Disclosed are micro-/nano-structured anti-biofilm surfaces, methods of making anti-biofilm surfaces, methods of reducing bacterial and fungal growth, and dental appliances having micro-/nano-structured anti-biofilm surfaces.
Pathogenic bacteria has long been and continues to be a threat to public health as it may cause morbidity and mortality. In recent years, the prevalence of multidrug-resistant bacteria has become a serious challenge in the clinical area. The trace of multidrug-resistant bacteria contamination can be easily found on inanimate surfaces and equipment in the intensive care unit (ICU) and surgery ward. In fact, medical equipment and high-contact communal surfaces such as a computer keyboard, curtains, entry doors and floors are the incubation sites for pathogenic biofilm formation. Both Gram-positive and Gram-negative bacteria can remain alive for months under humid and low temperature conditions.
Moreover, cross-transmission of bacteria from inanimate surfaces may play a significant role for ICU-acquired colonization and infections. Traditionally, chemical-based disinfection is used to remove the bacteria on various surfaces. However, regular cleaning with chlorine solution may not completely remove the multidrug-resistant bacteria containing a biofilm on dry surfaces. Furthermore, the chemical residue from the cleaning may be harmful to patients and the effectiveness does not last for a long period of time. Nanomaterials such as silver nanoparticles, silicon nanowires or carbon nanotubes have been proposed for antibacterial and biomedical applications, but the toxicity of these nanomaterials is a concern requiring further evaluation.
Recently, mechanobiological influence of micro/nanostructures on cells and bacteria has attracted much attention. Nanostructured surfaces have been proven effective to inhibit the bacteria growth on surfaces. The main mechanism is based on a biophysical bactericide model in which bacteria are neutralized by the mechanical puncturing and rupturing without using any chemical agent. Disinfectant-free bactericidal processes are favorable for clinical applications because they can reduce the risk of chemical residue contamination. However, most of the potential bactericidal nanostructures are fabricated on solid substrates such as silicon, titanium and aluminum because of the advances in photovoltaic devices in the past decades.
The following presents a simplified summary of the invention in order to provide a basic understanding of some aspects of the invention. This summary is not an extensive overview of the invention. It is intended to neither identify key or critical elements of the invention nor delineate the scope of the invention. Rather, the sole purpose of this summary is to present some concepts of the invention in a simplified form as a prelude to the more detailed description that is presented hereinafter.
One aspect of the invention relates to a dental appliance having antimicrobial properties, the dental device comprising nanostructures on at least a portion of a surface thereof, the nanostructures having a pyramid-like shape with a periodicity from to 0.25 μm to 5 μm, a height from 0.25 μm to 5 μm, and an aspect ratio from 0.5 to 5.
Another aspect of the invention relates to a method of inhibiting the formation of a biofilm on a medical device involving applying nanostructures on at least a portion of a surface of the medical device, the nanostructures having a pyramid-like shape with a periodicity from to 0.25 μm to 5 μm, a height from 0.25 μm to 5 μm, and an aspect ratio from 0.5 to 5.
Yet another aspect of the invention relates to a method of inhibiting the formation of a biofilm on a dental appliance involving applying nanostructures on at least a portion of a surface of the medical device, the nanostructures having a pyramid-like shape with a periodicity from to 0.25 μm to 5 μm, a height from 0.25 μm to 5 μm, and an aspect ratio from 0.5 to 5.
Still yet another aspect of the invention relates to a method of inhibiting the growth of bacteria and/or fungi on a dental appliance involving applying nanostructures on at least a portion of a surface of the medical device, the nanostructures having a pyramid-like shape with a periodicity from to 0.25 μm to 5 μm, a height from 0.25 μm to 5 μm, and an aspect ratio from 0.5 to 5.
To the accomplishment of the foregoing and related ends, the invention comprises the features hereinafter fully described and particularly pointed out in the claims. The following description and the annexed drawings set forth in detail certain illustrative aspects and implementations of the invention. These are indicative, however, of but a few of the various ways in which the principles of the invention may be employed. Other objects, advantages and novel features of the invention will become apparent from the following detailed description of the invention when considered in conjunction with the drawings.
It has been found that is difficult to apply bactericidal and/or fungicidal nanostructures to existing inanimate surfaces and equipment. In this regard, polymeric nanostructured films are promising alternative because the films can be readily attached to any surface as protection films of window. However, current reports on using polymer nanostructures for bactericide are limited by the small size of the nanostructured film, primarily fabricated with a biotemplating method, in which the polymeric nanostructured surfaces are obtained from the replicating the nanostructures from biospecies bodies, such as gecko skin. The small size of the animal's body surface limits the size of the replicated film. Furthermore, the variations on nanostructure geometry in the biospecies bodies hinder the development of large-scale process because of the high-cost for body sampling and complicated replication process.
