Antibacterial and Cytocompatible Nanoengineered Silk-Based

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Biological and Medical Applications of Materials and Interfaces

Antibacterial and cytocompatible nanoengineered silk-based materials for orthopedic implants and tissue engineering Babak Mehrjou, Shi Mo, Dorsa Dehghan-Baniani, Guomin Wang, Abdul Mateen Qasim, and Paul K Chu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b09066 • Publication Date (Web): 06 Aug 2019 Downloaded from pubs.acs.org on August 7, 2019

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ACS Applied Materials & Interfaces

Antibacterial and cytocompatible nanoengineered silk-based materials for orthopedic implants and tissue engineering Babak Mehrjou1, Shi Mo1, Dorsa Dehghan-Baniani2, Guomin Wang1, *, Abdul Mateen Qasim1, Paul K. Chu1, * 1

Department of Physics, Department of Materials Science and Engineering, and Department of Biomedical Engineering, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong

2

Division of Biomedical Engineering, Department of Chemical and Biological Engineering, The Hong Kong University of Science and Technology, Hong Kong *

Corresponding authors: [email protected] (P.K. Chu); [email protected] (G.M. Wang)

Abstract: Many post-surgical complications stem from bacteria colony formation on the surface of implants but usage of antibiotic agents may cause antimicrobial resistance (AMR). Therefore, there is a strong demand for biocompatible materials with intrinsic antibacterial resistance not requiring extraneous chemical agents. In this study, homogeneous nanocones were fabricated by oxygen plasma etching on the surface of natural, biocompatible Bombyx-mori silk films. The new hydroxyl bonds formed on the surface of the nanopatterned film by plasma etching increased the surface energy by around 176%. This hydrophilic nanostructure reduced bacterial attachment by more than 90% for both Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria and at the same time, proliferation of osteoblast cells was improved by 30%. The nanoengineered substrate 1 ACS Paragon Plus Environment

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and pristine silk were cultured for 6 h with three different bacteria concentrations of 107, 105, and 103 CFU mL-1 and cell proliferation on the nanopatterned samples was significantly higher due to limited bacteria attachment and prevention of biofilm formation. The concept and materials described here reveal a promising alternative to produce biomaterials with inherent biocompatibility and bacterial resistance simultaneously to mitigate post-surgical infections and minimize the use of antibiotics.

Keywords: Antibacterial resistance; Silk materials; Oxygen plasma etching; Nanocones; Osteoblast cells

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1. Introduction Since biofilm formation on orthopedic implants can cause serious problems and even patient fatality 1–3, there has been much effort to design and fabricate antibacterial biomaterials for tissue engineering applications. Most of the previous attempts have focused on delivery of antibiotics or antibacterial elements such as Ag 4,5, Au 6 and so on 7,8. However, some of these elements in high concentrations produce toxic effects in vivo and overuse of antibiotics can lead to antimicrobial resistance (AMR) 9–11. As bacteria grow swiftly on the suitable surface forming a robust biofilm within 6 h 12–14, it is imperative that implants can repel bacteria. In fact, such structures exist in nature. For example, lotus leaves and dragonfly wings repel bacteria efficiently

1,15,16.

The

mechanism is that the nanocolumns in these structures decrease the contact area between the bacteria and substrate and increase the pressure on the bacteria cell wall causing repulsion, cell wall rupturing, and even death of the bacteria

17–20.

Consequently, many types of bioinspired

structures have been designed to mimic these natural materials 21 by reducing the surface energy to form hydrophobic or superhydrophobic surfaces

12,22–27.

plasma etching have been used to prepare nanostructures

Although colloidal lithography and 28,29,

the combined use of these two

methods to modify natural materials such as silk to form the desirable nanostructures with the ability to kill and repel bacteria without the need of external chemical and biological reagents has seldom been reported. Titanium-based orthopedic implants are very common

30–32

but since they are not

biodegradable, a second surgery is often required for removal after tissue healing thus increasing the chance of infection. Therefore, bioresorbable and biodegradable materials such as synthetic and natural polymers have attracted attention in tissue engineering

