For patients undergoing orthopedic implant surgery, bacterial infections and infection-induced immune responses have always been life-threatening risks. Conventional biological materials are susceptible to biological contamination, which causes bacteria to invade the injured area and cause postoperative infection. Therefore, there is an urgent need to develop anti-infection and immune escape coatings for orthopedic implants. Here, we have developed an advanced surface modification technology for orthopedic implants called Lubricated Orthopedic Implant Surface (LOIS), which is inspired by the smooth surface of pitcher plant pitchers. LOIS has long-lasting and strong liquid repellency to a variety of liquids and biological substances (including cells, proteins, calcium and bacteria). In addition, we confirmed the mechanical durability against scratches and fixing force by simulating the inevitable damage during the in vitro surgery. The rabbit bone marrow inflammatory femoral fracture model was used to thoroughly study the anti-biological scaling and anti-infection ability of LOIS. We envision that LOIS, which has anti-biofouling properties and mechanical durability, is a step forward in infection-free orthopedic surgery.
Today, due to overall aging, the number of patients suffering from orthopedic diseases (such as elderly fractures, degenerative joint diseases, and osteoporosis) has greatly increased (1, 2). Therefore, medical institutions attach great importance to orthopedic surgery, including orthopedic implants of screws, plates, nails and artificial joints (3, 4). However, traditional orthopedic implants have been reported to be susceptible to bacterial adhesion and biofilm formation, which can cause surgical site infection (SSI) after surgery (5, 6). Once the biofilm is formed on the surface of the orthopedic implant, the removal of the biofilm becomes extremely difficult even with the use of large doses of antibiotics. Therefore, it usually leads to severe postoperative infections (7, 8). Due to the above problems, the treatment of infected implants should include reoperation, including removal of all implants and surrounding tissues; therefore, the patient will suffer severe pain and some risks (9, 10).
To solve some of these problems, drug-eluting orthopedic implants have been developed to prevent infection by eliminating bacteria attached to the surface (11, 12). However, the strategy still shows several limitations. It has been reported that long-term implantation of drug-eluting implants has caused damage to surrounding tissues and caused inflammation, which may lead to necrosis (13, 14). In addition, the organic solvents that may exist after the manufacturing process of drug-eluting orthopedic implants, which are strictly prohibited by the US Food and Drug Administration, require additional purification steps to meet its standards (15). Drug-eluting implants are challenging for the controlled release of drugs, and due to their limited drug loading, long-term application of the drug is not feasible (16).
Another common strategy is to coat the implant with an antifouling polymer to prevent biological matter and bacteria from adhering to the surface (17). For example, zwitterionic polymers have attracted attention due to their non-adhesive properties when in contact with plasma proteins, cells, and bacteria. However, it has some limitations related to long-term stability and mechanical durability, which hinder its practical application in orthopedic implants, especially because of mechanical scraping during surgical procedures (18, 19). In addition, due to its high biocompatibility, lack of need for removal surgery, and surface cleaning properties through corrosion, orthopedic implants made of biodegradable materials have been used (20, 21). During corrosion, the chemical bonds between the polymer matrix are broken down and detached from the surface, and the adherents clean the surface. However, anti-biological fouling by surface cleaning is effective in a short period of time. In addition, most absorbable materials including poly(lactic acid-glycolic acid copolymer) (PLGA), polylactic acid (PLA) and magnesium-based alloys will undergo uneven biodegradation and erosion in the body, which will negatively affect mechanical stability. (twenty two). In addition, the biodegradable plate fragments provide a place for bacteria to attach, which increases the chance of infection in the long run. This risk of mechanical degradation and infection limits the practical application of plastic surgery (23).
Superhydrophobic (SHP) surfaces that mimic the hierarchical structure of lotus leaves have become a potential solution for anti-fouling surfaces (24, 25). When the SHP surface is immersed in liquid, air bubbles will be trapped, thereby forming air pockets and preventing bacterial adhesion (26). However, recent studies have shown that the SHP surface has disadvantages related to mechanical durability and long-term stability, which hinders its application in medical implants. Moreover, the air pockets will dissolve and lose their anti-fouling properties, thus resulting in wider bacterial adhesion due to the large surface area of ​​the SHP surface (27, 28). Recently, Aizenberg and colleagues introduced an innovative method of anti-biofouling surface coating by developing a smooth surface inspired by Nepenthes pitcher plant (29, 30). The smooth surface shows long-term stability under hydraulic conditions, is extremely liquid repellent to biological liquids, and has self-repairing properties. However, there is neither a method to apply a coating to a complex-shaped medical implant, nor is it proven to support the healing process of damaged tissue after implantation.
Here, we introduce a lubricated orthopedic implant surface (LOIS), a micro/nano-structured orthopedic implant surface and tightly combined with a thin lubricant layer to prevent it from being associated with plastic surgery Bacterial infections, such as fracture fixation. Because the fluorine-functionalized micro/nano-level structure firmly fixes the lubricant on the structure, the developed LOIS can fully repel the adhesion of various liquids and maintain anti-fouling performance for a long time. LOIS coatings can be applied to materials of various shapes intended for bone synthesis. The excellent anti-biofouling properties of LOIS against biofilm bacteria [Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA)] and biological substances (cells, proteins and calcium) have been confirmed in vitro. The adhesion rate of extensive adhesion to the substrate is less than 1%. In addition, even after mechanical stress such as surface scratching occurs, the self-healing caused by the penetrating lubricant helps maintain its anti-fouling properties. The mechanical durability test results show that even after structural and chemical modification, the total strength will not be significantly reduced. In addition, an in vitro experiment that simulates the mechanical stress in the surgical environment was carried out to prove that LOIS can withstand various mechanical stresses that occur during plastic surgery. Finally, we used a rabbit-based in vivo femoral fracture model, which proved that LOIS has superior antibacterial properties and biocompatibility. Radiological and histological results confirmed that stable lubricant behavior and anti-biofouling properties within 4 weeks after implantation can achieve effective anti-infection and immune escape performance without delaying the bone healing process.
Figure 1A shows a schematic diagram of the developed LOIS, which is implanted with micro/nano-scale structures in the rabbit femoral fracture model to confirm its excellent anti-biological fouling and anti-infection properties. A biomimetic method is carried out to simulate the surface of a water pot plant, and to prevent biofouling by incorporating a lubricant layer within the micro/nano structure of the surface. The surface injected with lubricant can minimize the contact between biological substances and the surface. Therefore, due to the formation of stable chemical bonds on the surface, it has excellent antifouling performance and long-term stability. As a result, the anti-biofouling properties of the lubricating surface allow various practical applications in biomedical research. However, extensive research on how this special surface interacts in the body has not yet been completed. By comparing LOIS with naked substrates in vitro using albumin and biofilm bacteria, the non-adhesiveness of LOIS can be confirmed (Figure 1B). In addition, by rolling off the water droplets on the inclined bare substrate and the LOIS substrate (Figure S1 and Movie S1), the biological contamination performance can be demonstrated. As shown in the fluorescence microscope image, the exposed substrate incubated in a suspension of protein and bacteria showed a large amount of biological material adhering to the surface. However, due to its excellent anti-biofouling properties, LOIS hardly displays any fluorescence. In order to confirm its anti-biofouling and anti-infection properties, LOIS was applied to the surface of orthopedic implants for bone synthesis (plates and screws) and placed in a rabbit fracture model. Before implantation, the naked orthopedic implant and LOIS were incubated in a bacterial suspension for 12 hours. The pre-incubation ensures that a biofilm is formed on the surface of the exposed implant for comparison. Figure 1C shows a photo of the fracture site 4 weeks after implantation. On the left, a rabbit with a bare orthopedic implant showed a severe level of inflammation due to the formation of a biofilm on the surface of the implant. The opposite result was observed in rabbits implanted with LOIS, that is, the surrounding tissues of LOIS showed neither signs of infection nor signs of inflammation. In addition, the optical image on the left indicates the surgical site of the rabbit with the exposed implant, indicating that no multiple adhesives present on the surface of the exposed implant were found on the surface of the LOIS. This shows that LOIS has long-term stability and has the ability to maintain its anti-biological fouling and anti-adhesion properties.