In this disclosure, described herein is a facile molding process to fabricate flexible polystyrene (PS) antibacterial films with three-dimensional (3D) nanopyramid arrays on a surface. The geometry of the nanopyramid, i.e., their aspect ratio and pitch, can be precisely controlled by tuning the structure of the inverse nanopyramid template in the herein described fabrication process. The antibacterial film can be easily attached to any surface, such as curtains, walls in clinical wards, medical instruments, medical appliances, etc., to prevent the pathogenic bacteria from forming biofilm.
The fabrication method can be further developed into production scale process and the low cost feature of polystyrene enables large scale utilization in clinical applications. Particularly, the antibacterial effectiveness on Gram-negative bacterium Escherichia coli (E. coli) has been evaluated using scanning electron microscope (SEM) and confocal laser scanning microscope (CLSM). In this context, more than 90% reduction of E. coli colonization has been identified. The effectiveness can be maintained up to 168 hours without cleaning.
Furthermore, by tuning the aspect ratio of inverse nanopyramid nanostructures, systematic investigation of nanopyramid geometry on bacteriacidal performance has been performed. It has been found that the sharper pyramid surface has a better antibacterial effect in the initial stage, but the prolonged antibacterial effect is not as effective as less sharp nanopyramid surface. This disclosure helps to describe the mechanism of biophysical bactericidal effect with nanostructures.
The nanostructures have a pyramid-like shape that contributes to the antimicrobial properties. In this context, pyramid-like shape means that that the top portion is always narrower than the bottom portion of the structure. Examples of pyramid-like shapes not only include three sided pyramids, four sided pyramids, five sided pyramids, six sided pyramids, and so on, but also thorn-like shapes, spinule-like shapes, cone-shapes, and the like. The pyramid-like shape can be symmetrical or asymmetrical.
The nanostructures have a periodicity that contributes to the antimicrobial properties. In one embodiment, the nanostructures have a periodicity from to 0.25 μm to 5 μm. In another embodiment, the nanostructures have a periodicity from to 0.5 μm to 4 μm. In yet another embodiment, the nanostructures have a periodicity from to 1 μm to 3 μm.
The nanostructures have a spacing that contributes to the antimicrobial properties (that is, the distance from the base of one nanostructure to the base of an adjacent nanostructure). In one embodiment, the nanostructures have a spacing 100 nm to 5,000 nm. In another embodiment, the nanostructures have a spacing 200 nm to 4,000 nm. In yet another embodiment, the nanostructures have a spacing 300 nm to 2,500 nm.
The nanostructures have a height that contributes to the antimicrobial properties. In one embodiment, the nanostructures have a height from 0.25 μm to 5 μm. In another embodiment, the nanostructures have a height from 0.5 μm to 4 μm. In yet another embodiment, the nanostructures have a height from 1 μm to 3 μm.
The nanostructures have a bottom width that contributes to the antimicrobial properties. In one embodiment, the nanostructures have a bottom width from 250 nm to 5,000 nm. In another embodiment, the nanostructures have a bottom width from 500 nm to 4,000 nm. In yet another embodiment, the nanostructures have a bottom width from 750 nm to 2,500 nm. It is noted that the top portion is always narrower than the bottom portion of the pyramid-like nanostructures.
The nanostructures have an aspect ratio that contributes to the antimicrobial properties. In one embodiment, the nanostructures have an aspect ratio from 0.5 to 5. In another embodiment, the nanostructures have an aspect ratio from 0.75 to 4. In yet another embodiment, the nanostructures have an aspect ratio from 1 to 3.
It is noted that the molds for making the nanostructures have the same structure, periodicity, spacing, height, bottom width, and aspect ratio as the nanostructures as described above.