33–35

but unfortunately, most

polymers have poor mechanical strength. In this respect, Bombyx-mori silk possesses high 3 ACS Paragon Plus Environment


strength and is an interesting candidate for bone implants 36–38. The potential application of silk to biocarriers 39, bone implants 40, and so on 41–43 has been studied and elements like gold and silver 44,45,

antibiotics such as gentamycin, 46 and quaternary ammonium compounds (QAC) 47 have been

proposed to improve the antibacterial properties of silk. However, most of these processes require external chemicals and agents and to the best of our knowledge, there have been few attempts to improve the mechano-antimicrobial properties of silk without the use of extraneous chemicals. Considering the potential deleterious effects of chemicals, materials with inherent antibacterial ability and biocompatibility are highly desirable 48 but there are considerable challenges to produce biomaterials which possess the proper antibacterial properties, biocompatibility, biodegradability, and mechanical properties similar to those of biological tissues 49,50. In this work, hydrophilic and close-packed hexagonal arrays of nanocones are fabricated in silk samples by plasma etching to reduce bacteria attachment (both Gram-negative and Grampositive) and promote proliferation of osteoblasts simultaneously without using any chemical agents (Scheme 1). The nanopatterned arrays are biocompatible with hAMSCs (human adipose mesenchymal stem cells) and MC3T3 osteoblast cells. Co-culturing of bacteria and MC3T3 cells together shows that MC3T3 cells can grow and proliferate better on the bacteria-infected nanopatterned samples than the control one due to the notable reduction in bacteria attachment in the mimicked in vivo environment.


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Scheme 1. Schematic presentations: (a) Colloidal lithography and oxygen plasma etching producing close-packed hexagonal arrays of nanocones; (b) Co-culturing of MC3T3 cells and bacteria (E. coli and S. aureus) on the nanopatterned and pristine silk samples showing bacteria repulsion and cell proliferation on the nanoengineered sample and biofilm formation on the control sample.

2. Materials and methods 2.1.

Fabrication of nanopatterned silk films The silkworm (Bombix mori) cocoons (5 g) were boiled in 2 L of water containing 0.02 M

sodium carbonate (Na2CO3) for 30 mins. The degummed silk was rinsed with ultrapure water 3 times and dissolved in a 9.3 M solution of LiBr, followed by dialysis against ultrapure water for 48 h. The solution was centrifuged 2 times and 20 min each at 10,000 rpm and 4 ºC to remove impurities. 20 ml of the solution (concentration of 6 wt.%) were poured onto a 10 mm glass petri dish and dried under the hood for 48 h. The film with a thickness of about 100 µm was cut into 1×1 cm2 pieces and crosslinked by a methanol treatment for 1 h. They were then coated with 300 5 ACS Paragon Plus Environment


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nm polystyrene by colloidal lithography as described in the supporting information

51.

A

magnetron sputtering instrument (Phase II J, ATC Orion Sputtering System, AJA International Inc., USA) was used to etch the polystyrene nanospheres with an oxygen plasma for 20 minutes (7 mTorr and 50 W). 2.2. Characterization The surface morphology of the samples was characterized by atomic force microscopy (AFM, Veeco’s MultimodeV, Veeco, USA) and scanning electron microscopy (SEM, JSM 7001F, JEOL, Japan). To determine the chemical structure and states, attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR, Spectrum Two, PerkinElmer, USA)) and X-ray photoelectron spectroscopy (XPS, K-Alpha, Thermo Fischer Scientific, USA) were performed. The water contact angles before and after oxygen plasma etching were measured on the contact angle goniometer (Model 200-00-115, rame-hart instrument co., USA) and the surface energy was determined by the DROPimage standard software (Harmonic mode) and Owens-Wendt method 52.

The surface energy is composed of two dispersion (𝛾𝑑𝑠𝑣) and polar (𝛾𝑝𝑠𝑣) bonding forces as

shown in the following:

𝛾𝑑𝑊𝑎𝑡𝑒𝑟 𝛾𝑑𝑠𝑣 + 𝛾𝑝𝑊𝑎𝑡𝑒𝑟 𝛾𝑝𝑠𝑣 =

𝛾𝑑𝐷 ― 𝑀 𝛾𝑑𝑠𝑣 + 𝛾𝑝𝐷 ― 𝑀 𝛾𝑝𝑠𝑣 =

(1 + cos (𝜃𝑊𝑎𝑡𝑒𝑟))𝛾𝑊𝑎𝑡𝑒𝑟 2

(1 + cos (𝜃𝐷 ― 𝑀))𝛾𝐷 ― 𝑀 2

𝐸𝑞. 1

𝐸𝑞. 2

The surface energies of water and diiodomethane and the relative components were taken from the literature

