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Novel ordered TiO2 nanodot array on 316LSS with enhanced antibacterial properties
Journal Pre-proofs Novel ordered TiO2 nanodot array on 316LSS with enhanced antibacterial properties Yuanyuan Wei, Yiyang Ma, Jingjing Chen, Xuexia Yang, Shirong Ni, Feng Hong, Siyu Ni PII: DOI: Reference:
S0167-577X(20)30208-1 https://doi.org/10.1016/j.matlet.2020.127503 MLBLUE 127503
To appear in:
Materials Letters
Received Date: Revised Date: Accepted Date:
17 November 2019 11 January 2020 14 February 2020
Please cite this article as: Y. Wei, Y. Ma, J. Chen, X. Yang, S. Ni, F. Hong, S. Ni, Novel ordered TiO2 nanodot array on 316LSS with enhanced antibacterial properties, Materials Letters (2020), doi: https://doi.org/10.1016/ j.matlet.2020.127503
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier B.V.
Novel ordered TiO2 nanodot array on 316LSS with enhanced antibacterial properties Yuanyuan Weib＋, Yiyang Mad＋, Jingjing Chenb, Xuexia Yanga,b, Shirong Nic, Feng Honga,b, Siyu Nia,b,* aKey
Lab of Eco-Textile, Ministry of Education, Donghua University, North Renmin Road 2999, Shanghai 201620, P. R. China
bCollege
of Chemistry, Chemical Engineering and Biotechnology; Donghua University, North Renmin Road 2999, Shanghai 201620, P. R. China
cDepartment
d
of Pathology, Zhejiang Chinese Medical University, Hangzhou, 310053, China
Department of Orthopedic Surgery. Shanghai Jiao Tong University Affiliated Sixth People Hospital, Shanghai 200233, P. R. China ＋:These
two authors contributed equally to this work
Corresponding author*: E-mail: [email protected] Abstract: A novel ordered TiO2 nanodot array on 316LSS（NTA/316LSS）was first prepared by sol-gel in situ growth method. The surface microstructure, chemical components, antibacterial activity and cytocompatibility of the samples were characterized. The results showed that anatase TiO2 nanogranules (45 ± 5 nm) were embedded into nanopits (50 ± 5 nm) and arrayed in an ordered manner. The in vitro antibacterial test showed that there was a 96.3 ± 2.4% reduction in E. coli on the NTA/316LSS surface and a 98.1 ± 1.3% reduction in S. aureus compared to that on untreated 316LSS controls. Furthermore, NTA/316LSS showed good cytocompatibility in vitro. Our study revealed that such TiO2 nanodot surface can be designed to improve the use of 316LSS for various implant application. Keywords: surface, nanoparticles, ordered nanodot array, antibacterial property.
1
1. Introduction The 316L stainless steel (316LSS) is widely used in medical devices owing to its superior mechanical properties, corrosion resistance, and good processability [1]. However, medical device-induced infections pose a fatal threat to the life and safety of patients, and this has traditionally been the focus of programs to prevent healthcare-associated infections [2]. Therefore, it is essential to develop novel medical devices with inherently antibacterial properties and biocompatibility to prevent bacterial attachment and colonization. Surface functionalization can be utilized as an effective tool to improve the antibacterial performances of medical devices. For example, some antibacterial agents are coated or impregnated on the surface of 316LSS to prevent contamination of bacteria [3]. However, these methods are unable to provide a sustained and effective antibacterial action. In recent years, inorganic antibacterial agents have received considerable attention owing to their broad-spectrum antibacterial properties, high chemical stability, and biosafety [4]. Recently, Cu-bearing stainless steel scaffolds have been fabricated by selective laser melting (SLM). Elemental Cu addition endowed the scaffold with favorable antimicrobial properties [5]. Although the 316L-Cu scaffolds prepared using SLM are expensive and complicated, it has been proven that the introduction of an inorganic antimicrobial agent into the 316LSS surface is effective. Titanium dioxide (TiO2) is a type of broad-spectrum bactericide with excellent biocompatibility [6]. TiO2 produces hydroxyl radicals and reactive oxygen species (ROS), such as O2−. and .OH, under UV irradiation. These ROS can degrade the cell wall and the cytoplasmic membrane of the microorganism [7,8]. Previously, different-sized nanopits on 316LSS were prepared using an anodization procedure [9].
