In short, the medium was removed, and the cells were fixed with paraformaldehyde after 21 days of culture. After cell fixation, the cell/scaffold sample was washed with PBS, 40 mM ARS (Solarbio, G8550) was added and incubated at room temperature for 30 minutes. Then, the cell/scaffold samples were washed 3 times with distilled water, and the ARS stained samples were treated with 10% (w/v) CPC for 15 minutes at room temperature. Finally, transfer 150 µL of the solution to 96 wells, and measure the absorbance at 540 nm for calcium quantitative analysis. In order to further visually observe the influence of composite scaffolds on cell mineralization, PLGA, [email protected], [email protected]@PLGA, PLGA/Lys-g-GO and [email protected]/Lys-g-GO films were prepared and The treatment is carried out under similar conditions as the composite scaffold. After the cells grew on the surface of the film for 21 days, the cells were stained with ARS and observed under a microscope (Nikon, E80i, Japan).
After the cells were cultured on different composite scaffolds for 7 days, real-time quantitative PCR (RT-PCR) was used to detect the expression of genes related to bone formation in the cells. According to the manufacturer's protocol, total RNA was extracted using TRIzol reagent (Invitrogen, USA). Then, cDNA was synthesized using PrimeScriptTM RT kit (Takara Bio, Japan) according to the manufacturer's instructions. RT-PCR was performed by Mx3005 (Stratagene, USA) and osteogenic related genes, including glyceraldehyde 3-phosphate dehydrogenase (GAPDH), collagen type 1 (COL-I), runt-related transcription factor 2 (Runx2) and bone Pontin (OPN) was evaluated. The primer sequence is shown in Table 1. All experiments were performed in triplicate to obtain average data. Table 1 List of nucleotide sequences of genes and primers
Table 1 List of nucleotide sequences of genes and primers
In order to further test the effects of different composite scaffolds on the osteogenic differentiation of cells, a thin film was prepared and processed under similar conditions as the composite scaffold, and the cells were seeded on the surface of the thin film. After the cells were cultured for 7 days, the cells were fixed with paraformaldehyde and washed with PBS. Then, the cells were perforated with 0.2% (v/v) Triton X-100 (CAS No. 9002-93-1), and then incubated with 1% BSA solution. Add COL-I (Abcam, ab34710), Runx2 (Abcam, ab114133) and OPN (Abcam, ab63856) primary antibodies to the cells and incubate overnight at 4°C. After the primary antibody is incubated, FITC-labeled secondary antibody is added and incubated at room temperature for 60 minutes. The cells were then stained with DAPI for 1 minute, and the cells were observed under a fluorescence microscope (Nikon, E80i, Japan).
Male New Zealand white rabbits (2.5 kg) were randomly divided into five groups (PLGA, [email protection], [email protection], PLGA/Lys-g-GO and [email protection]/Lys-g-GO, each group Four animals)) and used for radial defect implantation of stents. All experimental animals were provided by Liaoning Changsheng Biotechnology Company and were raised in the animal room of the School of Public Health, Jilin University. In short, after the experimental animals were completely anesthetized, the rabbit forelimb hair was removed, and the skin was disinfected with iodine. Then, make a 3cm incision in the middle radius of the experimental animal, and use a chain saw to remove a radius of about 20mm. Then, trim the stent material and implant the defect. Finally, the epidermal wound was sutured and bandaged regularly. Within 7 days after the operation, penicillin was intramuscularly injected into all experimental animals at a daily dose of 400,000 units. The animals' defecation and wound infection were observed every day.
At 0, 4, and 12 weeks after surgery, the Digital Radio chart (Kodak, CR-400 plus, USA) was used to examine the effects of different composite scaffolds on bone regeneration. According to the X-ray results, the new bone regeneration was evaluated by Lane-Sandhu X-ray score (Table 2). At the same time, 12 weeks after the end of the experiment, the experimental animals were sacrificed and the radius tissue was removed. After removing the soft tissue of the periosteum, the radius tissue was fixed with paraformaldehyde, and the bone tissue was reconstructed in 3D with a CT scanner (General Electric, Imatron C300, USA). Table 2 Lane-Sandhu radiological image scoring system
Table 2 Lane-Sandhu radiography scoring system
At the 12th week after surgery, a sample of bone tissue was taken out and fixed with paraformaldehyde. Then, the bone sample was completely decalcified in 15% EDTA solution. After decalcification, the samples were embedded in paraffin and cut into tissue sections. Subsequently, the tissue sections were stained with Masson Trichrome, H&E and Sirius Red according to the standard protocol, and observed using an optical microscope (Nikon, E80i, Japan) and a polarizing microscope (Nikon, Eclipse Ci-L, Japan). The ratio of type I and type III collagen was quantitatively analyzed by Image-pro Plus 6.0 software (Media Cybernetics, USA).
The experimental animals were sacrificed at the 12th week after the operation, and a healthy New Zealand white rabbit without surgical treatment was used as a control group. Important internal organs, including the heart, liver, spleen and kidneys, were taken out of all experimental animals. Then, a series of sections were cut from the organ tissue and stained with H&E. Finally, observe the physiological structure of important organs of experimental animals under a microscope (Nikon, E80i, Japan) to evaluate the biological safety of the composite scaffold.
All quantitative data were analyzed by one-way analysis of variance using Origin 8.0 software (Origin Lab Corporation, USA). Data are expressed as mean ± SD. In all analyses, a P value of less than 0.05 was considered statistically significant.