52

as follows: 𝛾𝑊𝑎𝑡𝑒𝑟 = 72.8 𝑚𝑁/𝑚, 𝛾𝐷 ― 𝑀 = 50.8 𝑚𝑁/𝑚, 𝛾𝑑𝑊𝑎𝑡𝑒𝑟 = 21.8 𝑚𝑁/𝑚,

𝛾𝑝𝑊𝑎𝑡𝑒𝑟 = 51 𝑚𝑁/𝑚, 𝛾𝑑𝐷 ― 𝑀 = 49.5 𝑚𝑁/𝑚 and 𝛾𝑝𝐷 ― 𝑀 = 1.3 𝑚𝑁/𝑚.


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2.3.

Bacteria studies

2.3.1.

Adhesion tests

The nanopatterned and untreated silk samples were tested. For sterilization, 3 samples of each group were immersed for 20 min in 75% ethanol and dried in a biosafety cabinet. Two bacteria strains, Staphylococcus aureus (ATCC 29213) and Escherichia coli (ATCC 25922), were used in the assessment. The bacteria were cultured in the Lysogeny broth (LB) medium in an incubator under agitation at 220 rpm at 37 ºC overnight. The optical density (OD) of the bacteria was tuned at 0.1 (OD600 = 0.1) by adding adequate fresh medium and reactivated for another 3 h. To evaluate bacteria adhesion, the bacteria concentration was reduced to 2-3×105 CFU mL-1 and 500 µL of the solution were spread on the surface of each sample to mimic immersion. At time points of 6 and 24 h, the culture medium was removed. The samples were taken out, fixed with 2.5% glutaraldehyde overnight, washed successively with 10, 30, 50, 75 and 96% ethanol, and dried at room temperature before observation by SEM. 2.3.2. Bacteria live/dead staining In the bacteria live/dead staining test, each sample was washed twice with PBS for 5 min and stained with the live/dead backlight bacterial viability kit (L7007, Invitrogen, USA) for 15 min in darkness at room temperature. To reduce the background, the samples were washed 2-3 times at 37 ºC with PBS before examination under an inverted fluorescent microscope (Axio Observer Z1 Inverted Phase Contrast Fluorescence Microscope, Zeiss, Germany) using 420-480 nm and 480550 nm as the excitation wavelengths and 520-580 nm and 590-800 nm as the emission wavelengths for green and red fluorescent signals, respectively.



2.4.

Cell culturing

2.4.1. Osteoblasts and hAMSCs cells The osteoblast cell line (MC3T3-E1, Cell bank of the Chinese Academy of Sciences) and hAMSCs (Cat. No. PCS-00-011, ATCC) were used. The MC3T3 cells were cultured in a medium consisting of 90% Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Life Technologies Corporation, USA) and 10% fetal bovine serum (FBS, Gibco, Life Technologies Corporation, USA) and the hAMSCs were cultured in the basal growth medium (Cat. No. PCS-500-030, ATCC). Both types of cells were cultivated at 37 ºC in an incubator under 95% air and 5% CO2 at a relative humidity of about 90%. The cells were detached from the petri dish by Trypsin (Gibco, Life Technologies Corporation, USA) and 100 µL of the cell suspension with concentrations of about 25,000 MC3T3 mL-1 and 10,000 hAMSC mL-1 were introduced to each sample and immersed in 1 mL of the DMEM medium on a 24 well plate. The cell culture medium was changed every 2 days and all the experiments were performed in triplicate. 2.4.2. MTT, live/dead staining, and Cytoskeleton assays Cell proliferation was assessed quantitatively with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) for 3 samples of each group. After culturing for 1, 4, and 7 d, the medium was replaced by the MTT solution. The cells reacted with the MTT solution for 4 h and dimethyl sulphoxide (DMSO) was introduced to dissolve the formazan crystals. After 30 min, 100 µL of the solution were transferred to a 96 well plate and the optical density was measured on the multimode reader (BioTek, USA) at 570 nm. Besides the nanopatterned and pristine silk samples, pure DMSO was used as a negative control in the MTT assay. The measurements were performed in triplicate and the average values were used in the data analysis. In the live/dead staining analysis, propidium iodide and fluorescein diacetate (10 µg mL-1) 8 ACS Paragon Plus Environment