(A) Schematic diagram of LOIS and its implantation in a rabbit femoral fracture model. (B) Fluorescence microscopy image of protein and bacterial biofilm on bare surface and LOIS substrate. 4 weeks after implantation, (C) a photographic image of the fracture site and (D) an X-ray image (highlighted by a red rectangle). Image courtesy: Kyomin Chae, Yonsei University.
The sterilized, exposed negatively implanted rabbits showed a normal bone healing process without any signs of inflammation or infection. On the other hand, SHP implants pre-incubated in a bacterial suspension exhibit infection-related inflammation on the surrounding tissues. This can be attributed to its inability to inhibit bacterial adhesion for a long time (Figure S2). In order to prove that LOIS does not affect the healing process, but inhibits possible infections related to implantation, X-ray images of the exposed positive matrix and LOIS at the fracture site were compared (Figure 1D). The X-ray image of the bare positive implant showed persistent osteolysis lines, indicating that the bone was not completely healed. This suggests that the bone recovery process may be greatly delayed due to infection-related inflammation. On the contrary, it showed that the rabbits implanted with LOIS had healed and did not show any obvious fracture site.
In order to develop medical implants with long-term stability and functionality (including resistance to biofouling), many efforts have been made. However, the presence of various biological substances and the dynamics of tissue adhesion limits the development of their clinically reliable methods. In order to overcome these shortcomings, we have developed a micro/nano layered structure and chemically modified surface, which is optimized due to high capillary force and chemical affinity to keep the smoothest lubricant to the greatest extent. Figure 2A shows the overall manufacturing process of LOIS. First, prepare a medical grade stainless steel (SS) 304 substrate. Secondly, the micro/nano structure is formed on the SS substrate by chemical etching using hydrofluoric acid (HF) solution. In order to restore the corrosion resistance of SS, a nitric acid (HNO3) solution (31) is used to process the etched substrate. Passivation enhances the corrosion resistance of the SS substrate and significantly slows down the corrosion process that may reduce the overall performance of LOIS. Then, by forming a self-assembled monolayer (SAM) with 1H, 1H, 2H, 2H-perfluorooctyltriethoxysilane (POTS), the surface is chemically modified to improve the chemical interaction between the surface and the smooth lubricant Affinity. The surface modification significantly reduces the surface energy of the fabricated micro/nano-scale structured surface, which matches the surface energy of the smooth lubricant. This allows the lubricant to be completely wetted, thereby forming a stable lubricant layer on the surface. The modified surface exhibits enhanced hydrophobicity. The results show that the slippery lubricant exhibits stable behavior on LOIS due to the high chemical affinity and capillary force caused by the micro/nano structure (32, 33). The optical changes on the surface of SS after surface modification and lubricant injection were studied. The micro/nano layered structure formed on the surface can cause visual changes and darken the surface. This phenomenon is attributed to the enhanced light scattering effect on the rough surface, which increases the diffuse reflection caused by the light trapping mechanism (34). In addition, after the lubricant is injected, the LOIS becomes darker. The lubricating layer causes less light to be reflected from the substrate, thereby darkening the LOIS. In order to optimize the microstructure/nanostructure to show the smallest sliding angle (SA) to achieve anti-biofouling performance, scanning electron microscopy (SEM) and atomic pairs were used to perform different HF etching times (0, 3). , 15 and 60 minutes) Force Microscope (AFM) (Figure 2B). SEM and AFM images show that after a short time of etching (3 minutes of etching), the bare substrate has formed uneven nano-scale roughness. The surface roughness changes with the etching time (Figure S3). The time-varying curve shows that the surface roughness continues to increase and reaches a peak at 15 minutes of etching, and then only a slight decrease in roughness value is observed at 30 minutes of etching. At this point, the nano-level roughness is etched away, while the micro-level roughness develops vigorously, making the roughness change more stable. After etching for more than 30 minutes, a further increase in roughness is observed, which is explained in detail as follows: SS is composed of steel, alloyed with elements including iron, chromium, nickel, molybdenum and many other elements. Among these elements, iron, chromium and molybdenum play an important role in forming micron/nano-scale roughness on the SS by HF etching. In the early stages of corrosion, iron and chromium are mainly corroded because molybdenum has higher corrosion resistance than molybdenum. As the etching progresses, the etching solution reaches local oversaturation, forming fluorides and oxides caused by etching. Fluoride and oxide precipitate and eventually redeposit on the surface, forming a surface roughness in the micron/nano range (31). This micro/nano-level roughness plays an important role in the self-healing properties of LOIS. The dual scale surface produces a synergistic effect, greatly increasing the capillary force. This phenomenon allows the lubricant to penetrate the surface stably and contributes to self-healing properties (35). The formation of roughness depends on the etching time. Under 10 minutes of etching, the surface contains only nano-scale roughness, which is not enough to hold enough lubricant to have biofouling resistance (36). On the other hand, if the etching time exceeds 30 minutes, the nano-scale roughness formed by the redeposition of iron and chromium will disappear, and only the micro-scale roughness will remain due to molybdenum. The over-etched surface lacks nano-scale roughness and loses the synergistic effect of two-stage roughness, which negatively affects the self-healing characteristics of LOIS. SA measurements were performed on substrates with different etching times to prove anti-fouling performance. Various types of liquids were selected based on viscosity and surface energy, including deionized (DI) water, blood, ethylene glycol (EG), ethanol (EtOH) and hexadecane (HD) (Figure S4). The time-varying etching pattern shows that for various liquids with different surface energies and viscosities, the SA of LOIS after 15 minutes of etching is the lowest. Therefore, LOIS is optimized to etch for 15 minutes to form micron and nano-scale roughness, which is suitable for effectively maintaining the durability of the lubricant and excellent anti-fouling properties.
(A) Schematic diagram of the four-step manufacturing process of LOIS. The inset shows the SAM formed on the substrate. (B) SEM and AFM images, used to optimize the micro/nano structure of the substrate under different etching times. X-ray photoelectron spectroscopy (XPS) spectra of (C) Cr2p and (D) F1s after surface passivation and SAM coating. au, arbitrary unit. (E) Representative images of water droplets on bare, etched, SHP and LOIS substrates. (F) The contact angle (CA) and SA measurement of liquids with different surface tensions on SHP and LOIS. Data are expressed as mean ± SD.