In this study, the surface of GO was modified with L-lysine by chemical grafting. We first studied the chemical composition of Lys-g-GO nanoparticles by Fourier Infrared Spectroscopy (FITR). As shown in Figure 2A, the FITR spectrum of GO shows three characteristic peaks at 1052 cm-1, 3417 cm-1, and 1730 cm-1, representing the CO bond, the “OH stretch” band of the hydroxyl group, and the carboxylic acid. "C=O stretch" belt (black arrow). The results show that there are abundant oxygen-containing groups on the surface of GO. After grafting L-lysine to the surface of GO, the C=O bond representing the amide and carboxylic acid groups changed significantly in the range of 1620 ~ 1630 cm-1 (red arrow). In addition, the stretching peaks representing OH and NH also changed significantly at 3420 cm-1 (blue arrow). The results showed that L-lysine was successfully grafted onto the surface of GO. The TEM observation of the surface morphology of GO and Lys-g-GO showed that GO was flake and wrinkled, similar to peeling wrinkled flakes, as shown in Figure 2B. The surface of Lys-g-GO has many substances with similar crystal structure, which is rougher than the surface of GO. We speculate that the surface-attached amino acids may cause the formation of this morphology. The above FITR and TEM results proved that L-lysine was successfully grafted onto the surface of GO. Figure 2 (A) FITR spectra and (B) TEM images of GO and Lys-g-GO nanoparticles, all scale bars are 1μm. (C) Contact angle analysis of PLGA, PLGA/GO and PLGA/Lys-g-GO composite materials. (D) BSA adsorption efficiency of PLGA, PLGA/GO and PLGA/Lys-g-GO composite materials, *P <0.05, n=3.
Figure 2 (A) FITR spectra and (B) TEM images of GO and Lys-g-GO nanoparticles, all scale bars are 1μm. (C) Contact angle analysis of PLGA, PLGA/GO and PLGA/Lys-g-GO composite materials. (D) BSA adsorption efficiency of PLGA, PLGA/GO and PLGA/Lys-g-GO composite materials, *P <0.05, n=3.
First, we prepared PLGA/Lys-g-GO and PLGA/GO films by blending, and studied the effects of Lys-g-GO and GO on the physical and chemical properties of PLGA. As shown in Figure 2C, the water contact angle of PLGA is 95.5±2.61°, and the water contact angle of the composite material is significantly reduced to 85.8±3.17° after adding GO. There are abundant oxygen-containing functional groups such as hydroxyl, carboxyl and epoxy on the surface of GO, which gives GO good hydrophilicity. Compared with PLGA/GO, the surface hydrophilicity of PLGA/Lys-g-GO composite material is further improved, and the contact angle is reduced to 79.8±5.5°. The highly hydrophilic lysine can provide the material with hydrophilic groups such as amino and carboxyl groups. Grafting lysine on the surface of GO can increase the number of hydrophilic groups in the material to a certain extent. Figure 2D shows the protein adsorption capacity of each group within one hour. The protein adsorption capacity test using BSA as a model protein showed that the addition of Lys-g-GO to PLGA enhanced the protein adsorption capacity of the composite, which was significantly higher than that of the PLGA/GO group (P<0.05). We speculate that lysine increases The positive charge on the GO surface in the physiological environment enhances the electrostatic interaction between the material and the negatively charged BSA. The material's excellent protein absorption can quickly form a protein film on the surface, provide more binding sites, and improve cell adhesion and migration.
In order to further study the effect of GO and Lys-g-GO on the biological activity of PLGA materials, we planted MC3T3-E1 cells on the surface of different materials, and detected cell proliferation, adhesion and osteogenic differentiation by MTT and FITC staining methods, and PNPP determination, respectively. The results show that, compared with GO, Lys-g-GO has no significant effect on cell proliferation, but can significantly promote cell adhesion. Cell image analysis (Figure S1) showed that the cell growth status and area percentage of the PLGA/Lys-g-GO group were better than those of the PLGA/GO group after 3 days of growth (P <0.05). In addition, among these two kinds of nanoparticles, Lys-g-GO significantly improved the osteoinductive ability of PLGA. The ALP level of the PLGA/Lys-g-GO group was significantly higher than that of the PLGA group, especially on day 7 (P <0.05) (Figure S2). Many studies have found that lysine can regulate the expression of integrin genes in cells and promote the formation of extracellular matrix. 44 At the same time, hydroxylysine is the main component of collagen, which can promote the synthesis and mineralization of collagen in the extracellular matrix. Therefore, compared with unmodified GO, Lys-g-GO can further improve the hydrophilicity, protein absorption and osteogenic induction of PLGA.
Considering the excellent biological activity of Lys-g-GO, we prepared PLGA/Lys-g-GO composite scaffolds by particle leaching/phase change method using PLGA and Lys-g-GO as matrix materials. In addition, we applied the AuNPs-PDA functional coating to the surface of the stent by reducing the gold ions in the solution to AuNPs with polydopamine. As shown in Figure 3, all stents present a very typical through-hole structure, and the channels are connected to each other and run through the entire stent. In addition, the macroporous surface of the scaffold has a microchannel structure, ranging in size from nanometers to more than ten microns, which can effectively promote nutrient exchange inside the scaffold and has advantages over flat-pored surfaces in regulating cell functions. The pure PLGA stent is pure white. After adding Lys-g-GO, the overall appearance of the stent did not change significantly, but the color darkened slightly to light gray. After surface modification with polydopamine, the scaffold was further darkened to black, and the entire scaffold appeared dark brown after loading AuNPs. The surface of PLGA holes is relatively flat and has a dispersed microchannel structure. The addition of Lys-g-GO significantly increases the size and number of surface microchannels, which may be related to the layered PLGA material caused by the addition of nanoparticles, thereby forming more microchannel structures. [email protected] It has a similar morphology and structure to a single PLGA stent, but the surface of the stent is rougher and the diameter of the microchannel is reduced, which may be due to the polydopamine coating. After loading AuNPs, some agglomerated gold nanoparticles were found on the surface of the scaffold. Figure 3 (A) Macro view of PLGA (a), [Email Protection] (b), [Email Protection] (c), PLGA/Lys-g-GO (d) and [Email Protection] (A) And (B and C) SEM images are protected]/Lys-g-GO (e) bracket. The strip lengths are 400 μm (B) and 40 μm (C).