2
Owing to its special nanoporous structure, it is feasible to load the pits with suitable antibacterial agents to elicit antibacterial activity. Sol-gel technology has a number of advantages over traditional preparation methods of nanomaterials, such as higher homogeneities, mild and controllable reaction process. Hence, TiO2 nanogranules were first decorated into 316LSS nanopits by the sol-gel in situ growth method, and then the antibacterial activity of the obtained samples against both E. coli and S. aureus was investigated. In particular, to the best of our knowledge, there has not been report on the preparation of ordered TiO2 nanodot array on 316LSS as well as their antibacterial activity. 2. Materials and methods 2.1 Sample preparation and characterization The 316LSS nanopit specimens were fabricated as previously described [9]. The sol-gel in situ growth method was used to decorate TiO2 nanoparticles into 316LSS nanopits. Solution A was prepared by mixing 13 mL of tetrabutyl titanate, 11 mL of ethanol, and 3 mL of acetic acid with continuous stirring at 0C. Solution B was prepared by gradually adding 2.5 mL of distilled water acidified using nitric acid into 5.0 mL of ethanol with continuous stirring. Solution B was gradually added into solution A under continuous magnetic stirring. The reaction mixture was stirred for 0.5 h at 0C. Subsequently, the 316LSS samples were placed in a vessel containing an appropriate amount of TiO2 sol for 3 h at room temperature. Then, the samples were taken out and gently rinsed with absolute ethanol to remove the excess sol from the surface. Finally, the samples were dried at 65C for 5 h and calcined at 350C for 5 h. The obtained sample was named NTA/316LSS. In addition, the TiO2 films on degreased flat 316LSS (termed NTF/316LSS) were prepared using the same procedure. Field emission scanning electron microscopy (FESEM, Hitachi S-4800) was conducted to assess surface morphologies of the specimens.
3
The diameter of sample was measured by ImageJ software (Rawak Software Inc., Stuttgart, Germany). Energy dispersive spectroscopy (EDS, IE 300X) was used to determine the elemental composition. The surface composition of the specimens was analyzed by X-ray diffraction (XRD, Rigaku). 2.2 Antibacterial assays Antibacterial properties of the samples against S. aureus and E. coli were evaluated using the plate-counting method. Each sample was soaked in a 1-mL bacterial suspension with a concentration of 106 CFU mL−1. UV light (365 nm, 25 W) was used to irradiate the bacterial suspension. Bacterial colonies were counted on the plate to evaluate the antibacterial performance of the samples. 2.3 Cell proliferation assays To examine the in vitro cytocompatibility of the samples, rat bone marrow mesenchymal stem cells (rBMSCs) proliferation assay was conducted using cell counting kit-8 (CCK-8). The absorbance was measured at 450 nm with a microplate reader (ELX800, Bio-Tek, USA). Morphology of rBMSCs seeded on the different samples for one day were observed by confocal laser scanning microscopy (CLSM, TPS SP8, Leica, Germany) and FESEM.
Figure 1 The summary of the total experimental procedures.
4
Figure 2 (A) FESEM image of the 316LSS nanopits; (B) FESEM image of the NTA/316LSS; (C) EDS analysis of the NTA/316LSS; (D) XRD pattern of the NTA/316LSS. 3. Results Figure 1 summarizes the total experimental procedures. Figure 2A shows the typical surface FESEM images of 316LSS subsequent to the anodized treatment. It is observed that the surface of 316LSS exhibited highly ordered pits of hexagonal configuration with an average diameter of 50 ± 5 nm. Figure 2B clearly shows that the outlines of 316LSS nanopits were covered by TiO2 nanogranules. Every pit was evenly filled with TiO2 granules and formed an ordered TiO2 dot structure. The diameter of single TiO2 nanogranules in the pits was approximately 45 ± 5 nm. In recent years, microstructural design (i.e., defect structures) on the surface of the substrate has become an effective strategy for preparing low-dimensional nano-materials such as dot array structures [10]. The defect structures preferentially nucleate compared with the plane of the substrate during crystal growth. Furthermore, the growth rate of the stable atomic cluster in defect structures is larger than that of the atomic cluster on a flat surface [11]. In this study, well-arranged pit defects on the surface of 316LSS are prepared using the anodization method. Thus, it is reasonable to speculate that the TiO2 sol prefers to nucleate and grow in each pit zone of 316LSS. Then,
5
ordered TiO2 dot array is subsequently formed on the surface of 316LSS. EDS analysis (Fig. 2C) shows that the dominant elements on NTA/316LSS surfaces are O, Fe, Ni, and Ti. XRD was performed to investigate the surface phase structure of the prepared samples (Fig. 2D). All of the diffraction peaks at 25.27°, 37.88°, 48.12°, 53.97°, 62.14°, and 70.31° were well matched with the standard pattern of anatase TiO2 (JCPDS 21-1272).