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were added to the culture medium (1:1000) and incubated with the cells for 30 min. The medium was removed and the cells were washed with PBS prior to observation under the fluorescent microscope. In the cytoskeleton assay, the samples were washed 2 times with PBS and fixed with 3.7% formaldehyde in PBS for 5 minutes. Before staining with the Phalloidin solution (PhalloidinFITC, catalog No. P5282, Sigma Aldrich, USA), the cells were permeabilized with 0.1% Triton X-100 (Sigma Aldrich, USA) in PBS for 5 min and washed with PBS. Afterwards, the cells were stained with 50 µg mL-1 fluorescent Phalloidin-FITC in PBS for 40 min at room temperature in the dark. To stain the cell nuclei, the DAPI solution (D9542, Sigma Aldrich, USA) was incubated with the cells for 5 min. Each step mentioned above was carried out twice and unbonded reagents were extracted by rinsing with PBS for 5 minutes before examination by inverted fluorescent microscopy. 2.4.3. MC3T3/Bacteria co-culturing The silk-based substrates (control and nanopatterned) were cultured with 50 µL of E. coli and S. aureus separately with 3 different bacteria concentrations of 107, 105 and 103 CFU mL-1 for 6 h. They were washed with PBS twice to remove unattached bacteria and underwent MC3T3 cell culturing. 100 µL of the MC3T3 cell suspension with a concentration of 2.5×104 cells mL-1 were used for the pre-infected samples and after 24 h, the samples were fixed with 2.5% glutaraldehyde and dehydrated with 10%, 30%, 50%, 75%, and 96% ethanol sequentially before observation by SEM. In live/dead staining of the cells cultured on the pre-infected samples, propidium iodide and fluorescein diacetate for dying cells and L7007 for dying bacteria were added to the DMEM medium with a ratio of 1 to 1000 (1 µL in 1 mL). After removing the culture medium, 1 mL of the dye-containing medium was added to the wells and cultured again for 20 min in darkness. To 9 ACS Paragon Plus Environment


reduce the background, the samples were washed 3 times with warm PBS which also removed unattached cells or bacteria. 2.5.

Statistical analysis All the experiments were done in triplicate and the data were reported as mean ± standard

deviation (SD). The statistical significance of the results was determined by analysis of the variance (ANOVA single factor) and set at P < 0.05 (*) and P < 0.01 (**).


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3. Results and discussion The close-packed hexagonal nanosphere arrays (Fig. 1-a) are used as a mask in oxygen plasma etching. As shown in the SEM image (Fig. 1b), the nanocones are distributed evenly on the surface of the silk film and AFM shows that the height and center-to-center distance of the nanocones are about 400 nm and 250 nm, respectively (Figs. 1c and 1d). The surface roughness Rq increases from 2.48 nm on the pristine silk film to 135 nm on the nanopatterned sample and the surface area of the latter increases by more than 4 times. The surface angles decrease from 60º ± 1 to 10º ± 1 after nanopatterning (Figs. 1e and 1f). In general, if nanofeatures are introduced

12,22,24

, air is

trapped and the wettability diminishes. However, in this study, O2 plasma etching changes the surface chemistry of silk rendering it more hydrophilic as shown in Table 1. The surface energy results calculated by both DROPimage standard software (harmonic state) and Owens-Wendt 52

method

indicate that the surface energy after plasma etching increases from 47±1 mN m-1 to

more than 130±2 mN m-1 revealing that O2 plasma etching produces a nearly superhydrophilic surface.

Table 1. Surface energy and contact angles of water and diiodomethane on the untreated and O2plasma etched silk samples.

Contact angles, θ (degree) Surface energy ( mN m-1 )

Materials Water

Diiodomethane

Untreated silk

60±2

48±2

47±1

O2-plasma etched silk

10±1

0

130±2



a

c

e

b

d

f

Fig. 1. SEM images: (a) Polystyrene particles and (b) Arrays of silk nanocones; (c) AFM image and (d) Line scan of the silk nanocones; Water contact angles: (e) Plasma etched silk and (f) Untreated silk film. The scale bars in the SEM pictures is 500 nm.