Then, in order to confirm the change in the chemical properties of the surface, X-ray photoelectron spectroscopy (XPS) was used to study the change in the chemical composition of the substrate surface after each surface coating. Figure 2C shows the XPS measurement results of the HF etched surface and the HNO 3 treated surface. The two main peaks at 587.3 and 577.7 eV can be attributed to the Cr-O bond existing in the chromium oxide layer, which is the main difference from the HF etched surface. This is mainly due to the consumption of iron and chromium fluoride on the surface by HNO3. The HNO3-based etching allows chromium to form a passivating oxide layer on the surface, which makes the etched SS again resistant to corrosion. In Figure 2D, XPS spectra were obtained to confirm that fluorocarbon-based silane was formed on the surface after the SAM coating, which has extremely high liquid repellency even for EG, blood and EtOH. The SAM coating is completed by reacting silane functional groups with hydroxyl groups formed by plasma treatment. As a result, a significant increase in CF2 and CF3 peaks was observed. The binding energy between 286 and 296 eV indicates that the chemical modification has been successfully completed by the SAM coating. SHP shows relatively large CF2 (290.1 ​​eV) and CF3 (293.3 eV) peaks, which are caused by the fluorocarbon-based silane formed on the surface. Figure 2E shows representative optical images of contact angle (CA) measurements for different groups of deionized water in contact with bare, etched, SHP, and LOIS. These images show that the etched surface becomes hydrophilic due to the micro/nano structure formed by chemical etching so that deionized water is absorbed into the structure. However, when the substrate is coated with SAM, the substrate exhibits strong water repellency, so a surface SHP is formed and the contact area between water and the surface is small. Finally, a decrease in CA was observed in LOIS, which can be attributed to the penetration of lubricant into the microstructure, thereby increasing the contact area. In order to prove that the surface has excellent liquid repellency and non-adhesive properties, the LOIS was compared with the SHP substrate by measuring CA and SA using various liquids (Figure 2F). Various types of liquids were selected based on viscosity and surface energy, including deionized water, blood, EG, EtOH and HD (Figure S4). CA measurement results show that when CA tends to HD, the reduction value of CA, where CA has the lowest surface energy. In addition, the LOIS of the overall CA is low. However, the SA measurement shows a completely different phenomenon. Except for the ionized water, all liquids adhere to the SHP substrate without slipping off. On the other hand, LOIS shows a very low SA, where when all the liquid is tilted at an angle lower than 10° to 15°, all the liquid will roll off. This strongly shows that the non-adhesiveness of LOIS is better than that of SHP surface. In addition, LOIS coatings are also applied to various types of materials, including titanium (Ti), polyphenylsulfone (PPSU), polyoxymethylene (POM), polyether ether ketone (PEEK) and bioabsorbable polymers (PLGA), They are implantable orthopedic materials (Figure S5)). The sequential images of the droplets on the material treated by LOIS show that the anti-biofouling properties of LOIS are the same on all substrates. In addition, the measurement results of CA and SA show that the non-adhesive properties of LOIS can be applied to other materials.
In order to confirm the anti-fouling properties of LOIS, various types of substrates (including bare, etched, SHP and LOIS) were incubated with Pseudomonas aeruginosa and MRSA. These two bacteria were selected as representative hospital bacteria, which can lead to the formation of biofilms, leading to SSI (37). Figure 3 (A and B) shows the fluorescence microscope images and the colony forming unit (CFU) measurement results of the substrates incubated in the bacterial suspension for short-term (12 hours) and long-term (72 hours), respectively. In a short period of time, bacteria will form clusters and grow in size, covering themselves with mucus-like substances and preventing their removal. However, during the 72-hour incubation, the bacteria will mature and become easy to disperse to form more colonies or clusters. Therefore, it can be considered that 72-hour incubation is long-term and is the appropriate incubation time to form a strong biofilm on the surface (38). In a short period of time, the etched surface and the surface of the SHP exhibited bacterial adhesion, which was reduced by about 25% to 50% compared to the bare substrate. However, due to its excellent anti-biofouling performance and stability, LOIS did not show bacterial biofilm adhesion in the short and long term. The schematic diagram (Figure 3C) describes the explanation of the anti-biological fouling mechanism of the etching solution, SHP and LOIS. The assumption is that the etched substrate with hydrophilic properties will have a larger surface area than the bare substrate. Therefore, more bacterial adhesion will occur on the etched substrate. However, compared with the bare substrate, the etched substrate has significantly less biofilm formed on the surface. This is because water molecules bind firmly to the hydrophilic surface and act as a lubricant for water, thus interfering with the adhesion of bacteria in the short term (39). However, the layer of water molecules is very thin and soluble in bacterial suspensions. Therefore, the water molecular layer disappears for a long time, leading to extensive bacterial adhesion and proliferation. For SHP, due to its short-term non-wetting properties, bacterial adhesion is inhibited. The reduced bacterial adhesion can be attributed to air pockets trapped in the layered structure and lower surface energy, thereby minimizing contact between the bacterial suspension and the surface. However, extensive bacterial adhesion was observed in SHP because it lost its anti-fouling properties for a long time. This is mainly due to the disappearance of air pockets due to hydrostatic pressure and the dissolution of air in water. This is mainly due to the disappearance of air pockets due to dissolution and the layered structure that provides a larger surface area for adhesion (27, 40). Unlike these two substrates that have an important effect on long-term stability, the lubricating lubricant contained in LOIS is injected into the micro/nano structure and will not disappear even in the long term. Lubricants filled with micro/nano structures are very stable and are strongly attracted to the surface due to their high chemical affinity, thereby preventing bacterial adhesion for a long time. Figure S6 shows a reflection confocal microscope image of a lubricant-infused substrate immersed in phosphate buffered saline (PBS). Continuous images show that even after 120 hours of slight shaking (120 rpm), the lubricant layer on the LOIS remains unchanged, indicating long-term stability under flow conditions. This is due to the high chemical affinity between the fluorine-based SAM coating and the perfluorocarbon-based lubricant, so that a stable lubricant layer can be formed. Therefore, the anti-fouling performance is maintained. In addition, the substrate was tested against representative proteins (albumin and fibrinogen), which are in plasma, cells closely related to immune function (macrophages and fibroblasts), and those related to bone formation. The content of calcium is very high. (Figure 3D, 1 and 2, and Figure S7) (41, 42). In addition, the fluorescence microscope images of the adhesion test for fibrinogen, albumin and calcium showed different adhesion characteristics of each substrate group (Figure S8). During bone formation, newly formed bone and calcium layers may surround the orthopedic implant, which not only makes removal difficult, but may also cause unexpected harm to the patient during the removal process. Therefore, low levels of calcium deposits on bone plates and screws are beneficial for orthopedic surgery that requires implant removal. Based on the quantification of the attached area based on the fluorescence intensity and the cell count, we confirmed that LOIS shows excellent anti-biofouling properties for all biological substances compared with other substrates. According to the results of in vitro experiments, the anti-biological fouling LOIS can be applied to orthopedic implants, which can not only inhibit infections caused by biofilm bacteria, but also reduce inflammation caused by the body’s active immune system.