Figure 3 (A) Photographs of colonies of E. coli and S. aureus treated with different samples; (B) Antibacterial rate of the different samples; (C)and(D) CLSM and FESEM images of NTA/316LSS after 1 day of incubation; (E) CCK-8 assay results; (Date are expressed as mean ± standard deviation (n=5); *p<0.05; **p<0.01). Figure 3A shows typical images of the bacterial colonies on different specimens. The sample with nano-TiO2 showed a significant antibacterial activity compared with that of the flat 316LSS control (p < 0.01). Interestingly, compared to the untreated 316LSS surface, NTA/316LSS showed the highest antibacterial effect. The bactericidal rates for E. coli and S. aureus were 96.3 ± 2.4% and 98.1 ± 1.3%, respectively (Fig. 3B). In addition, it is observed that the bactericidal rates of NTF/316LSS against E. coli and S. aureus were 86.5 ± 4.1% and 88.0 ± 2.2%, respectively. This is mainly because E. coli is relatively
6
more resistant to photocatalytic disinfection due to its cell wall structure, which restricts absorption of many molecules to movements through the cell membrane [12]. In previous studies, it has been shown that the antibacterial activity of TiO2 is attributed to its photocatalytic induction under UV irradiation [13]. In general, anatase TiO2 exhibits better photocatalytic activity than those of other crystalline phases [14]. In addition to phase structures, TiO2 nanomorphology considerably affects its photocatalytic efficiency owing to the large specific surface area, which offers many active sites for producing ROS and bacterial contact [15]. The high concentration of generated O2 and peroxide or .OH can inactivate bacteria [13]. Thus, in this work, the as-prepared TiO2 nanodot array structure could be the major factor contributing to improved antibacterial activity. Herein, rBMSCs were cultured on the specimens to investigate their in vitro cytocompatibility. Cells appeared to interact well with the surfaces of each specimen. Figure 3C and 3D shows that rBMSCs spread well and exhibited intimated contact with the NTA/316LSS after 1 day of incubation. There were no significant changes of cell morphology between each group. Figure 3E shows that there were no cytotoxic effects from any of the surfaces. In particular, the NTA/316LSS sample stood out in cell attachment and proliferation. 4. Conclusions In this study, a novel ordered TiO2 nanodot array on 316LSS was first prepared by facile sol-gel in situ growth method. In vitro antibacterial results showed that NTA/316LSS exhibited better antimicrobial activity against E. coli and S. aureus compared with that of other samples. The in vitro cytocompatibility assays showed that NTA/316LSS promoted adhesion and proliferation of rBMSCs. To summarize, this study shows the potential of using NTA/316LSS in medical devices owing to its antibacterial properties
7
and good cytocompatibility. Acknowledgements The work was jointly supported by the Natural Science Foundation of Shanghai (No. 19ZR1401000) and the Opening Project of State Key Laboratory of High Performance Ceramics and Superfine Microstructure (Grant No. SKL201709SIC).