Three characteristic bands at 3,000-3,600 cm-1 (amide A, N-H stretching), 1,600-1,700 cm-1 (amide I, C=O stretching), and 1,500-1,600 cm-1 (amide II, C-N stretching) 53,54 are observed from the pristine silk sample as shown in Fig. 2a. The ATR-FTIR spectra before and after plasma 12 ACS Paragon Plus Environment

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treatment disclose notable differences. In zone I (3,000-3,600 cm-1), two peaks related to amide A and O-H stretching overlap with each other but after oxygen plasma etching, the peak in this region becomes a little wider and the intensity decreases. Another important change between 2,800-3,000 cm-1 is attributed to reduction of C-H stretching after plasma etching and the band between 1,350-1,450 cm-1 (zone III) related to C-O bond is different as well. All in all, the surface chemistry of silk changes after O2 plasma etching due to addition of O-H which is responsible for the increased surface energy and hydrophilicity. The chemical change after oxygen plasma etching is shown in Fig. 2b. XPS is performed for further investigation (Fig. 3). Fig. 3a reveals increase in the amount of oxygen from 20.4 to 33.4 wt% and more details are shown by the C 1s and O 1s high-resolution data. C-O-H is related to new bond formation (Figs. 3b and 3c). The peaks at 532.7 eV and 285.7 eV in the O 1s and C 1s spectra of the O2 plasma etched sample are related to C-O-H, whereas the C=O peak of both the O2 plasma etched and pristine silk samples has the same position in the C 1s (530.9 eV) and O 1s (287.3 eV) spectra. As aforementioned, the large increase in the surface area (more than 4 times) after plasma etching and newly formed O-H bonds produce a nearly superhydrophilic state on the nanoengineered surface.



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a

b

Fig. 2. (a) ATR-FTIR spectra of the untreated silk (upper) and O2 plasma etched samples (lower); (b) Proposed bonds changes on the surface of the O2 plasma etched silk film.


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a

c

b

a

a

d

e

b

b a

a

Fig. 3. Full survey XPS spectra: (a) Untreated and O2 plasma etched silk samples; High-resolution spectra of (b) C 1s and (c) O 1s of the untreated silk sample; (d) C 1s and (e) O 1s spectra of the O2 plasma etched silk sample.



Fig. 4.

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Fluorescent images of hAMSCs on (a) Pristine silk and (b) Nanopatterned film;

Cytoskeleton staining of MC3T3 cells on the (c) Pristine silk and (d) Nanopatterned film after culturing for 24 h; (e) MTT results of the pristine silk and oxygen plasma etched films after 1, 4 and 7 days (Scale bar = 500 µm).

The biocompatibility is assessed with hAMSCs and the live/dead staining of hAMSCs results show that both the pristine and nanopatterned samples are biocompatible. 16 ACS Paragon Plus Environment

a

b

Enhanced cell

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attachment and proliferation are observed from the latter (Figs. 4a and 4b). Plasma treatment has been used to enhance the cell response on silk 53,55. Cytoskeleton staining is also done on both the pristine and nanoengineered silk samples to reveal the shape of the cells and as shown in Figs. 4c and 4d, the nuclei are blue and the microtubules and actin filaments of the cells appear green. To evaluate osteogenesis on both samples, the MTT assay was implemented after culturing the MC3T3 cells for 1, 4 and 7 days as shown in Fig. 4e. Although more pressure is applied to the cell walls by the nanocones because of the reduced contact area, as reported previously 12,56, the cells can tolerate the pressure and the patterns are engulfed by them. This is mainly because of the larger cells compared to the nanofeatures. Another reason for better cell proliferation on the nanopatterned samples is the higher surface energy as the superhydrophilic state is more suitable for living organisms to attach and proliferate. The bacterial adhesion and antibacterial behavior on the nanopatterned and pristine silk samples is examined after culturing for 30 min (Figure S1) and 6 and 24 h in the presence of E. coli and S. aureus. Although the bacteria attachment mechanism is quite complicated, it can be divided into two phases: (a) an initial, reversible physical phase and (b) an irreversible, chemical and cellular phase. In the first stage, many factors such as the electrostatic charge, gravity, surface roughness, and surface energy affect the process 57. This stage usually takes 6 h and if bacteria attach to the surface during this time, they can grow rapidly forming a biofilm and spreading to other surfaces 12. The robust biofilm is the primary culprit leading to post-surgical infection. The SEM and fluorescent microscopy images of the bacteria on the nanopatterned and pristine silk samples after 6 and 24 h in Fig. 5 show that bacteria attachment on the nanopatterned silk is reduced by more than 90% for both Gram-negative and Gram-positive bacteria.