(A) Fluorescence microscope images of each group (naked, etched, SHP and LOIS) incubated in Pseudomonas aeruginosa and MRSA suspensions for 12 and 72 hours. (B) The number of adherent CFU of Pseudomonas aeruginosa and MRSA on the surface of each group. (C) Schematic diagram of the anti-biological fouling mechanism of short-term and long-term etching, SHP and LOIS. (D) (1) The number of fibroblasts adhered to each substrate and fluorescence microscope images of the cells adhered to the bare and LOIS. (2) Adhesion test of immune-related proteins, albumin and calcium involved in the bone healing process (* P <0.05, ** P <0.01, *** P <0.001 and **** P <0.0001). ns, not important.
In the case of unavoidable concentrated stresses, mechanical durability has always been the main challenge for the application of antifouling coatings. Traditional anti-sewage gel methods are based on polymers with low water solubility and fragility. Therefore, they are usually susceptible to mechanical stress in biomedical applications. Therefore, mechanically durable antifouling coatings remain a challenge for applications such as orthopedic implants (43, 44). Figure 4A(1) demonstrates the two main types of stress applied to orthopedic implants, including scratching (shear stress) and compression with the optical image of the damaged implant produced by the forceps. For example, when the screw is tightened with a screwdriver, or when the surgeon holds the bone plate tightly with tweezers and applies compressive force, the plastic bone plate will be damaged and scratched on both the macro and micro/nano scales (Figure 4A, 2) . In order to test whether the manufactured LOIS can withstand these damages during plastic surgery, nanoindentation was performed to compare the hardness of the bare substrate and the LOIS on the micro/nano scale to study the mechanical properties of the micro/nano structure Impact (Figure 4B). The schematic diagram shows the different deformation behavior of LOIS due to the presence of micro/nano structures. A force-displacement curve was drawn based on the results of nanoindentation (Figure 4C). The blue image represents the bare substrate, which shows only slight deformation, as seen by the maximum indentation depth of 0.26-μm. On the other hand, the gradual increase in nanoindentation force and displacement observed in LOIS (red curve) may show signs of reduced mechanical properties, resulting in a nanoindentation depth of 1.61μm. This is because the micro/nano structure present in the LOIS provides a deeper advancement space for the tip of the nanoindenter, so its deformation is greater than that of the bare substrate. Konsta-Gdoutos et al. (45) believes that due to the presence of nanostructures, nanoindentation and micro/nano roughness lead to irregular nanoindentation curves. The shaded area corresponds to the irregular deformation curve attributed to the nanostructure, while the non-shaded area is attributed to the microstructure. This deformation may damage the microstructure/nanostructure of the holding lubricant and negatively affect its anti-fouling performance. In order to study the impact of damage on LOIS, inevitable damage to micro/nano structures was replicated in the body during plastic surgery. By using blood and protein adhesion tests, the stability of the anti-biofouling properties of LOIS after in vitro can be determined (Figure 4D). A series of optical images shows the damage that occurred near the holes of each substrate. A blood adhesion test was performed to demonstrate the effect of mechanical damage on the anti-biofouling coating (Figure 4E). Like SHP, the anti-fouling properties are lost due to damage, and LOIS exhibits excellent anti-fouling properties by repelling blood. This is because, because the surface energy is driven by the capillary action covering the damaged area, the flow in the microstructured lubricant lubricant restores the anti-fouling properties (35). The same trend was observed in the protein adhesion test using albumin. In the damaged area, the adhesion of protein on the surface of SHP is widely observed, and by measuring its area coverage, it can be quantified as half of the adhesion level of the bare substrate. On the other hand, LOIS maintained its anti-biofouling properties without causing adhesion (Figure 4, F and G). In addition, the surface of the screw is often subjected to strong mechanical stress, such as drilling, so we studied the ability of the LOIS coating to remain intact on the screw in vitro. Figure 4H shows optical images of different screws, including bare, SHP and LOIS. The red rectangle represents the target area where strong mechanical stress occurs during bone implantation. Similar to the protein adhesion test of the plate, a fluorescence microscope is used to image the protein adhesion and measure the coverage area to prove the integrity of the LOIS coating, even under strong mechanical stress (Figure 4, I and J). The LOIS-treated screws exhibit excellent anti-fouling performance, and almost no protein adheres to the surface. On the other hand, protein adhesion was observed in bare screws and SHP screws, where the area coverage of SHP screws was one third of that of bare screws. In addition, the orthopedic implant used for fixation must be mechanically strong to withstand the stress applied to the fracture site, as shown in Figure 4K. Therefore, a bending test was performed to determine the effect of chemical modification on mechanical properties. In addition, this is done to maintain the fixed stress from the implant. Apply vertical mechanical force until the implant is fully folded and a stress-strain curve is obtained (Figure 4L, 1). Two properties including Young’s modulus and flexural strength were compared between bare and LOIS substrates as indicators of their mechanical strength (Figure 4L, 2 and 3). Young’s modulus indicates the ability of a material to withstand mechanical changes. The Young’s modulus of each substrate is 41.48±1.01 and 40.06±0.96 GPa, respectively; the observed difference is about 3.4%. In addition, it is reported that the bending strength, which determines the toughness of the material, is 102.34±1.51 GPa for the bare substrate and 96.99±0.86 GPa for SHP. The bare substrate is approximately 5.3% higher. The slight decrease in mechanical properties may be caused by the notch effect. In the notch effect, the micro/nano roughness may act as a set of notches, leading to local stress concentration and affecting the mechanical properties of the implant (46). However, based on the fact that the stiffness of human cortical bone is reported to be between 7.4 and 31.6 GPa, and the measured LOIS modulus exceeds that of human cortical bone (47), the LOIS is sufficient to support the fracture and its overall The mechanical properties are minimally affected by surface modification.
(A) Schematic diagram of (1) the mechanical stress applied to the orthopedic implant during the operation, and (2) the optical image of the damaged orthopedic implant. (B) Schematic diagram of nano-mechanical properties measurement by nanoindentation and LOIS on the bare surface. (C) Nanoindentation force-displacement curve of bare surface and LOIS. (D) After in vitro experiments, simulate the optical images of different types of orthopedic plates (the damaged area is highlighted with a red rectangle) to simulate the mechanical stress caused during the operation. (E) Blood adhesion test and (F) protein adhesion test of the damaged orthopedic plate group. (G) Measure the area coverage of the protein adhering to the plate. (H) Optical images of different types of orthopedic screws after the in vitro experiment. (I) Protein adhesion test to study the integrity of different coatings. (J) Measure the area coverage of the protein adhering to the screw. (K) The movement of the rabbit is intended to generate a fixed stress on the fractured bone. (L) (1) Bend test results and optical images before and after bending. The difference in (2) Young’s modulus and (3) bending strength between bare implant and SHP. Data are expressed as mean ± SD (*P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001). Image courtesy: Kyomin Chae, Yonsei University.