Figure 3 (A) Macro view of PLGA (a), [Email Protection] (b), [Email Protection] (c), PLGA/Lys-g-GO (d) and [Email Protection] (A) And (B and C) SEM images are protected]/Lys-g-GO (e) bracket. The strip lengths are 400 μm (B) and 40 μm (C).
We further analyzed the surface chemical composition of different scaffolds to confirm the functional modification. Figures 4A and B show the XPS high-resolution scanning spectra of the stent surface. The elements on the surface of different stents have obvious peak intensity changes. The prominent peaks on the surface of the PLGA scaffold are C1S and O1S. After surface modification of the PLGA scaffold with polydopamine, a prominent N1S peak appeared at 400 eV, which was caused by the introduction of surface amines. The element composition of the PLGA/Lys-g-GO scaffold is the same as that of the PLGA scaffold, and there is no obvious N1S peak, which may be related to the low content of Lys-g-GO. The Au1S peak was detected after loading AuNPs on the stent. The high-resolution spectra of Au confirmed the existence of zero-valent gold, indicating that AuNP had been deposited on the surface of the stent. We detected the crystal structure of different scaffold samples by XRD. Figure 4C shows the XRD patterns of different scaffold samples. We found peaks in the range of 13-27 at 2θ positions of different samples, which are characteristic peaks of PLGA. After modifying the scaffold with polydopamine, the XRD pattern did not change significantly, indicating that polydopamine had no significant effect on the crystal structure of the scaffold. After adding Lys-g-GO to the stent, the XRD spectrum of the PLGA/Lys-g-GO sample shows a characteristic peak at position 11 (black arrow), and the corresponding interlayer spacing is about 0.79 nm, which is typical for GO The characteristic peak indicates the presence of Lys-g-GO in the scaffold. After AuNPs were loaded on the stent, the characteristic peaks of gold appeared at 38, 44, 65, and 78 (yellow arrows). XPS and XRD results confirmed that the surface modification of the stent was completed. Figure 4 (A) XPS, (C) XRD pattern and (B) PLGA (a), [email protection] (b), [email protection] (c), PLGA/Lys-Detailed diagram of Au 4f count g-GO (d) and [email protection]/Lys-g-GO (e) brackets.
Figure 4 (A) XPS, (C) XRD pattern and (B) PLGA (a), [email protection] (b), [email protection] (c), PLGA/Lys-Detailed diagram of Au 4f count g-GO (d) and [email protection]/Lys-g-GO (e) brackets.
Surface hydrophilicity will significantly affect the biocompatibility of bone implant materials. The hydrophilic surface can effectively promote cell infiltration and adhesion. In this study, we evaluated the wettability of the scaffold material by measuring the contact angle of water droplets on the surface of different materials. As shown in Figure 5A and B, after adding Lys-g-GO nanoparticles, the contact angle of PLGA was significantly reduced. After surface modification with polydopamine, the contact angle of the stent was further reduced to 29.1±6.27°, which may be due to the abundant hydrophilic catechol and nitrogen-containing groups in polydopamine. After the stent was loaded with AuNPs, we found that the contact angle of the stent surface increased to a certain level (43.3±5.06°), which may be due to the hydrophobic AuNPs affecting the binding of elements on the stent surface with water to some extent. Studies have shown that when the material contact angle is between 0° and 40°, the cell adhesion ability is significantly improved. 45 [email protected]/Lys-g-GO The surface contact angle of the scaffold is 38.7±7.85°, which may create more suitable environments for cell growth. Figure 5 (A and B) Contact angle analysis, (C) compression strength and (D) PLGA (a), [email protection] (b), [email protection] (c), PLGA/Lys-g- Elastic modulus GO (d) and [email protection]/Lys-g-GO (e) bracket. * P<0.05, the error bar represents the standard deviation of n=3.
Figure 5 (A and B) Contact angle analysis, (C) compression strength and (D) PLGA (a), [email protection] (b), [email protection] (c), PLGA/Lys-g- Elastic modulus GO (d) and [email protection]/Lys-g-GO (e) bracket. * P<0.05, the error bar represents the standard deviation of n=3.