References [1] P. Qi, Y. Yang, K. Xiong, et al,. ACS Biomater. Sci. Eng. 1 (7) (2015) 513-24. [2] S. S. Magill, J. R. Edwards, W. Bamberg, et al,. New Engl J. Med. 370 (13) (2014) 1198-208. [3] N. Mohd Daud, I. F. Saeful Bahri, N. Malek, et al,. Colloids Surf. B Biointerfaces. 145 (2016) 130-39. [4] S. Sonia, R. Jayasudha, N. D. Jayram, et al,. Curr. Appl. Phys. 16 (2016) 914-21. [5] Q. Wang, L. Ren, X. Li, et al,. Mater. Sci. Eng C. 68 (2016) 519-22. [6] T. Verdier, M. Coutand, A. Bertron, et al,. Coatings. 4 (2014) 670-86. [7] D. Campoccia, L. Montanaro, C. R. Arciola,. Biomaterials. 34 (2013) 8533-554. [8] Y. Song, Y. Miao, C. Song,. New Phytol. 201 (4) (2014) 1121-140. [9] S. Ni, L. Sun, B. Ercan, et al,. J. Biomed. Mater. Res. 102 (6) (2014) 1297-303. [10] X.T. Shen, X.C. Wang, F. H. Sun, et al,. Relat. Mater. 73 (2017) 7-14. [11] T. Zhang, Q. Feng, Z. Yu, et al,. Int. J. Refract. Met. Hard Mater. 84 (2019) 105016. [12] H. M. Yadav, S. V. Otari, V. B. Koli, et al,. J. Photochem. Photobiol., A. 280 (2014) 32-8. [13] B. Pant, M. Park, S. J. Park,. Coatings. 9 (10) (2019) 1-19. [14] T. Di, J. Zhang, B. Cheng, et al,. Sci. China Chem. 61 (3) (2018) 1-7. [15] H. A. Foster, I. B. Ditta, S. Varghese, et al,. Appl. Microbiol. Biotechnol. 90 (6) (2011) 1847-868.
8
Highlights An ordered TiO2 nanodot array was prepared by sol-gel in situ growth method. NTA/316LSS exhibited better antimicrobial activity against E. coli and S. aureus. NTA/316LSS showed good cytocompatibility in vitro.
No conflict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for submission. I would like to declare on behalf of my co-authors that the work described was original research that has not been published previously, and not under consideration for publication elsewhere, in whole or in part. All the authors listed have approved the manuscript that is enclosed.
Credit Author Statement All authors discussed the results and contributed to the final manuscript, provided critical feedback and helped shape the research, analysis and manuscript. Authors contributed to the design and implementation of the research, to the analysis of the results and to the writing of the manuscript.
Figure Captions: Fig.1: The summary of the total experimental procedures. Fig.2: (A) FESEM image of the 316LSS nanopits; (B) FESEM image of the NTA/316LSS; (C) EDS analysis of the NTA/316LSS; (D) XRD pattern of the NTA/316LSS. Fig.3: (A) Photographs of colonies of E. coli and S. aureus treated with different samples; (B)
9
Antibacterial rate of the different samples; (C)and(D) CLSM and FESEM images of NTA/316LSS after 1 day of incubation; (E) CCK-8 assay results; (Date are expressed as mean ± standard deviation (n=5); *p<0.05; **p<0.01).
Fig.1
10
Fig.2
Fig.3
11
S0167-577X(20)30208-1 https://doi.org/10.1016/j.matlet.2020.127503 MLBLUE 127503
To appear in:
Materials Letters
Received Date: Revised Date: Accepted Date:
17 November 2019 11 January 2020 14 February 2020
Please cite this article as: Y. Wei, Y. Ma, J. Chen, X. Yang, S. Ni, F. Hong, S. Ni, Novel ordered TiO2 nanodot array on 316LSS with enhanced antibacterial properties, Materials Letters (2020), doi: https://doi.org/10.1016/ j.matlet.2020.127503
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier B.V.
Novel ordered TiO2 nanodot array on 316LSS with enhanced antibacterial properties Yuanyuan Weib＋, Yiyang Mad＋, Jingjing Chenb, Xuexia Yanga,b, Shirong Nic, Feng Honga,b, Siyu Nia,b,* aKey
Lab of Eco-Textile, Ministry of Education, Donghua University, North Renmin Road 2999, Shanghai 201620, P. R. China
bCollege
of Chemistry, Chemical Engineering and Biotechnology; Donghua University, North Renmin Road 2999, Shanghai 201620, P. R. China
cDepartment
d
of Pathology, Zhejiang Chinese Medical University, Hangzhou, 310053, China
Department of Orthopedic Surgery. Shanghai Jiao Tong University Affiliated Sixth People Hospital, Shanghai 200233, P. R. China ＋:These
two authors contributed equally to this work
Corresponding author*: E-mail: [email protected] Abstract: A novel ordered TiO2 nanodot array on 316LSS（NTA/316LSS）was first prepared by sol-gel in situ growth method. The surface microstructure, chemical components, antibacterial activity and cytocompatibility of the samples were characterized. The results showed that anatase TiO2 nanogranules (45 ± 5 nm) were embedded into nanopits (50 ± 5 nm) and arrayed in an ordered manner. The in vitro antibacterial test showed that there was a 96.3 ± 2.4% reduction in E. coli on the NTA/316LSS surface and a 98.1 ± 1.3% reduction in S. aureus compared to that on untreated 316LSS controls. Furthermore, NTA/316LSS showed good cytocompatibility in vitro. Our study revealed that such TiO2 nanodot surface can be designed to improve the use of 316LSS for various implant application. Keywords: surface, nanoparticles, ordered nanodot array, antibacterial property.