Antimicrobial properties encompass the ability to kill microbes and/or inhibit the growth/reproduction/spread of microbes and/or reduce the growth/reproduction/spread of microbes. Microbes include bacteria and fungi, such as yeasts. Non-limiting examples of fungi genera include Candida, Cladosporium, Aureobasidium, Saccharomycetales, Aspergillus, Fusarium, and Cryptococcus. Non-limiting examples of fungi include Candida albicans, Candida parapsilosis, Candida tropicalis, Candida glabrata, Candida krusei, Candida guilliermondii, and Candida lusitaniae, Histoplasma capsulatum, Cryptococcus neoformans, Cryptococcus gattii, Aspergillus fumigatus, Coccidioides immitis, and Coccidioides posadasii. Non-limiting examples of bacteria genera include Actinomyces, Arachnia, Bacteroides, Bifidobacterium, Eubacterium, Fusobacterium, Gemella, Granulicatella, Lactobacillus, Leptotrichia, Peptococcus, Peptostreptococcus, Propionibacterium, Selenomonas, Streptococcus, Treponema, and Veillonella. Non-limiting examples of bacteria include Escherichia coli, Actinobacillus actinomycetemcomitans, Streptococcus salivarius, Streptococcus mutans, and Streptococcus sanguinis.
Described herein is an effective way to keep the surface of dental appliances clean, to inhibit attachment of microorganisms and physically kill those that manage to attach to the material surface without the need for chemical agents. This application is highly valuable especially for those aged individuals who are denture-wearers and have reduced manual dexterity to maintain an excellent oral hygiene, as it aims to overcome the limitations (namely, lack of anti-biofilm property) of existing appliances made of the un-modified material.
The subject matter herein imparts an anti-biofilm activity to a common polymeric dental material that is used for making dentures. This anti-biofilm and antifungal property are very useful for prevention of denture-induced stomatitis, which condition is caused or aggravated by microorganisms, especially fungi. More specifically, dental compositions described herein are useful for preparing dental appliances and articles that repel or inactivate one or more microbes (especially fungi/yeast) in the oral environment.
The application of biomimetic nanostructures on the dental devices, such as prostheses, is considered as an alternative and effective way to inhibit biofilm formation through mechanical penetration and physical shredding of adherent bacteria on their surfaces. No chemical agent is needed to maintain an excellent antibacterial effect for an extended period time.
Described herein is a relatively simple method of incorporating antimicrobial properties into dental prostheses, without the need to use chemical agents. The nano-textured surface is easy to maintain and keep clean.
The nano-textured surface is highly valuable to the dental clinical applications, especially for dental rehabilitation devices and appliances. As the population of the aged increases, there is a growing demand on high quality dental appliances such as removable partial and complete dentures. Despite the sophistication in design and advances in material science, polymeric dental appliances remain as one of the mainstream solutions to replace missing teeth and oral structures, to restore function and appearance for patients. However, these appliances are not immune from the formation of bacteria-laden biofilm on their surfaces. Patients may then suffer from mucosal diseases due to the aggregation of biofilm on the surface of the protheses. Currently, antibiotics or chemical antimicrobial agents are used to manage the mucosal condition or to prevent bacterial growth on the prostheses, but such practice may lead to more serious health problems such as antibiotic resistance and body side effects. The application of biomimetic surface nanostructures as described herein is considered as an effective way to inhibit biofilm formation; as no chemical agent is needed.
Described herein are anti-bacterial, nano-structural surfaces especially suitable for dental clinical applications, among other medical applications. The surfaces can maintain an excellent antibacterial effect and that antimicrobial activity can be refreshed by a simple procedure. Dental prostheses with such nano-textured surfaces can benefit many elderly individuals who may be lacking the necessary manual dexterity to maintain the cleanliness of the dental appliances in time.
Described herein are the optimal nanostructures that can effectively inhibit the growth of biofilm for dental clinical applications. On one hand, the surfaces can be readily applied to common dental polymeric materials (polymethyl methacrylate, in particular) and, hence, be used in clinical application as described above. On another hand, the methods herein can apply the principle to identify appropriate dental materials including plastics and metals which are suitable for building nanostructured, biocompatible and anti-bacterial surfaces on top.
Common dental devices that are made of polymethyl methacrylate, such as complete or partial dentures, and removable orthodontic retainers, but other materials can be used. Also described herein is the application to restorative resin composite material and dental implants with micro-/nano-structures, for a lasting anti-biofilm property to enhance the longevity of these treatment modalities.
The micro-/nano-structures can be fabricated on different dental materials with a flat surface. Nevertheless, the majority of dental prostheses can have undulated or curved surfaces, and the micro-/nano-structures can be fabricated thereon as well.
The following examples illustrate the subject invention. Unless otherwise indicated in the following examples and elsewhere in the specification and claims, all parts and percentages are by weight, all temperatures are in degrees Centigrade, and pressure is at or near atmospheric pressure.
Fabrication of i-Pyramid Template with Different Aspect Ratio
A clean <100> oriented Si wafer with 100 nm thermally grown silicon oxide on the surface was spin-coated with a photoresist AZ7908 and patterned with photolithography to obtain a regular square array of pits with a pitch of 1.5 μm.