On the

nanopatterned samples, bacteria cannot attach to form a biofilm within 6 h and the shape of E. coli 17 ACS Paragon Plus Environment


changes from that of a rod to cocci. Size minimization in a means is to tolerate the harsh environmental conditions and avoid death 58–60. After 6 h, the inset in Figure 5d shows that some individual E. coli bacteria have a round shape indicating that they try to attach to the surface by deflecting the silk nanocones. After 24 h, the attached bacteria spread limitedly (grey area in Fig. 5e) but on the other hand, the control sample (Figs. 5b) is fully covered by E. coli. This difference is quite obvious in the fluorescent images (Figs. 5c and5 f). The S. aureus cells are smaller and have thicker cell walls than E. coli enabling them to attach and proliferate better on a rough surface. Smaller and round S. aureus may be able to find a more suitable place in comparison with rodshaped E. coli. The silk nanocones are flexible under wet conditions and so can deflect the cones and tolerate the pressure more easily with the thick peptidoglycan membrane consequently preventing the cell walls from rupturing. On the untreated silk sample (Fig. 5-h), a thick biofilm is formed by S. aureus after 24 h (denser than that formed by E. coli), but on the nanopatterned sample as shown in Fig. 5k, they attach in a limited way. By comparing E. coli and S. aureus after culturing for 24 h (Figs. 5h, 5k, 5i, and 5l), a larger percentage of the surface is covered by S. aureus but in general, reduction of more than 90% in bacteria attachment is achieved. The fluorescent microscopy images of the control and nanopatterned samples infected by E. coli and S. aureus after 24 h (Figs. 5c, f, I, and l) show that S. aureus attach more on both samples. No dead bacteria are seen since the antibacterial properties of silk nanocones are related to bacteria repulsion and dead bacteria are usually washed away easily during staining and rinsing. The difference in surface coverage is calculated by ImageJ software and shown in Figure S2.


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b

c

d

e

f

g

h

i

j

k

l

Fig. 5. SEM and fluorescent images of E. coli cultured on pristine silk after 6 and 24 h (a-c, respectively); SEM and fluorescent images of E. coli cultured on nanopatterned silk after 6 and 24 h (d-f, respectively); SEM and fluorescent images of S. aureus cultured on pristine silk after 6 and 24 h (g-i, respectively); SEM and fluorescent images of S. aureus cultured on nanopatterned silk after 6 and 24 h (j-l, respectively); The scale bars in the SEM, inset, and fluorescent images are 20, 2, and 50 µm, respectively.



Pristine silk

a

107 CFU mL-1

105 CFU mL-1

103 CFU mL-1

Nanopatterned

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b

c

a

a

d

e

f

g

h

i

j

k

l

Fig. 6. SEM and fluorescent images of the cells cultured on the pre-infected pristine silk samples (a-f) and nanopatterned silk (g-l) with E. coli with different concentrations of 103, 105, and 107 CFU ml-1. The scale bars in the SEM and fluorescent images are 10 and 250 µm, respectively.


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In real situations, bacteria and cells coexist and surface adhesion becomes a race. case, cell proliferation on pre-infected samples can mimic the conditions in vivo

61–63.

In this In our

experiments, after the samples are pre-infected with S. aureus and E. coli, MC3T3 cells are cultured on them to study colonization of bacteria. As described previously, the nanopatterned samples repels bacteria by more than 90% and hence, it is expected that cells can grow better. In the presence of the smallest concentration of E. coli (103 CFU mL-1), the cells cannot proliferate on the pristine silk sample (Fig. 6a). Although some living cells are detected, most of them have a round shape indicating that they are not in good conditions (Fig. 6d) in contrast to those on the nanopatterned sample (Fig. 6g) on which a thick layer of proliferated cells is observed. For the high and medium concentrations of 107 and 105 CFU mL-1 (Figs. 6b and 6c), the cells cannot attach to the surface of the pristine silk sample because it is covered with a thick layer of bacteria and a large number of dead cells can be observed from the fluorescent images (Figs. 6e and 6f). On the other hand, on the nanopatterned samples cultured with the medium bacteria concentration (Fig. 6h), the cells manage to attach to the surface, although the number of living cells decreases (Fig. 6k). At the higher concentration (Fig. 6i), colonies formed by bacteria interfere with cell attachment and only one round-shape cell is observed to attach to the surface but without any spreading and proliferation (Fig. 6l). S. aureus proliferate better than E. coli as shown in Figs. 7a-7c.