In clinical situations, most bacterial contact with biological materials and wound sites comes from mature, mature biofilms (48). Therefore, the US Centers for Disease Control and Prevention estimates that 65% of all human infections are related to biofilms (49). In this case, it is necessary to provide an in vivo experimental design that provides consistent biofilm formation on the surface of the implant. Therefore, we developed a rabbit femoral fracture model in which orthopedic implants were pre-incubated in a bacterial suspension and then implanted in rabbit femurs to study the anti-fouling properties of LOIS in vivo. Due to the following three important facts, bacterial infections are induced by pre-culture rather than direct injection of bacterial suspensions: (i) The immune system of rabbits is naturally stronger than that of humans; therefore, injection of bacterial suspensions and planktonic bacteria is possible It has no effect on the formation of biofilms. (Ii) Planktonic bacteria are more susceptible to antibiotics, and antibiotics are usually used after surgery; finally, (iii) the planktonic bacteria suspension may be diluted by the animal’s body fluids (50). By pre-culturing the implant in a bacterial suspension before implantation, we can thoroughly study the harmful effects of bacterial infection and foreign body reaction (FBR) on the bone healing process. The rabbits were sacrificed 4 weeks after implantation, because the osseointegration essential for the bone healing process will be completed within 4 weeks. Then, the implants were removed from the rabbits for downstream studies. Figure 5A shows the proliferation mechanism of bacteria. The infected orthopedic implant is introduced into the body. As a result of pre-incubation in bacterial suspension, six of the six rabbits implanted with naked implants were infected, while none of the rabbits implanted with LOIS-treated implants were infected. Bacterial infections proceed in three steps, including growth, maturation and dispersion (51). First, the attached bacteria reproduce and grow on the surface, and then the bacteria form a biofilm when they excrete extracellular polymer (EPS), amyloid and extracellular DNA. Biofilm not only interferes with the penetration of antibiotics, but also promotes the accumulation of antibiotic-degrading enzymes (such as β-lactamase) (52). Finally, the biofilm spreads the mature bacteria into the surrounding tissues. Therefore, infection occurs. In addition, when a foreign body enters the body, an infection that can cause a strong immune response can cause severe inflammation, pain, and decreased immunity. Figure 5B provides an overview of the FBR caused by the insertion of an orthopedic implant, rather than the immune response caused by a bacterial infection. The immune system recognizes the inserted implant as a foreign body, and then causes the cells and tissues to react to encapsulate the foreign body (53). In the early days of FBR, a supply matrix was formed on the surface of orthopedic implants, which resulted in the adsorption of fibrinogen. The adsorbed fibrinogen then forms a highly dense fibrin network, which promotes the attachment of leukocytes (54). Once the fibrin network is formed, acute inflammation will occur due to the infiltration of neutrophils. In this step, a variety of cytokines such as tumor necrosis factor-α (TNF-α), interleukin-4 (IL-4) and IL-β are released, and monocytes begin to infiltrate the implantation site and differentiate into giant cells. Phage (41, 55, 56). Reducing FBR has always been a challenge because excessive FBR can cause acute and chronic inflammation, which can lead to fatal complications. In order to assess the impact of bacterial infections in the tissues surrounding the bare implant and LOIS, hematoxylin and eosin (H&E) and Masson trichrome (MT) staining were used. For rabbits implanted with bare substrates, severe bacterial infections progressed, and H&E tissue slides clearly showed abscesses and necrosis caused by inflammation. On the other hand, the extremely strong anti-biofouling surface LOIS inhibits bacterial adhesion, so it shows no signs of infection and reduces inflammation (Figure 5C). The results of MT staining showed the same trend. However, MT staining also showed edema in rabbits implanted with LOIS, indicating that recovery is about to occur (Figure 5D). In order to study the degree of immune response, immunohistochemical (IHC) staining was performed using cytokines TNF-α and IL-6 related to immune response. A naked negative implant that was not exposed to bacteria was compared with a LOIS that was exposed to bacteria but not infected to study the healing process in the absence of bacterial infection. Figure 5E shows an optical image of an IHC slide that expresses TNF-α. The brown area represents the immune response, indicating that the immune response in LOIS is slightly reduced. In addition, the expression of IL-6 in LOIS was significantly less than the negative expression of sterile naked (Figure 5F). The expression of cytokine was quantified by measuring the area of ​​antibody staining corresponding to the cytokine (Figure 5G). Compared with the rabbits exposed to the negative implants, the expression levels of the rabbits implanted with LOIS were lower, showing a meaningful difference. The decrease in cytokine expression indicates that the long-term, stable anti-fouling properties of LOIS are not only related to the inhibition of bacterial infections, but also to the decrease of FBR, which is induced by macrophages adhering to the substrate (53, 57 , 58). Therefore, the reduced immune response due to the immune evasion properties of LOIS may solve the side effects after implantation, such as excessive immune response after plastic surgery.
(A) A schematic diagram of the mechanism of biofilm formation and spread on the surface of an infected orthopedic implant. eDNA, extracellular DNA. (B) Schematic diagram of immune response after orthopedic implant insertion. (C) H&E staining and (D) MT staining of the surrounding tissues of orthopedic implants with bare positive and LOIS. IHC of immune-related cytokines (E) TNF-α and (F) IL-6 are stained images of naked-negative and LOIS-implanted rabbits. (G) Quantification of cytokine expression by area coverage measurement (** P <0.01).
The biocompatibility of LOIS and its effect on bone healing process were examined in vivo using diagnostic imaging [x-ray and micro-computed tomography (CT)] and osteoclast IHC. Figure 6A shows the bone healing process involving three different stages: inflammation, repair, and remodeling. When a fracture occurs, inflammatory cells and fibroblasts will penetrate into the fractured bone and begin to grow into the vascular tissue. During the repair phase, the ingrowth of vascular tissue spreads near the fracture site. Vascular tissue provides nutrients for the formation of new bone, which is called callus. The final stage of the bone healing process is the remodeling stage, in which the size of the callus is reduced to the size of normal bone with the help of an increase in the level of activated osteoclasts (59). Three-dimensional (3D) reconstruction of the fracture site was performed using micro-CT scans to observe the differences in the level of callus formation in each group. Observe the cross-section of the femur to observe the thickness of the callus surrounding the fractured bone (Figure 6, B and C). X-rays were also used to examine the fracture sites of all groups every week to observe the different bone regeneration processes in each group (Figure S9). Callus and mature bones are shown in blue/green and ivory, respectively. Most soft tissues are filtered out with a preset threshold. Nude positive and SHP confirmed the formation of a small amount of callus around the fracture site. On the other hand, the exposed negative of LOIS and the fracture site are surrounded by thick callus. Micro-CT images showed that the formation of callus was hindered by bacterial infection and infection-related inflammation. This is because the immune system prioritizes the healing of septic injuries caused by infection-related inflammation, rather than bone recovery (60). IHC and Tartrate-resistant Acid Phosphatase (TRAP) staining were performed to observe osteoclast activity and bone resorption (Figure 6D) (61). Only a few activated osteoclasts stained purple were found in naked positives and SHP. On the other hand, many activated osteoclasts were observed near the naked positive and mature bones of LOIS. This phenomenon indicates that in the presence of osteoclasts, the callus around the fracture site is undergoing a violent remodeling process (62). The bone volume and osteoclast expression area of ​​the callus were measured to compare the level of callus formation around the fracture site in all groups, so as to quantify the micro-CT scan and IHC results (Figure 6E, 1 and 2). As expected, the naked negatives and callus formation in LOIS were significantly higher than in the other groups, indicating that positive bone remodeling occurred (63). Figure S10 shows the optical image of the surgical site, the MT staining result of the tissue collected near the screw, and the TRAP staining result highlighting the screw-bone interface. In the bare substrate, strong callus and fibrosis formation was observed, while the LOIS-treated implant showed a relatively unadhered surface. Similarly, compared to naked negatives, lower fibrosis was observed in rabbits implanted with LOIS, as indicated by the white arrows. In addition, the firm edema (blue arrow) can be attributed to the immune evasion properties of LOIS, thereby reducing severe inflammation. The non-stick surface around the implant and reduced fibrosis suggest that the removal process is easier, which usually results in other fractures or inflammation. The bone healing process after screw removal was evaluated by the osteoclast activity at the screw-bone interface. Both the bare bone and the LOIS implant interface absorbed similar levels of osteoclasts to further bone healing, indicating that the LOIS coating has no negative effect on bone healing or immune response. In order to confirm that the surface modification performed on the LOIS does not interfere with the bone healing process, X-ray examination was used to compare the bone healing of the rabbits with exposed negative ions and 6 weeks of LOIS implantation (Figure 6F). The results showed that compared with the uninfected nude positive group, LOIS showed the same degree of bone healing, and there were no obvious signs of fracture (continuous osteolysis line) in both groups.