1
1. Introduction The 316L stainless steel (316LSS) is widely used in medical devices owing to its superior mechanical properties, corrosion resistance, and good processability [1]. However, medical device-induced infections pose a fatal threat to the life and safety of patients, and this has traditionally been the focus of programs to prevent healthcare-associated infections [2]. Therefore, it is essential to develop novel medical devices with inherently antibacterial properties and biocompatibility to prevent bacterial attachment and colonization. Surface functionalization can be utilized as an effective tool to improve the antibacterial performances of medical devices. For example, some antibacterial agents are coated or impregnated on the surface of 316LSS to prevent contamination of bacteria [3]. However, these methods are unable to provide a sustained and effective antibacterial action. In recent years, inorganic antibacterial agents have received considerable attention owing to their broad-spectrum antibacterial properties, high chemical stability, and biosafety [4]. Recently, Cu-bearing stainless steel scaffolds have been fabricated by selective laser melting (SLM). Elemental Cu addition endowed the scaffold with favorable antimicrobial properties [5]. Although the 316L-Cu scaffolds prepared using SLM are expensive and complicated, it has been proven that the introduction of an inorganic antimicrobial agent into the 316LSS surface is effective. Titanium dioxide (TiO2) is a type of broad-spectrum bactericide with excellent biocompatibility [6]. TiO2 produces hydroxyl radicals and reactive oxygen species (ROS), such as O2−. and .OH, under UV irradiation. These ROS can degrade the cell wall and the cytoplasmic membrane of the microorganism [7,8]. Previously, different-sized nanopits on 316LSS were prepared using an anodization procedure [9].
2
Owing to its special nanoporous structure, it is feasible to load the pits with suitable antibacterial agents to elicit antibacterial activity. Sol-gel technology has a number of advantages over traditional preparation methods of nanomaterials, such as higher homogeneities, mild and controllable reaction process. Hence, TiO2 nanogranules were first decorated into 316LSS nanopits by the sol-gel in situ growth method, and then the antibacterial activity of the obtained samples against both E. coli and S. aureus was investigated. In particular, to the best of our knowledge, there has not been report on the preparation of ordered TiO2 nanodot array on 316LSS as well as their antibacterial activity. 2. Materials and methods 2.1 Sample preparation and characterization The 316LSS nanopit specimens were fabricated as previously described [9]. The sol-gel in situ growth method was used to decorate TiO2 nanoparticles into 316LSS nanopits. Solution A was prepared by mixing 13 mL of tetrabutyl titanate, 11 mL of ethanol, and 3 mL of acetic acid with continuous stirring at 0C. Solution B was prepared by gradually adding 2.5 mL of distilled water acidified using nitric acid into 5.0 mL of ethanol with continuous stirring. Solution B was gradually added into solution A under continuous magnetic stirring. The reaction mixture was stirred for 0.5 h at 0C. Subsequently, the 316LSS samples were placed in a vessel containing an appropriate amount of TiO2 sol for 3 h at room temperature. Then, the samples were taken out and gently rinsed with absolute ethanol to remove the excess sol from the surface. Finally, the samples were dried at 65C for 5 h and calcined at 350C for 5 h. The obtained sample was named NTA/316LSS. In addition, the TiO2 films on degreased flat 316LSS (termed NTF/316LSS) were prepared using the same procedure. Field emission scanning electron microscopy (FESEM, Hitachi S-4800) was conducted to assess surface morphologies of the specimens.
3
The diameter of sample was measured by ImageJ software (Rawak Software Inc., Stuttgart, Germany). Energy dispersive spectroscopy (EDS, IE 300X) was used to determine the elemental composition. The surface composition of the specimens was analyzed by X-ray diffraction (XRD, Rigaku). 2.2 Antibacterial assays Antibacterial properties of the samples against S. aureus and E. coli were evaluated using the plate-counting method. Each sample was soaked in a 1-mL bacterial suspension with a concentration of 106 CFU mL−1. UV light (365 nm, 25 W) was used to irradiate the bacterial suspension. Bacterial colonies were counted on the plate to evaluate the antibacterial performance of the samples. 2.3 Cell proliferation assays To examine the in vitro cytocompatibility of the samples, rat bone marrow mesenchymal stem cells (rBMSCs) proliferation assay was conducted using cell counting kit-8 (CCK-8). The absorbance was measured at 450 nm with a microplate reader (ELX800, Bio-Tek, USA). Morphology of rBMSCs seeded on the different samples for one day were observed by confocal laser scanning microscopy (CLSM, TPS SP8, Leica, Germany) and FESEM.