At a low bacteria

concentration of 103 CFU mL-1, the cells are fully covered by bacteria. The cells attach to the surface first and are then gradually taken over by colonies of bacteria due to the faster growing rates of bacteria (Fig. 7a). At larger bacteria concentrations, the cells cannot attach to the surface on the control sample and die (Figs. 7b, 7c, 7e, and 7f). In comparison, on the nanopatterned sample pre-infected with a small bacteria concentration, the cells proliferate much better even 21 ACS Paragon Plus Environment


though some dead cells can be observed from the SEM image (Fig. 7g) and at least they are not covered by S. aureus like the control sample. If the bacteria concentration is increased, the number of live cells decreases (Figs. 7h, 7i, 7k, and 7l). The fluorescent images reveal some live (stretched) and unhealthy cells (round) but the surface is primarily covered by a large population of bacteria indicated by small green dots in Figs. 7 k and 7l.

105 CFU mL-1

Pristine silk

103 CFU mL-1

Nanopatterned

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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107 CFU mL-1

a

b

c

d

e

f

g

h

i

j

k

l

Fig. 7. SEM and fluorescent images of the cells cultured on the pre-infected pristine silk (a-fy) and nanopatterned silk (g-l) with S. aureus with different concentrations of 103, 105, and 107 CFU ml-1. The scale bars in the SEM and fluorescent images are 10 and 250 µm, respectively 22 ACS Paragon Plus Environment

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Co-culturing of MC3T3 cells and bacteria on the pristine and nanoengineered silk samples show that if bacteria do not attach to the surface during the first 6 h, cells can find enough suitable regions to attach and proliferate. On the pristine silk sample, no pressure is applied to the bacteria cell walls by the substrate and consequently, the whole surface is covered by bacteria and there is no space for cells to attach. However, the nanoengineered sample is not a good choice for bacteria to seed as the pressure on the cell walls created by the nanocones is high enough to repel them from the surface. However, since cells are larger than bacteria, they tolerate the pressure better and so attach and proliferate on the surface. Our results show that the nanoengineered silk-based materials have intrinsic cytocompatibility and bacteria resistance not requiring external chemical agents.

More

importantly, the nanoengineered film is based on a naturally-derived protein polymer with other outstanding properties including biocompatibility, mechanical properties, adjustable degradation, availability, and ease of processing which are important factors when designing biomaterials 36,37,40. Up to now, researchers have mostly focused on the antibacterial properties 64 and cell proliferation 40

separately on artificial materials 12,22. In this respect, our results show that natural materials such

as silk-based substances can be engineered to deliver excellent biological and antibacterial performance and have large potential in tissue engineering applications. 4. Conclusion The antibacterial properties of silk-based materials are enhanced by producing close-packed hexagonal arrays of nanocones by plasma etching. By changing the surface chemistry with plasma treatment, the materials become more hydrophilic as reflected by more than 100% increase in the surface energy. The nanopatterned silk film enhances attachment and proliferation of hAMSCs and simultaneously reduces bacteria attachment by more than 90% (both Gram-negative and 23 ACS Paragon Plus Environment


Gram-positive) due to the pressure exerted onto the bacteria cell walls by the nanocones. The cytocompatibility and bacterial resistance of the nanopatterned silk samples pre-infected with 3 different bacteria concentrations of 103, 105, and 107 CFU mL-1 are studied in conjunction with MC3T3 cells. The cells manage to find suitable and free surface on the nanopatterned samples as a result of effective bacteria repulsion. On the other hand, even in the presence of the smallest bacteria concentration, the cells on the control sample cannot attach and proliferate efficiently. The nanoengineered silk-based materials not only repel bacteria (both Gram-negative and Grampositive), but also promote proliferation of hAMSC and MC3T3 cells and the dual functionalities are highly desirable for biomaterials.

Acknowledgements: This work was financially supported by Hong Kong Research Grants Council (RGC) General Research Funds (GRF) No. CityU 11205617.

Supporting Information: Fabrication of polystyrene coatings on the silk samples; SEM, AFM, FTIR and water contact angle characterization details; SEM images of E. coli cultured on the pristine silk and nanopatterned silk samples after 30 min; Surface coverage of control and nanopatterned samples after culturing with E. coli and S. aureus for 6 and 24 h.


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