The stent can not only fill the bone defect, but also provide mechanical support during the treatment of the bone defect. Therefore, the mechanical strength of the stent is very important for bone repair, especially when applied to weight-bearing parts. We analyzed the compressive strength and elastic modulus of different stent materials through a mechanical tester. As shown in Figure 5C and D, the compressive strength and elastic modulus of the pure PLGA stent are 1.68±0.22MPa and 3.06±1.02MPa, respectively. After adding Lys-g-GO, the compressive strength and elastic modulus of the stent increased to 2.79±0.15MPa and 7.44±2.94Mpa, respectively. The results show that Lys-g-GO nanoparticles can effectively improve the mechanical properties of the scaffold. GO can interact with polymers through π-π interactions, van der Waals forces and hydrogen bonds, stabilize the material structure, improve interface interactions, and effectively enhance the mechanical strength of the material. 46 The compressive strength of [email protected], [email protection] and [email protection]/Lys-g-GO bracket are 1.93±0.22, 1.8±0.16 and 2.68±0.21 MPa, respectively. The elastic modulus of [email protected], [email protected] and [email protected]/Lys-g-GO brackets are 3.1±1.49, 3.9±2.25 and 7.2±1.47MPa, respectively. The compressive strength and elastic modulus of the stents modified with polydopamine or loaded with AuNPs did not change significantly (P> 0.05). The results show that the AuNPs-PDA functional coating has no negative effect on the intrinsic mechanical strength of the stent, and the compressive strength of the stent is similar to that of human cancellous bone, which can be used as an ideal bone substitute material.
The ideal bone implant material should also have a certain antibacterial ability, so as to reduce the use of antibiotics, prevent bacterial infections, and improve the success rate of surgery. Recently, many studies have found that GO and AuNPs can improve the antibacterial properties of implant materials to a certain extent. 47,48 Therefore, in this study, Staphylococcus aureus and Escherichia coli were used as experimental strains to test the effect of AuNPs-the effect of PDA functional coating and Lys-g-GO nanoparticles on the antibacterial properties of PLGA materials. In our previous research, PLGA material was proved to have no obvious antibacterial activity, therefore, PLGA was used as an experimental control group. 49 As shown in Figure 6A, the [email protection] group showed an OD value similar to that of the PLGA group, indicating that the PDA coating has no significant effect on the antibacterial properties of the material. After AuNPs were loaded on the surface of the PLGA scaffold, the OD value of the bacterial solution decreased to a certain extent, but the downward trend was not obvious. There was a significant difference only when the scaffold was co-cultured with E. coli (P <0.05). Among all stents, the PLGA/Lys-g-GO and [email protected]/Lys-g-GO groups showed the highest bacterial inhibition, which was significantly different from the PLGA group (P <0.05), indicating Lys-g-GO can enhance the antibacterial activity of PLGA materials. In order to observe the antibacterial properties of different scaffold materials more intuitively, bacteria were separated from the scaffold samples and inoculated on LB agar plates, and the antibacterial effects of the scaffold materials were observed according to the colony formation. As shown in Figure 6B, a large number of colonies were found in the PLGA and [email protection] groups, covering almost the entire LB culture plate. The [email protected], PLGA/Lys-g-GO and [email protected]/Lys-g-GO groups performed much better in reducing the number of bacteria than the PLGA and [email protected] groups. Bacterial inhibition was most obvious in the PLGA/Lys-g-GO and [Email Protection]/Lys-g-GO groups. Figure 6 (A) Comparison of antibacterial efficiency of different composite scaffolds, (B) Photographs of colonies formed by different composite scaffolds treated E. coli and Staphylococcus aureus. PLGA (a), [email protection] (b), [email protection] (c), PLGA/Lys-g-GO (d) and [email protection]/Lys-g-GO (e). * P<0.05, the error bar represents the standard deviation of n=3.
Figure 6 (A) Comparison of antibacterial efficiency of different composite scaffolds, (B) Photographs of colonies formed by different composite scaffolds treated E. coli and Staphylococcus aureus. PLGA (a), [email protection] (b), [email protection] (c), PLGA/Lys-g-GO (d) and [email protection]/Lys-g-GO (e). * P<0.05, the error bar represents the standard deviation of n=3.
According to the above experimental results, both AuNPs-PDA functionalized coating and Lys-g-GO can improve the antibacterial activity of PLGA stents to varying degrees. However, compared with Lys-g-GO, AuNPs-PDA coating has less antibacterial effect. Some studies have found that free AuNPs can enter the bacteria through the cell membrane and destroy the internal structure of bacteria by inducing oxidative stress. 50 However, the antibacterial activity of AuNPs is directly related to the concentration of AuNPs. Some studies have found that AuNPs concentrations higher than 3.125 and 6.25μg/mL have an inhibitory effect on bacteria. 51 Therefore, we speculate that the content of AuNPs loaded on the surface of the PLGA stent through the PDA coating is low and cannot achieve an effective antibacterial concentration. Compared with the AuNPs-PDA functional coating, Lys-g-GO has a more significant improvement in the antibacterial properties of the stent material. The antibacterial mechanism of GO materials is mainly divided into two ways: mechanical damage and induction of oxidative stress. First, GO can use its sharp edge structure to cut through the bacterial cell wall. This mechanical action will destroy the structural integrity of the bacteria. 52 However, this mechanical action mainly occurs in the GO suspension state. When GO is uniformly distributed in the polymer material, this mechanical damage effect is significantly reduced. Another reason why GO has excellent antibacterial properties is that GO can induce oxidative stress in bacteria, thereby increasing the pressure on the bacterial cell membrane and destroying the bacterial structure. 53 We speculate that this property of GO is the main reason for the improvement of bacteria. Antibacterial properties of the stent material. In addition, lysine molecules can increase the number of positive charges of GO, so the Lys-g-GO position on the surface of the scaffold can easily trap negatively charged bacteria, which further enhances the antibacterial properties of the scaffold material. More importantly, we found that AuNPs-PDA coating and Lys-g-GO have a synergistic effect on improving the antibacterial properties of the stent material. In general, the [email protected]/Lys-g-GO composite stent has good antibacterial properties, which can prevent the occurrence of infections and significantly increase the success rate of implant surgery.