Then, the patterned wafer was etched with benzoxazolinone (BOA) to remove the exposed Si oxide layer, which functions as an etching mask in the subsequent i-pyramid formation. After removing the photoresist in acetone, the wafer was then put into the 15% tetramethylammonium hydroxide (TMAH) solution at 50° C. for 50 mins. The highly regular array of inverted pyramids (i-pyramids) was formed by anisotropic etching of the patterned Si wafer. Finally, 400 nm, 800 nm or 1200 nm of chromium were sputtered to wafers respectively to adjust the aspect ratio of the i-pyramids using Nano-master NSC3000 Sputtering System (SPT-NSC3000).
Fabrication of Nanopyramid Films
Polystyrene (PS) with average molecular weight of around 192,000 was purchased from Sigma Aldrich. The material (10 g) was dissolved in 100 mL of toluene to obtain a PS solution. Then, the solution was poured onto the i-pyramid wafer. The PS solution was first heat cured at 90° C. for 3 hours and then was held at 120° C. for the next 30 mins. After that, the PS nanopyramid film was readily peeled off from the i-pyramid wafer, owing to the anti-sticking property of chromium. Groups of specimens, Nanopyramid type A (NPA), Nanopyramid type B (NPB) and Nanopyramid type C (NPC), are obtained corresponding to the nanopyramid films obtained from templates coated with 400 nm, 800 nm or 1200 nm of chromium respectively.
Characterization and Bacterial Cell Viability Analysis
SEM images were taken by a JEOL JSM-7100F SEM operated at 10 kV. Water contact angle was measured using USA KINO contact angle meter SL200 KB with water droplet volume of 5 μL. For the bacterial cell viability analysis, confocal laser scanning microscopy (CLSM) was taken to visualize the relative proportion of live and dead cells on the nanopyramid surface after staining with the LIVE/DEAD BacLight Bacterial Viability Kit (L-7012 Invitrogen, Molecular Probes, Eugene, Oreg., USA) according to the manufacturer's protocol. This proprietary staining kit contains a mixture of SYTO 9 and propidium iodide fluorescent dyes that make live bacteria show up in green and dead bacteria in red color. Nine randomly assigned regions of each specimen with field measured 200 μm×200 μm were imaged using a CLSM (IX81 FluoView FV1000, Olympus, Tokyo, Japan). All CLSM images were imported into the computer and the amount of live and dead bacterial cells on the surfaces were determined using the image analysis software (ImageJ, National Institute of Health, Bethesda, Md., USA).
Referring to
With the process described above, the nanopyramid PS films obtained have a number of the distinct features that makes them attractive as antibacterial surface.
b-d showed the SEM images of the NPA, NPB and NPC PS film. The positive nanopyramids with a pitch of 1.5 μm was highly ordered and showed poor wetting (very high contact angle) by water. The highly ordered and tunable surface structures also provide an excellent and versatile platform to investigate interactions, at a small scale, between various nanotopography-geometry combinations and bacteria. There was wide-ranging selectivity for different morphologies provided by varying thickness of Cr sputtered coating on the Si mold, which is an effective means to control the aspect ratio of the nanoscale structures. Therefore, by modulating the Cr thickness, the relief of the structures could be altered with increasing protuberance sharpness. Water contact angle is one of the key factors that determines the bacterial adhesion on a surface. Typically, a high water contact angle suggests a low surface energy. The lower the surface energy, the more difficult for bacteria adhere to the surface. Therefore, water contact angle is used to compare surfaces for their antibacterial potential.
To verify the antibacterial effect of the nano-patterned surface, the three groups of molded PS films together with a smooth (non-textured control) sample were placed in an Escherichia coli (E. coli) suspension of concentration 1×109 cells/mL. Four incubation times (1 hour, 24 hours, 72 hours and 168 hours) were examined in such aqueous environment.