(A) Schematic diagram of bone healing process after fracture. (B) The difference in the degree of callus formation of each surface group and (C) the cross-sectional image of the fracture site. (D) TRAP staining to visualize osteoclast activity and bone resorption. Based on TRAP activity, the formation of external callus of cortical bone was quantitatively analyzed by (E) (1) micro-CT and (2) osteoclast activity. (F) 6 weeks after implantation, X-ray images of the fractured bone of the exposed negative (highlighted by the red dashed rectangle) and LOIS (highlighted by the blue dashed rectangle). Statistical analysis was performed by one-way analysis of variance (ANOVA). * P <0.05. ** P <0.01.
In short, LOIS provides a new type of antibacterial infection strategy and immune escape coating for orthopedic implants. Conventional orthopedic implants with SHP functionalization exhibit short-term anti-biofouling properties, but cannot maintain their properties for a long time. The superhydrophobicity of the substrate traps air bubbles between the bacteria and the substrate, thereby forming air pockets, thereby preventing bacterial infection. However, due to the diffusion of air, these air pockets are easily removed. On the other hand, LOIS has well proven its ability to prevent biofilm-related infections. Therefore, due to the anti-rejection properties of the lubricant layer injected into the layered micro/nano structure surface, infection-related inflammation can be prevented. Various characterization methods including SEM, AFM, XPS and CA measurements are used to optimize LOIS manufacturing conditions. In addition, LOIS can also be applied to various biological materials commonly used in orthopedic fixation equipment, such as PLGA, Ti, PE, POM and PPSU. Then, LOIS was tested in vitro to prove its anti-biofouling properties against bacteria and biological substances related to immune response. The results show that it has excellent antibacterial and anti-biofouling effects compared to the bare implant. In addition, LOIS shows mechanical strength even after applying mechanical stress, which is unavoidable in plastic surgery. Due to the self-healing properties of the lubricant on the surface of the micro/nano structure, LOIS successfully maintained its anti-biological fouling properties. In order to study the biocompatibility and antibacterial properties of LOIS in vivo, LOIS was implanted into rabbit femur for 4 weeks. No bacterial infection was observed in rabbits implanted with LOIS. In addition, the use of IHC demonstrated a reduced level of local immune response, indicating that LOIS does not inhibit the bone healing process. LOIS exhibits excellent antibacterial and immune evasion properties, and has been proven to effectively prevent biofilm formation before and during orthopedic surgery, especially for bone synthesis. By using a rabbit bone marrow inflammatory femoral fracture model, the effect of biofilm-related infections on the bone healing process induced by pre-incubated implants was deeply studied. As a future study, a new in vivo model is needed to study possible infections after implantation to fully understand and prevent biofilm-related infections during the entire healing process. In addition, osteoinduction is still an unresolved challenge in integration with LOIS. Further research is needed to combine selective adhesion of osteoinductive cells or regenerative medicine with LOIS to overcome the challenge. Overall, LOIS represents a promising orthopedic implant coating with mechanical robustness and excellent anti-biofouling properties, which can reduce SSI and immune side effects.
Wash the 15mm x 15mm x 1mm 304 SS substrate (Dong Kang M-Tech Co., Korea) in acetone, EtOH and DI water for 15 minutes to remove contaminants. In order to form a micro/nano-level structure on the surface, the cleaned substrate is immersed in a 48% to 51% HF solution (DUKSAN Corp., South Korea) at 50°C. The etching time varies from 0 to 60 minutes. Then, the etched substrate was cleaned with deionized water and placed in a 65% HNO3 (Korea DUKSAN Corp.) solution at 50°C for 30 minutes to form a chromium oxide passivation layer on the surface. After passivation, the substrate is washed with deionized water and dried to obtain a substrate with a layered structure. Next, the substrate was exposed to oxygen plasma (100 W, 3 minutes), and immediately immersed in a solution of 8.88 mM POTS (Sigma-Aldrich, Germany) in toluene at room temperature for 12 hours. Then, the substrate coated with POTS was cleaned with EtOH, and annealed at 150°C for 2 hours to obtain a dense POTS SAM. After SAM coating, a lubricant layer was formed on the substrate by applying a perfluoropolyether lubricant (Krytox 101; DuPont, USA) with a loading volume of 20 μm/cm 2. Before use, filter the lubricant through a 0.2 micron filter. Remove excess lubricant by tilting at a 45° angle for 15 minutes. The same manufacturing procedure was used for orthopedic implants made of 304 SS (locking plate and cortical locking screw; Dong Kang M-Tech Co., Korea). All orthopedic implants are designed to fit the geometry of the rabbit femur.
The surface morphology of the substrate and orthopedic implants was inspected by field emission SEM (Inspect F50, FEI, USA) and AFM (XE-100, Park Systems, South Korea). The surface roughness (Ra, Rq) is measured by multiplying the area of ​​20 μm by 20 μm (n=4). An XPS (PHI 5000 VersaProbe, ULVAC PHI, Japan) system equipped with an Al Kα X-ray source with a spot size of 100μm2 was used to analyze the surface chemical composition. A CA measurement system equipped with a dynamic image capture camera (SmartDrop, FEMTOBIOMED, ​​South Korea) was used to measure liquid CA and SA. For each measurement, 6 to 10 μl of droplets (deionized water, horse blood, EG, 30% ethanol, and HD) are placed on the surface to measure CA. When the inclination angle of the substrate increases at a speed of 2°/s (n = 4), the SA is measured when the droplet falls.