Changes in the surface properties of the scaffold can interact with cells and affect cell adhesion, proliferation, differentiation, and other behaviors. The combination of AuNPs-PDA coating and Lys-g-GO can significantly change the surface morphology of the PLGA stent and improve its hydrophilicity, mechanical strength and antibacterial properties. We planted MC3T3-E1 cells on the surface of different scaffolds and tested cell proliferation to confirm whether these changes in material properties have a positive effect on bone regeneration. Figure 7A shows the proliferation of cells on the surface of different scaffolds. After culturing on different scaffolds for 1 day, there was no obvious change in cell proliferation in each group. On day 3, the cell proliferation rate of the [email protected] group was significantly higher than that of the PLGA and PLGA/Lys-g-GO groups (P <0.05), indicating that polydopamine modification can effectively increase the cell proliferation rate. However, after loading AuNPs on the surface of the scaffold, the cell proliferation rate decreased slightly (P> 0.05). After 7 days of cell culture, the cell proliferation of each group was similar to that on day 3. However, the cell proliferation of the [email protected]/Lys-g-GO group increased to a certain extent, and the cell proliferation rate was similar to that of the [email protected] group, which was significant It was higher than that in the PLGA/Lys-g-GO group (P <0.05). Figure 7 (A) Cell proliferation and cell proliferation of MC3T3-E1 cells cultured on PLGA (a), [email protection] (b), [email protection] (c), PLGA/Lys-g-GO (d) (B) Shape and [email protection]/Lys-g-GO (e) bracket. The length of the scale bar is 200 μm, *P<0.05, and the error bar represents the standard deviation of n=3.
Figure 7 (A) Cell proliferation and cell proliferation of MC3T3-E1 cells cultured on PLGA (a), [email protection] (b), [email protection] (c), PLGA/Lys-g-GO (d) (B) Shape and [email protection]/Lys-g-GO (e) bracket. The length of the scale bar is 200 μm, *P<0.05, and the error bar represents the standard deviation of n=3.
In each group, we made a patch of scaffold material and planted MC3T3-E1 cells to further observe the effect of scaffold material on cell adhesion. After the cells were cultured for 3 days, we observed the adhesion and spread of the cells on the surface of the material. As shown in Figure 7B, although the cells grow well on the surface of the pure PLGA material, the number of cells in a single field of view is obviously low, and most of the cells are not fully expanded. After modifying the surface of the material with polydopamine, compared with the PLGA and PLGA/Lys-g-GO groups, the adherent cells showed a greater number, larger adhesion area and better spreading state. In the [email protected], [email protected] and [email protected]/Lys-g-GO groups, the cells were almost completely covered by the material and stacked on top of each other, and the interaction between the cells was more prominent, indicating strong cell adhesion behavior.
The above results indicate that polydopamine can improve cell adhesion and proliferation, which may be related to the increase in hydrophilicity and the abundant catechol-based adhesion groups in polydopamine. 54,55 However, we found that cell proliferation decreased slightly after loading AuNPs. Scaffolding surface. Loading AuNPs is a hydrophobic metal substance, which can reduce the hydrophilicity of the surface of the material and the exposure of the catechol-based adhesive groups on the surface of the scaffold to a certain extent, which may affect the early adhesion of cells within a specific range. Echoes the proliferation. In addition, we found that the cell proliferation of the [email protected]/Lys-g-GO group was slightly lower than that of the [email protected] group in the early stage of proliferation, but was significantly improved in the late stage of proliferation. According to the experimental results, Lys-g-GO can also effectively enhance cell proliferation and adhesion behavior, which may have a special synergistic effect with polydopamine coating, which overcomes the adverse effects of AuNPs to a certain extent. The experimental results of cell proliferation and adhesion show that the PLGA/Lys-g-GO scaffold modified with AuNPs-PDA functionalized coating can significantly enhance cell adhesion and proliferation.
Cell mineralization is a marker of mature bone differentiation, which is widely used to determine the degree of bone cell differentiation and the secretion of mineralized extracellular matrix. We evaluated the effects of different scaffold materials on the mineralization of MC3T3-E1 cells through ARS staining and calcium quantification. As shown in Figure 8A, the PLGA group showed the lowest OD value among all groups, indicating that the cellular calcium deposition in the PLGA scaffold was the lowest. After modification with polydopamine, the OD value of the [email protected] group was significantly higher than that of the PLGA group (P<0.05), indicating an effective increase in cellular calcium deposition. After loading AuNPs on the surface of the scaffold, the cell calcium deposition content further increased, and the OD value was close to the PLGA/Lys-g-GO group, and was significantly higher than the [email protected] group (P<0.05). Due to the limitation of the three-dimensional shape of the scaffold, we cannot directly observe cell mineralization. Therefore, we made different scaffold materials into film sheets and implanted cells. After 21 days of culture, ARS staining was observed. As shown in Figure 8B, the PLGA group had lighter staining and fewer calcium nodules, while the [email protected] group staining intensity and red calcium nodule density increased significantly, indicating that polydopamine can promote calcium deposition in the extracellular matrix. Polydopamine can adsorb Ca and P ions on the surface of the material through its adhesion ability, thereby accelerating the rate of cell mineralization. 56 After loading AuNPs, the number of mineralized nodules further increased. Among the sample groups, the [email protected]/Lys-g-GO group showed the largest staining area, and the red mineralized nodules almost covered the entire field of view, indicating that the AuNPs-PDA functionalized coating and Lys-g-GO showed Synergistic effect in improving cellular calcium deposition. Figure 8 (A) Corresponding quantitative evaluation of calcium content and mineral deposits in different composite stents. (B) Alizarin red staining of MC3T3-E1 cells cultured on different membranes on the 21st day. PLGA (a), [email protection] (b), [email protection] (c), PLGA/Lys-g-GO (d) and [email protection]/Lys-g-GO (e). The length of the scale bar is 200 μm, P<0.05, and the error bar represents the standard deviation of n=3.