Figure 1 The summary of the total experimental procedures.
4
Figure 2 (A) FESEM image of the 316LSS nanopits; (B) FESEM image of the NTA/316LSS; (C) EDS analysis of the NTA/316LSS; (D) XRD pattern of the NTA/316LSS. 3. Results Figure 1 summarizes the total experimental procedures. Figure 2A shows the typical surface FESEM images of 316LSS subsequent to the anodized treatment. It is observed that the surface of 316LSS exhibited highly ordered pits of hexagonal configuration with an average diameter of 50 ± 5 nm. Figure 2B clearly shows that the outlines of 316LSS nanopits were covered by TiO2 nanogranules. Every pit was evenly filled with TiO2 granules and formed an ordered TiO2 dot structure. The diameter of single TiO2 nanogranules in the pits was approximately 45 ± 5 nm. In recent years, microstructural design (i.e., defect structures) on the surface of the substrate has become an effective strategy for preparing low-dimensional nano-materials such as dot array structures [10]. The defect structures preferentially nucleate compared with the plane of the substrate during crystal growth. Furthermore, the growth rate of the stable atomic cluster in defect structures is larger than that of the atomic cluster on a flat surface [11]. In this study, well-arranged pit defects on the surface of 316LSS are prepared using the anodization method. Thus, it is reasonable to speculate that the TiO2 sol prefers to nucleate and grow in each pit zone of 316LSS. Then,
5
ordered TiO2 dot array is subsequently formed on the surface of 316LSS. EDS analysis (Fig. 2C) shows that the dominant elements on NTA/316LSS surfaces are O, Fe, Ni, and Ti. XRD was performed to investigate the surface phase structure of the prepared samples (Fig. 2D). All of the diffraction peaks at 25.27°, 37.88°, 48.12°, 53.97°, 62.14°, and 70.31° were well matched with the standard pattern of anatase TiO2 (JCPDS 21-1272).
Figure 3 (A) Photographs of colonies of E. coli and S. aureus treated with different samples; (B) Antibacterial rate of the different samples; (C)and(D) CLSM and FESEM images of NTA/316LSS after 1 day of incubation; (E) CCK-8 assay results; (Date are expressed as mean ± standard deviation (n=5); *p<0.05; **p<0.01). Figure 3A shows typical images of the bacterial colonies on different specimens. The sample with nano-TiO2 showed a significant antibacterial activity compared with that of the flat 316LSS control (p < 0.01). Interestingly, compared to the untreated 316LSS surface, NTA/316LSS showed the highest antibacterial effect. The bactericidal rates for E. coli and S. aureus were 96.3 ± 2.4% and 98.1 ± 1.3%, respectively (Fig. 3B). In addition, it is observed that the bactericidal rates of NTF/316LSS against E. coli and S. aureus were 86.5 ± 4.1% and 88.0 ± 2.2%, respectively. This is mainly because E. coli is relatively
6
more resistant to photocatalytic disinfection due to its cell wall structure, which restricts absorption of many molecules to movements through the cell membrane [12]. In previous studies, it has been shown that the antibacterial activity of TiO2 is attributed to its photocatalytic induction under UV irradiation [13]. In general, anatase TiO2 exhibits better photocatalytic activity than those of other crystalline phases [14]. In addition to phase structures, TiO2 nanomorphology considerably affects its photocatalytic efficiency owing to the large specific surface area, which offers many active sites for producing ROS and bacterial contact [15]. The high concentration of generated O2 and peroxide or .OH can inactivate bacteria [13]. Thus, in this work, the as-prepared TiO2 nanodot array structure could be the major factor contributing to improved antibacterial activity. Herein, rBMSCs were cultured on the specimens to investigate their in vitro cytocompatibility. Cells appeared to interact well with the surfaces of each specimen. Figure 3C and 3D shows that rBMSCs spread well and exhibited intimated contact with the NTA/316LSS after 1 day of incubation. There were no significant changes of cell morphology between each group. Figure 3E shows that there were no cytotoxic effects from any of the surfaces. In particular, the NTA/316LSS sample stood out in cell attachment and proliferation. 4. Conclusions In this study, a novel ordered TiO2 nanodot array on 316LSS was first prepared by facile sol-gel in situ growth method. In vitro antibacterial results showed that NTA/316LSS exhibited better antimicrobial activity against E. coli and S. aureus compared with that of other samples. The in vitro cytocompatibility assays showed that NTA/316LSS promoted adhesion and proliferation of rBMSCs. To summarize, this study shows the potential of using NTA/316LSS in medical devices owing to its antibacterial properties
7
and good cytocompatibility. Acknowledgements The work was jointly supported by the Natural Science Foundation of Shanghai (No. 19ZR1401000) and the Opening Project of State Key Laboratory of High Performance Ceramics and Superfine Microstructure (Grant No. SKL201709SIC).