Pseudomonas aeruginosa [American Type Culture Collection (ATCC) 27853] and MRSA (ATCC 25923) were purchased from ATCC (Manassas, Virginia, USA), and the stock culture was maintained at -80°C . Before use, the frozen culture was incubated in trypsin-thawed soybean broth (Komed, Korea) at 37°C for 18 hours and then transferred twice to activate it. After incubation, the culture was centrifuged at 10,000 rpm for 10 minutes at 4°C and washed twice with a PBS (pH 7.3) solution. The centrifuged culture is then subcultured on blood agar plates (BAP). MRSA and Pseudomonas aeruginosa were prepared overnight and cultured in Luria-Bertani broth. The concentration of Pseudomonas aeruginosa and MRSA in the inoculum was quantitatively determined by the CFU of the suspension in serial dilutions on agar. Then, adjust the bacterial concentration to 0.5 McFarland standard, which is equivalent to 108 CFU/ml. Then dilute the working bacterial suspension 100 times to 106 CFU/ml. To test the antibacterial adhesion properties, the substrate was sterilized at 121°C for 15 minutes before use. The substrate was then transferred to 25 ml of bacterial suspension and incubated at 37°C with vigorous shaking (200 rpm) for 12 and 72 hours. After incubation, each substrate was removed from the incubator and washed 3 times with PBS to remove any floating bacteria on the surface. In order to observe the biofilm on the substrate, the biofilm was fixed with methanol and stained with 1 ml of crimidine orange for 2 minutes. Then a fluorescence microscope (BX51TR, Olympus, Japan) was used to take pictures of the stained biofilm. In order to quantify the biofilm on the substrate, the attached cells were separated from the substrate by the bead vortex method, which was considered to be the most suitable method to remove attached bacteria (n = 4). Using sterile forceps, remove the substrate from the growth medium and tap the well plate to remove excess liquid. Loosely attached cells were removed by washing twice with sterile PBS. Each substrate was then transferred to a sterile test tube containing 9 ml of 0.1% protein ept saline (PSW) and 2 g of 20 to 25 sterile glass beads (0.4 to 0.5 mm in diameter). It was then vortexed for 3 minutes to detach the cells from the sample. After vortexing, the suspension was serially diluted 10-fold with 0.1% PSW, and then 0.1 ml of each dilution was inoculated on BAP. After 24 hours of incubation at 37°C, the CFU was counted manually.
For the cells, mouse fibroblasts NIH/3T3 (CRL-1658; American ATCC) and mouse macrophages RAW 264.7 (TIB-71; American ATCC) were used. Use Dulbecco’s modified Eagle medium (DMEM; LM001-05, Welgene, Korea) to culture mouse fibroblasts and supplement with 10% calf serum (S103-01, Welgene) and 1% penicillin-streptomycin (PS ; LS202-02, Welgene (Welgene). Use DMEM to culture mouse macrophages, supplemented with 10% fetal bovine serum (S001-01, Welgene) and 1% PS. Place the substrate in a six-well cell culture plate , And inoculate the cells at 105 cells/cm2. The cells were incubated overnight at 37°C and 5% CO2. For cell staining, the cells were fixed with 4% paraformaldehyde for 20 minutes and placed in 0.5% Triton X Incubate for 5 minutes in -100. Immerse the substrate in 50nM tetramethylrhodamine at 37°C for 30 minutes. After the incubation process, use the substrate with 4′,6-diamino-2-phenylindole (H -1200, Vector Laboratories, UK) VECTASHIELD fixation medium (n = 4 per cell). For protein, fluorescein, fluorescein isothiocyanate-albumin (A9771, Sigma-Aldrich, Germany) and human plasma The Alexa Fluor 488-conjugated fibrinogen (F13191, Invitrogen, USA) was dissolved in PBS (10 mM, pH 7.4). The concentrations of albumin and fibrinogen were 1 and 150 μg/ml, respectively. After the substrate Before immersing in the protein solution, rinse them with PBS to rehydrate the surface. Then immerse all the substrates in a six-well plate containing the protein solution and incubate at 37°C for 30 and 90 minutes. After incubation, The substrate was then removed from the protein solution, washed gently with PBS 3 times, and fixed with 4% paraformaldehyde (n = 4 for each protein). For calcium, sodium chloride (0.21 M) and potassium phosphate (3.77 mM) ) Was dissolved in deionized water. The pH of the solution was adjusted to 2.0 by adding hydrochloride solution (1M). Then calcium chloride (5.62 mM) was dissolved in the solution. By adding 1M tris(hydroxymethyl)-amino Methane adjusts the pH of the solution to 7.4. Immerse all substrates in a six-well plate filled with 1.5× calcium phosphate solution and remove from the solution after 30 minutes. For staining, 2 g Alizarin Red S (CI 58005) Mix with 100 ml of deionized water. Then, use 10% ammonium hydroxide to adjust the pH to 4. Dye the substrate with Alizarin Red solution for 5 minutes, and then shake off the excess dye and blot. After the shaking process, remove the substrate. The material is dehydrated, then immersed in acetone for 5 minutes, then immersed in an acetone-xylene (1:1) solution for 5 minutes, and finally washed with xylene (n = 4). Fluorescence microscope (Axio Imager) with ×10 and ×20 objective lenses is used. . A2m, Zeiss, Germany) images all substrates. ImageJ/FIJI (https://imagej.nih.gov/ij/) was used to quantify the adhesion data of biological substances on each group of four different imaging areas. Convert all images to binary images with fixed thresholds for substrate comparison.
A Zeiss LSM 700 confocal microscope was used to monitor the stability of the lubricant layer in the PBS in reflection mode. The fluorine-based SAM-coated glass sample with an injected lubricating layer was immersed in a PBS solution, and tested using an orbital shaker (SHO-1D; Daihan Scientific, South Korea) under mild shaking conditions (120 rpm). Then take the sample and monitor the loss of lubricant by measuring the loss of reflected light. To acquire fluorescence images in reflection mode, the sample is exposed to a 633 nm laser and then collected, because the light will be reflected back from the sample. The samples were measured at time intervals of 0, 30, 60, and 120 hours.
In order to determine the influence of the surface modification process on the nanomechanical properties of orthopedic implants, a nanoindenter (TI 950 TriboIndenter, Hysitron, USA) equipped with a three-sided pyramid-shaped Berkovich diamond tip was used to measure nanoindenedione. The peak load is 10 mN and the area is 100μmx 100μm. For all measurements, the loading and unloading time is 10 s, and the holding time under peak indentation load is 2 s. Take measurements from five different locations and take the average. In order to evaluate the mechanical strength performance under load, a transverse three-point bending test was performed using a universal testing machine (Instron 5966, Instron, USA). The substrate is compressed at a constant rate of 10 N/s with an increased load. The Bluehill Universal software program (n = 3) was used to calculate the flexural modulus and maximum compressive stress.
In order to simulate the operation process and the related mechanical damage caused during the operation, the operation process was performed in vitro. The femurs were collected from the executed New Zealand white rabbits. The femur was cleaned and fixed in 4% paraformaldehyde for 1 week. As described in the animal experiment method, the fixed femur was surgically operated. After the operation, the orthopedic implant was immersed in blood (horse blood, KISAN, Korea) for 10 s to confirm whether blood adhesions occurred after the mechanical injury was applied (n = 3).