Figure 8 (A) Corresponding quantitative evaluation of calcium content and mineral deposits in different composite stents. (B) Alizarin red staining of MC3T3-E1 cells cultured on different membranes on the 21st day. PLGA (a), [email protection] (b), [email protection] (c), PLGA/Lys-g-GO (d) and [email protection]/Lys-g-GO (e). The length of the scale bar is 200 μm, P<0.05, and the error bar represents the standard deviation of n=3.
Bone regeneration involves processes such as cell adhesion, proliferation, osteogenic differentiation and calcium deposition, and these processes are regulated by a variety of genes. For example, the osteogenic transcription factor (Runx-2) is a typical gene expressed in the early stage of osteogenic differentiation. 57 Osteopontin (OPN) is an important component of the extracellular matrix of bone, which can be expressed in large quantities in the middle and late stages of cell osteogenesis. 27,58 Collagen type I (COL-I) is expressed in the early stage of extracellular matrix secretion, and its expression level gradually increases with cell differentiation and maturation. 59 Therefore, in this study, Runx-2, OPN and selected COL-I genes were used to detect the expression of bone-related genes in cells by RT-PCR. As shown in Figure 9A, after 7 days of culture of different scaffold cells, compared with the PLGA group, the expression level of COL-I in each group increased to a certain extent. The expression of COL-I in the [email protected]/Lys-g-GO group was significantly higher than that in the PLGA group (P <0.05). There was no significant difference in Runx-2 expression between the PLGA and [email protection] groups, indicating that polydopamine had no significant effect on the induction of early cell osteogenic differentiation. Compared with the [email protected] and PLGA groups, the expression level of Runx-2 in the [email protected] group was significantly increased (P <0.05). After modification with AuNPs-PDA functionalized coating and Lys-g-GO, the level of Runx-2 of the cells was further increased. Comparing the PLGA/Lys-g-GO group, we found that the [email protected] group had higher Runx-2 levels, indicating that AuNPs is slightly better than Lys-g-GO in regulating early osteoblast differentiation. Regarding the expression of OPN, we found that the expression of OPN in the [email protected] group was significantly higher than that in the PLGA group (P<0.05). After using AuNPs-PDA coating and Lys-g-GO to modify the scaffold, the expression of OPN in the cells further increased, and the expression level was significantly higher than that in the [email protected] and PLGA groups (P <0.05). Figure 9 (A) Quantitative real-time PCR analysis and (B) COL-I, Runx-2 and OPN expression of MC3T3-E1 cells seeded on PLGA (a), [email protection] (b), [email] Immunofluorescence image protection] (c), PLGA/Lys-g-GO (d) and [email protection]/Lys-g-GO (e) composite scaffolds. DAPI staining of the nucleus (blue) and FITC-conjugated secondary antibody of the protein (green). *P<0.05, the error bar represents the standard deviation of n=3, and the length of the scale bar is 200 μm.
Figure 9 (A) Quantitative real-time PCR analysis and (B) COL-I, Runx-2 and OPN expression of MC3T3-E1 cells seeded on PLGA (a), [email protection] (b), [email] Immunofluorescence image protection] (c), PLGA/Lys-g-GO (d) and [email protection]/Lys-g-GO (e) composite scaffolds. DAPI staining of the nucleus (blue) and FITC-conjugated secondary antibody of the protein (green). *P<0.05, the error bar represents the standard deviation of n=3, and the length of the scale bar is 200 μm.
We use protein immunofluorescence staining to detect the protein expression of osteogenic factors (COL-I, OPN and Runx2) to further examine the osteogenic differentiation ability of cells on the surface of different scaffolds. As shown in Figure 9B, the COL-I expression in the PLGA group was the lowest, while the COL-I protein expression in the other groups was higher. After loading AuNPs, more Runx2 expression was observed in the fluorescence image of the sample, which was significantly higher than that of other groups. In terms of OPN protein expression, we found that the protein expression of the [email protected] group was higher than that of the PLGA group. After loading AuNPs or adding Lys-g-GO, the OPN expression in the sample was further improved. In particular, a large area of OPN protein expression appeared in the [email protected]/Lys-g-GO group. The expression analysis of proteins and genes related to osteogenic differentiation shows that the two are highly consistent, indicating that [email protected]/Lys-g-GO scaffold can effectively promote osteogenic differentiation of cells.
The above experimental results show that both AuNPs-PDA coating and Lys-g-GO can effectively improve the osteoinductive ability of the scaffold and promote the expression of genes and proteins related to osteogenic differentiation. Many studies have shown that GO has a strong osteoinductive ability, and the co-culture of GO and cells can effectively improve the osteogenic differentiation ability of cells. Our research once again proves this point. 60,61 AuNPs can induce cellular stress response to enter cells, activate the p38 MAPK signaling pathway, and induce the up-regulation of genes related to osteoblast differentiation. 62 In addition, polydopamine improves the hydrophilicity and adhesion of the stent, promotes the deposition of calcium and phosphorus ions, and enhances the osteoinduction of the stent.