References [1] P. Qi, Y. Yang, K. Xiong, et al,. ACS Biomater. Sci. Eng. 1 (7) (2015) 513-24. [2] S. S. Magill, J. R. Edwards, W. Bamberg, et al,. New Engl J. Med. 370 (13) (2014) 1198-208. [3] N. Mohd Daud, I. F. Saeful Bahri, N. Malek, et al,. Colloids Surf. B Biointerfaces. 145 (2016) 130-39. [4] S. Sonia, R. Jayasudha, N. D. Jayram, et al,. Curr. Appl. Phys. 16 (2016) 914-21. [5] Q. Wang, L. Ren, X. Li, et al,. Mater. Sci. Eng C. 68 (2016) 519-22. [6] T. Verdier, M. Coutand, A. Bertron, et al,. Coatings. 4 (2014) 670-86. [7] D. Campoccia, L. Montanaro, C. R. Arciola,. Biomaterials. 34 (2013) 8533-554. [8] Y. Song, Y. Miao, C. Song,. New Phytol. 201 (4) (2014) 1121-140. [9] S. Ni, L. Sun, B. Ercan, et al,. J. Biomed. Mater. Res. 102 (6) (2014) 1297-303. [10] X.T. Shen, X.C. Wang, F. H. Sun, et al,. Relat. Mater. 73 (2017) 7-14. [11] T. Zhang, Q. Feng, Z. Yu, et al,. Int. J. Refract. Met. Hard Mater. 84 (2019) 105016. [12] H. M. Yadav, S. V. Otari, V. B. Koli, et al,. J. Photochem. Photobiol., A. 280 (2014) 32-8. [13] B. Pant, M. Park, S. J. Park,. Coatings. 9 (10) (2019) 1-19. [14] T. Di, J. Zhang, B. Cheng, et al,. Sci. China Chem. 61 (3) (2018) 1-7. [15] H. A. Foster, I. B. Ditta, S. Varghese, et al,. Appl. Microbiol. Biotechnol. 90 (6) (2011) 1847-868.
8
Highlights An ordered TiO2 nanodot array was prepared by sol-gel in situ growth method. NTA/316LSS exhibited better antimicrobial activity against E. coli and S. aureus. NTA/316LSS showed good cytocompatibility in vitro.
No conflict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for submission. I would like to declare on behalf of my co-authors that the work described was original research that has not been published previously, and not under consideration for publication elsewhere, in whole or in part. All the authors listed have approved the manuscript that is enclosed.
Credit Author Statement All authors discussed the results and contributed to the final manuscript, provided critical feedback and helped shape the research, analysis and manuscript. Authors contributed to the design and implementation of the research, to the analysis of the results and to the writing of the manuscript.
Figure Captions: Fig.1: The summary of the total experimental procedures. Fig.2: (A) FESEM image of the 316LSS nanopits; (B) FESEM image of the NTA/316LSS; (C) EDS analysis of the NTA/316LSS; (D) XRD pattern of the NTA/316LSS. Fig.3: (A) Photographs of colonies of E. coli and S. aureus treated with different samples; (B)
9
Antibacterial rate of the different samples; (C)and(D) CLSM and FESEM images of NTA/316LSS after 1 day of incubation; (E) CCK-8 assay results; (Date are expressed as mean ± standard deviation (n=5); *p<0.05; **p<0.01).
Fig.1
10
Fig.2
Fig.3
11