Furthermore, the regular nanostructure arrays separated individual bacterium, trapping between the protuberances, so that interaction between bacteria was significantly disturbed. For surfaces with the nanostructures with highest aspect ratio (e.g. on NPC PS film (
Confocal laser scanning microscopy (CLSM) was used to quantify the effectiveness of bacteria annhilation on the different surfaces after 1, 24, 72 and 168 hours E. coli incubation. A mixture of SYTO 9 and propidium iodide stains were used as the fluorescent dyes to visualize the live and dead E. coli cells in CLSM. Cells with intact cell membranes staining green are considered to be viable while cells with damaged membranes staining red are considered to be non-viable. For the planar (control) sample, it could be clearly seen that the density of live E. coli cells increased with time in an exponential manner, and that an overwhelming majority of them remained vital and alive. Apparently, the surface density of cells on the flat surface is significantly greater than that on the nano-patterned surfaces throughout the period of incubation, regardless of type of nanostructures, as shown in
To systematically analyze the bactericidal effect on all three types of nanostructures, namely NPA, NPB and NPC, the result from the confocal laser scanning microscopy has been summarized in
Initially, E. coli cells adhered to the planar, smooth PS surface and then started reproduction process so that the colonization rate increased exponentially in the first 24 hours. However, the life cycle of E. coli came to an end after 24 hours and hence the amount of live E. coli decreased. Compared to the planar control, the three nanostructured samples showed excellent antibacterial performance. The colonization for live E. coli over 168 hours remained at a low level. Most of the specimens showed colonization below 4.0×103 cells/cm2. Generally speaking, the growth and attachment of live E. coli cells on the nanostructured surface was inhibited in the first 72-hour, after which the growth picked up again, but the amount remained very low compared with the control.
One explanation for the presence of some bacteria attaching onto the nanostructured surface might be that the “valleys” of the nanopatterns have been filled up and the nanostructures were flattened by some adherent dead bacteria; and some later arriving cells managed to grow on the flattened surface. Bactericidal effect was demonstrated by the nanostructured surface through the biophysical action (perforating and rupturing of the bacteria) at least in the first 72 hours. For longer periods of incubation, SEM images showed that the dead E. coli cells were plentiful within the gap of the nanostructures (
See
Besides studying the exact number of live and dead E. coli, it is also worthwhile to calculate the proportion of the live E. coli in all adhered bacteria, so that the bactericidal mechanism of adhered bacteria could be studied.
Specifically, NPC reaches its best antibacterial efficiency of 97.7% at 24-hr incubation and then progressively decrease to 90.7% at 168-hr incubation time. NPA and NPB showed a more similar trend that they reached the peak at 72-hr incubation to around 96% and decreased to around 94% at 168-hr incubation but NPA started with a lower efficiency of 88.3% while NPB started with 91.6%. The trend could be explained by the progressive weakening of the combined action of anti-adhesion and biophysical bactericidal effect with the incubation time. Both effects were weakened due to the flattening of nanostructures by adhered dead bacteria. The initial increase in the efficiency was mainly contributed by the biophysical bactericidal effect as mentioned previously. The bactericidal effect dramatically dropped afterwards because the nanostructures were covered to form flattened surface. The performance was expected to further decrease after 168-hr use. After all, the nanopyramid films definitely showed an excellent antibacterial performance compared to the planar surface.
Notice that the i-pyramids became sharper with a higher aspect ratio (
Corresponding to the features of i-pyramids for each Cr mold, the surface feature of NP400 was low-rise and pyramid-like (
The mean Ra values of the nanoscale pyramidal surfaces ranged from 43.7±2.7 nm (for N1), 53.1±8.8 nm (for N2) to 108.0±13.4 nm (for N3), all of which were statistically different from the control (15.9±2.9 nm for the control) (
Referring to
Materials:
S mutans
S sangunis
C albicans
In summary, described herein is a facile process to fabricate large-scale, flexible nanostructured films with regular nano-engineered templates. It has been proved that these films possess antibacterial effect and can inhibit the growth of biofilm on their surfaces. The antibacterial mechanism and performance were quantitatively examined by SEM and CLSM analysis. These nanostructured surfaces showed excellent and effective bactericidal performance with >90% reduction of E. coli colonization on the surface, compared with the control flat sample. Moreover, that effectiveness can be maintained up to 168 hours without cleaning. The reported nano-patterned films can be applied in clinical applications to reduce the risk of pathogenic infection.
With respect to any figure or numerical range for a given characteristic, a figure or a parameter from one range may be combined with another figure or a parameter from a different range for the same characteristic to generate a numerical range.
Other than in the operating examples, or where otherwise indicated, all numbers, values and/or expressions referring to quantities of ingredients, reaction conditions, etc., used in the specification and claims are to be understood as modified in all instances by the term “about.”
While the invention is explained in relation to certain embodiments, it is to be understood that various modifications thereof will become apparent to those skilled in the art upon reading the specification. Therefore, it is to be understood that the invention disclosed herein is intended to cover such modifications as fall within the scope of the appended claims.
Filing Document | Filing Date | Country | Kind |
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PCT/CN2020/084693 | 4/14/2020 | WO |
Number | Date | Country | |
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62833871 | Apr 2019 | US |