We established a rabbit radius defect model and used different scaffolds to repair the defect model to further evaluate the bone repair ability of different scaffold materials. As shown in Figure 10A, we created a 20 mm defect in the radius of the rabbit and repaired the defect with different scaffold materials. We first observed the healing of the radius by X-rays in different experimental animals. As shown in Figure 10B, at 0 weeks after surgery, all the experimental animals had a neat and clear defect with approximately the same length in the radius; the PLGA group observed less new bone formation in the 4th week, and failed to complete the defect connection in the subsequent 12 weeks. And repair. The results show that the bone repair ability of the PLGA scaffold is limited. At 4 weeks, the PLGA/Lys-g-GO group and [email protected]/Lys-g-GO group showed more new bone tissue at the defect site. Compared with the PLGA group, the defect site had initially connected. Although a large amount of new bone tissue was found in the defect site of the [email protected] group, the amount of bone tissue was slightly less than that of the PLGA/Lys-g-GO group and the [email protected]/Lys-g-GO group. At 12 weeks, the defect sites in the [email protected] group, PLGA/Lys-g-GO group, and [email protected]/Lys-g-GO group were filled with new bone, the bone edges were convergent, and the morphology was close to normal bone tissue. The defect in the [email protected] group was not completely filled, but the new bone mass was slightly higher than that in the PLGA group. Then, we performed Lane-Sandhu X-ray scoring for bone regeneration in different groups. Figure 10D shows that the [email protected]/Lys-g-GO group has the highest repair effect score, while the PLGA group has the lowest repair effect score. The repair effect is scored as follows: [email protected]/Lys-g-GO (7.25±2.21)>PLGA/Lys-g-GO (6.75±2)>[email protected] (6±1.41)>[email protected] (3.25) ±1.25)>PLGA (2.75±1.5). We collected radius samples from experimental animals at 12 weeks postoperatively and performed three-dimensional reconstruction with a CT scanner, as shown in Figure 10C. [email protected]/Lys-g-GO, PLGA/Lys-g-GO, [email protected] groups have more significant repair effects, and the radius shape is close to normal bone tissue. Bone defects can be seen in the radial center of the PLGA group and the [email protection] group. Figure 10 (A) Animal experiment process diagram, (B) X-ray, (C) CT reconstructed image and (D) Lane-Sandhu radiographic score, PLGA (a), [email protected] (b), [email protected] (c), PLGA/Lys-g-GO (d) and [email protection]/Lys-g-GO (e) stent treatment group. *P<0.05, the error bar represents the standard deviation of n=4.
Figure 10 (A) Animal experiment process diagram, (B) X-ray, (C) CT reconstructed image and (D) Lane-Sandhu radiographic score, PLGA (a), [email protected] (b), [email protected] (c), PLGA/Lys-g-GO (d) and [email protection]/Lys-g-GO (e) stent treatment group. *P<0.05, the error bar represents the standard deviation of n=4.
We analyzed the bone formation and collagen deposition at the defect site by HE, Masson, and Sirius red staining. As shown in Figure 11A, the scaffolds of each experimental group were broken into pieces and scattered in the tissue. After 12 weeks, the formation of new bone was poor in the PLGA group and [email protected] group. The tissues at the defect site were mainly hyperplastic connective tissue, and only a small amount of bone tissue was observed. In contrast, the restoration effect of [email protected]/Lys-g-GO group, PLGA/Lys-g-GO group and [email protected] group was significantly better than the other two groups. There are more new bones in the bone defect, and more laminar bones, indicating that the implant restoration effect is excellent. Figure 11B shows the results of Masson staining. A small amount of blue collagen fibers can be seen in the PLGA group. In contrast, we found more blue collagen fibers in the [email protection] group, indicating that surface modification with polydopamine can promote collagen secretion in bone defects. More mature collagen fibers appeared in the PLGA/Ly-g-GO and [email protection] groups, accompanied by osteoid matrix. In addition, we found that the [email protected]/Lys-g-GO group contained the largest areas of collagen fibers, with blue collagen fibers covering most of the defective tissue. Figure 11 Histological analysis using H&E (A), Mason's Tricolor (B), Sirius Red (C) staining, and COL-I/COL-III area ratio (D). PLGA (a), [email protection] (b), [email protection] (c), PLGA/Lys-g-GO (d) and [email protection]/Lys-g-GO (e). Magnification, 200 times.
Figure 11 Histological analysis using H&E (A), Mason's Tricolor (B), Sirius Red (C) staining, and COL-I/COL-III area ratio (D). PLGA (a), [email protection] (b), [email protection] (c), PLGA/Lys-g-GO (d) and [email protection]/Lys-g-GO (e). Magnification, 200 times.
Among the organic components of normal bone tissue, the content of COL-I is more than 90%. COL-I can provide binding sites for bone tissue biomineralization, promote cell differentiation, and determine the performance of new bone to a large extent. Therefore, COL-I can assess the maturity of new bones to a certain extent. In this study, Sirius Red staining was used to evaluate the collagen composition in the sample tissue. As shown in Figure 11C, the amount of COL-I in each group was higher than that of COL-III, indicating that the defect has been repaired through the initial stage of fracture. However, the red/yellow area is larger in the [email protected]/Lys-g-GO group and the PLGA/Lys-g-GO group, the red/yellow area is smaller, and the green area in the PLGA group is larger, [email protection ] Group. As shown in Figure 11D, the COL-I/COL-III ratios of PLGA, [email protected], [email protected], PLGA/Lys-g-GO and [email protected]/Lys-g-GO groups are approximately 4.5±1.54, 3.95±1.11, 5.27±1.33, 6.74±1.73 and 9.53±1.23. Analysis of the ratio of COL-I and COL-III shows that the ratio of COL-I/COL-III in the [email protection]/Lys-g-GO group is significantly higher than that in the PLGA and [email protection] groups (P < 0.05), indicating that the collagen content of the new tissue is close to that of the normal bone tissue. Histological examination results show that the [email protected]/Lys-g-GO scaffold implant can achieve a good repair effect on bone defects, and the new tissue composition is close to normal bone tissue.