A total of 24 male New Zealand white rabbits (weight 3.0 to 3.5kg, average age 6 months) were randomly divided into four groups: nude negative, nude positive, SHP and LOIS. All procedures involving animals were performed in accordance with the ethical standards of the Institutional Animal Care and Use Committee (IACUC approved, KOREA-2017-0159). The orthopedic implant consists of a locking plate with five holes (length 41 mm, width 7 mm and thickness 2 mm) and cortical locking screws (length 12 mm, diameter 2.7 mm) for fracture fixation. Except for those plates and screws used in the bare-negative group, all plates and screws were incubated in MRSA suspension (106 CFU/ml) for 12 hours. The naked-negative group (n=6) was treated with naked surface implants without exposure to bacterial suspension, as a negative control for infection. The bare positive group (n = 6) was treated with a bare surface implant exposed to bacteria as a positive control for infection. The SHP group (n = 6) was treated with bacterially exposed SHP implants. Finally, the LOIS group was treated with bacterial-exposed LOIS implants (n = 6). All animals are kept in a cage, and a lot of food and water are provided. Before the operation, the rabbits were fasted for 12 hours. The animals were anesthetized by intramuscular injection of xylazine (5mg/kg) and intravenous injection of paclitaxel (3mg/kg) for induction. After that, deliver 2% isoflurane and 50% to 70% medical oxygen (flow rate 2 L/min) through the respiratory system to maintain anesthesia. It is implanted through a direct approach to the lateral femur. After hair removal and povidone-iodine disinfection of the skin, an incision about 6 cm long was made on the outside of the left middle femur. By opening the gap between the muscles covering the femur, the femur is fully exposed. Place the plate in front of the femoral shaft and fix it with four screws. After fixation, use a saw blade (1 mm thick) to artificially create a fracture in the area between the second hole and the fourth hole. At the end of the operation, the wound was washed with saline and closed with sutures. Each rabbit was injected subcutaneously with enrofloxacin (5 mg/kg) diluted one-third in saline. Postoperative X-rays of the femur were taken in all animals (0, 7, 14, 21, 28, and 42 days) to confirm the osteotomy of the bone. After deep anesthesia, all animals were killed by intravenous KCl (2 mmol/kg) on ​​28 and 42 days. After execution, the femur was scanned by micro-CT to observe and compare the bone healing process and new bone formation between the four groups.
After execution, the soft tissues that were in direct contact with the orthopedic implants were collected. The tissue was fixed in 10% neutral buffered formalin overnight and then dehydrated in EtOH. The dehydrated tissue was embedded in paraffin and sectioned at a thickness of 40 μm using a microtome (400CS; EXAKT, Germany). In order to visualize the infection, H&E staining and MT staining were performed. In order to check the host response, the sectioned tissue was incubated with rabbit anti-TNF-α primary antibody (AB6671, Abcam, USA) and rabbit anti-IL-6 (AB6672; Abcam, USA), and then treated with horseradish. Oxidase. Apply the avidin-biotin complex (ABC) staining system to the sections according to the manufacturer’s instructions. In order to appear as a brown reaction product, 3,3-diaminobenzidine was used in all parts. A digital slide scanner (Pannoramic 250 Flash III, 3DHISTECH, Hungary) was used to visualize all slices, and at least four substrates in each group were analyzed by ImageJ software.
X-ray images were taken in all animals after surgery and every week to monitor fracture healing (n=6 per group). After execution, high-resolution micro-CT was used to calculate the formation of callus around the femur after healing. The obtained femur was cleaned, fixed in 4% paraformaldehyde for 3 days, and dehydrated in 75% ethanol. The dehydrated bones were then scanned by using micro-CT (SkyScan 1173, Brooke Micro-CT, Kandy, Belgium) to generate 3D voxel images (2240×2240 pixels) of the bone sample. Use 1.0 mm Al filter to reduce signal noise and apply high resolution to all scans (E = 133 kVp, I = 60 μA, integration time = 500 ms). Nrecon software (version 1.6.9.8, Bruker microCT, Kontich, Belgium) was used to generate a 3D volume of the scanned sample from the acquired 2D lateral projection. For analysis, the 3D reconstructed image is divided into 10mm×10mm×10mm cubes according to the fracture site. Calculate the callus outside the cortical bone. DataViewer (version 1.5.1.2; Bruker microCT, Kontich, Belgium) software was used to digitally redirect the scanned bone volume, and CT-Analyzer (version 1.14.4.1; Bruker microCT, Kontich, Belgium) software was used for analysis. The relative x-ray absorption coefficients in mature bone and callus are distinguished by their density, and then the volume of callus is quantified (n = 4). In order to confirm that the biocompatibility of LOIS does not delay the bone healing process, additional X-ray and micro-CT analysis were performed in two rabbits: the naked-negative and LOIS groups. Both groups were executed in the 6th week.
The femurs from sacrificed animals were collected and fixed in 4% paraformaldehyde for 3 days. The orthopedic implant is then carefully removed from the femur. The femur was decalcified for 21 days by using 0.5 M EDTA (EC-900, National Diagnostics Corporation). Then the decalcified femur was immersed in EtOH to make it dehydrated. The dehydrated femur was removed in xylene and embedded in paraffin. Then the sample was sliced ​​with an automatic rotary microtome (Leica RM2255, Leica Biosystems, Germany) with a thickness of 3 μm. For TRAP staining (F6760, Sigma-Aldrich, Germany), the sectioned samples were deparaffinized, rehydrated and incubated in TRAP reagent at 37°C for 1 hour. Images were acquired using a slide scanner (Pannoramic 250 Flash III, 3DHISTECH, Hungary) and quantified by measuring the area coverage of the stained area. In each experiment, at least four substrates in each group were analyzed by ImageJ software.
Statistical significance analysis was performed by using GraphPad Prism (GraphPad Software Inc., USA). Unpaired t-test and one-way analysis of variance (ANOVA) were used to test the differences between the evaluation groups. The significance level is indicated in the figure as follows: *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001; NS, no significant difference.
For supplementary materials for this article, please see http://advances.sciencemag.org/cgi/content/full/6/44/eabb0025/DC1
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Choe Kyung Min, Oh Young Jang, Park Jun Joon, Lee Jin Hyuk, Kim Hyun Cheol, Lee Kyung Moon, Lee Chang Kyu, Lee Yeon Taek, Lee Sun-uck, Jeong Morui
The antibacterial and immune escape coatings of orthopedic implants can reduce infections and immune responses caused by infections.
Choe Kyung Min, Oh Young Jang, Park Jun Joon, Lee Jin Hyuk, Kim Hyun Cheol, Lee Kyung Moon, Lee Chang Kyu, Lee Yeon Taek, Lee Sun-uck, Jeong Morui
The antibacterial and immune escape coatings of orthopedic implants can reduce infections and immune responses caused by infections.
©2021 American Association for the Advancement of Science. all rights reserved. AAAS is a partner of HINARI, AGORA, OARE, CHORUS, CLOCKSS, CrossRef and COUNTER. ScienceAdvances ISSN 2375-2548.