Imaging and histological experiments confirmed that Lys-g-GO and AuNPs-PDA functional coatings can significantly improve the bone repair ability of PLGA scaffolds. However, this nanomaterial can enter other parts of the body through the circulation of body fluids. Therefore, the biological safety of scaffold materials also needs to be studied. The rabbit heart, liver, spleen and kidney were observed histopathologically at 12 weeks after operation. As shown in Figure 12, the HE results of the heart tissue slices in the control group showed that the arrangement of myocardial fibers was normal, the myocardial interstitium had no obvious changes, and the myocardial cells had no changes such as granular degeneration and steatosis. The results of liver HE stained sections showed that the liver lobules were structurally complete, the liver cells were arranged in an orderly manner, and there was no degeneration or cirrhosis of the liver cells. The HE results of splenic tissue sections showed that the red and white pulp morphology was normal, and there was no significant increase in sinus endothelial cells and multinucleated white blood cells. HE results showed that the size and number of glomeruli were normal, and there was no obvious inflammatory cell infiltration in the renal interstitium. Compared with the viscera of the normal rabbits in the control group, there were no abnormal cells or structural changes in the viscera of the experimental groups, indicating that the scaffold materials in each group did not cause obvious damage to the internal organs. The above experimental results show that the [email protected]/Lys-g-GO composite porous scaffold prepared by us has excellent biocompatibility and bone repair ability. Figure 12 H&E stained sections of important organs (heart, liver, spleen, kidney) of experimental animals at 12 weeks postoperatively. PLGA (A), P[email protected] (B), [email protected] (C), PLGA/Lys-g-GO (D) and [email protected]/Lys-g-GO (E). The length of the scale bar is 200 μm.
Figure 12 H&E stained sections of important organs (heart, liver, spleen, kidney) of experimental animals at 12 weeks postoperatively. PLGA (A), P[email protected] (B), [email protected] (C), PLGA/Lys-g-GO (D) and [email protected]/Lys-g-GO (E). The length of the scale bar is 200 μm.
The treatment of large bone defects has always been the focus of orthopedic research. Although treatments such as autologous bone transplantation are effective for bone regeneration, many shortcomings limit its development, such as immune response, limited donor sources, and pathogen transfer. With the continuous development of tissue engineering technology, more and more researchers have begun to pay attention to the use of synthetic degradable polymers to solve the disadvantages of traditional bone defect treatment and promote the repair of bone defects. The ideal bone repair material should have good hydrophilicity, mechanical strength, antibacterial properties and osteoinductive ability, provide a suitable environment for cell growth and proliferation, and promote the healing rate of bone tissue. In this study, Lys-g-GO nanoparticles and AuNPs-PDA coating were used to optimize the physical and chemical properties of the PLGA scaffold and improve its tissue repair ability. The results show that the combination of AuNPs-PDA functional coating and Lys-g-GO nanoparticles can significantly improve the hydrophilicity, mechanical strength and antibacterial properties of PLGA stents. Lys-g-GO and AuNPs-PDA coatings show a synergistic effect in promoting cell proliferation, adhesion, osteogenic differentiation and calcium deposition. The results of animal experiments show that the [email protected]/Lys-g-GO composite porous scaffold exhibits excellent bone repair capabilities and effectively improves the formation of new bone and collagen deposition at the defect site. In addition, the scaffold material has good biocompatibility, and has no obvious toxic and side effects to the important organs of rabbits (liver, spleen, kidney, heart). Therefore, we believe that the scaffold material we prepared has good clinical development prospects and may play an important role in bone tissue regenerative medicine.
In this study, AuNPs-PDA coated modified PLGA/Lys-g-GO composite scaffolds with loose skeletons were prepared. The scaffold has a channel-like pore structure and full 3D interconnection, and the pore surface has a honeycomb-like micro-pore structure, which is suitable for cell infiltration and nutrient transport. The application of Lys-g-GO nanoparticles and AuNPs-PDA coating significantly enhanced the hydrophilicity, mechanical properties and antibacterial properties of the PLGA scaffold, resulting in good osteogenic activity. In vivo results show that the [email protection]/Lys-g-GO composite scaffold can effectively repair rabbit radius defects. The deposit of mineralized collagen and the increase in bone content confirm the successful regeneration of bone tissue. This study shows that the use of Lys-g-GO nanoparticles and AuNPs-PDA coating for functional modification of bone implants is a promising strategy, [email protection]/Lys-g-GO composite scaffold It has shown promise in the treatment of bone defects in clinical applications.
The original data used and/or analyzed during the current research period can be obtained from the corresponding author upon reasonable request.
The use and care of animals was approved by the Animal Care and Use Institutional Committee of Jilin University, China (Ethics Approval Number 2019132), and were carried out in accordance with the university’s guidelines. In addition, all animal experiments comply with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (NIH Publication No. 8023, revised in 1978).
This work was supported by the National New Drug Research and Development Major Program; Jilin Provincial Department of Science and Technology Fund Key Research and Development Project (20200404137YY); Jilin Province Educational Science Research Project (JJKH20211168KJ); Jilin Province Development and Reform Commission Project (2019C051-6); Jilin Provincial Department of Finance Major Directly under the special project (approval number: 2020SCZT065).
There is no conflict of interest to declare.
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