Chlorhexidine-grafted phenolamine coating to improve antibacterial property of the titanium surface_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

DING Sixie , HONG Huilei , XU Linghan , WANG Xiaowei , ZHANG Weibo , LI Xiangyang ,  WANG Yinlong ,  CHEN Jialong

Stomatologic Hospital and College, Key Laboratory of Oral Diseases Research of Anhui Province, Anhui Medical University, Hefei Anhui, 230032, P. R. China;

Corresponding author：

WANG Yinlong, Email: wangylah@sina.com; CHEN Jialong, Email: jialong_dt@126.com

Keywords：

Chlorhexidine; titanium; phenolamine-crosslinking; coating; antibacterial property

DOI：

10.7507/1002-1892.202108095

Video：

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Objective To investigate the physicochemical properties of pure titanium surface grafted with chlorhexidine (CHX) by phenolamine coating, and to evaluate its antibacterial activity and osteoblast-compatibility in vitro. Methods Control group was obtained by alkali and thermal treatment, and then immersed in the mixture of epigallocatechin-3-gallate/hexamethylene diamine (coating group). Phenolamine coating was deposited on the surface, and then it was immersed in CHX solution to obtain the grafted surface of CHX (grafting group). The surface morphology was observed by scanning electron microscope, the surface element composition was analyzed by X-ray photoelectron spectroscopy, and the surface hydrophilicity was measured by water contact angle test. Live/dead bacterial staining, nephelometery, and inhibition zone method were executed to evaluate the antibacterial property. Cytotoxicity was evaluated by MTT assay and cell fluorescence staining. Bacteria-MC3T3-E1 cells co‐culture was conducted to evaluate the cell viability on the samples under the circumstance with bacteria. Results Scanning electron microscope observation results showed that deposits of coating group and grafting group increased successively and gradually covered the porous structure. X-ray photoelectron spectroscopy results showed the peak of N1s enhanced and the peak of Cl2p appeared in grafting group. Water contact angle test results showed that the hydrophilic angle of three groups increased in turn, and there was significant difference between groups (P<0.05). Live/dead bacteria staining results showed that the grafting group had the least amount of bacteria adhered to the surface and the proportion of dead bacteria was high. The grafting group had a transparent inhibition zone around it and the absorbance (A) value did not increase, showing significant difference when compared with control group and coating group (P<0.05). MTT assay and cell fluorescence staining results showed that the number of adherent cells on the surface of the grafting group was the least, but the adherent cells had good proliferation activity. Bacteria-cell co-culture results showed that there was no bacteria on the surface of grafting group but live cells adhered well. Conclusion CHX-grafted phenolamine coating has the ability to inhibit bacterial adhesion and proliferation, and effectively protect cell adhesion and proliferation in a bacterial environment.

Citation： DING Sixie, HONG Huilei, XU Linghan, WANG Xiaowei, ZHANG Weibo, LI Xiangyang, WANG Yinlong, CHEN Jialong. Chlorhexidine-grafted phenolamine coating to improve antibacterial property of the titanium surface. Chinese Journal of Reparative and Reconstructive Surgery, 2022, 36(3): 335-342. doi: 10.7507/1002-1892.202108095 Copy

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14.	Villegas M, Alonso-Cantu C, Rahmani S, et al. Antibiotic-impregnated liquid-infused coatings suppress the formation of methicillin-resistant Staphylococcus aureus biofilms. ACS Appl Mater Interfaces, 2021, 13(24): 27774-27783.
15.	Jennings JA, Carpenter DP, Troxel KS, et al. Novel antibiotic-loaded point-of-care implant coating inhibits biofilm. Clin Orthop Relat Res, 2015, 473(7): 2270-2282.
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17.	Cui J, Shao Y, Zhang H, et al. Development of a novel silver ions-nanosilver complementary composite as antimicrobial additive for powder coating. Chem Eng J, 2021, 420: 127633. doi: 10.1016/j.cej.2020.127633.
18.	Wang S, Yang Y, Li W, et al. Study of the relationship between chlorhexidine-grafted amount and biological performances of micro/nanoporous titanium surfaces. ACS Omega, 2019, 4(19): 18370-18380.
19.	Zhou L, Li QL, Wong HM. A novel strategy for caries management: Constructing an antibiofouling and mineralizing dual-bioactive tooth surface. ACS Appl Mater Interfaces, 2021, 13(26): 31140-31152.
20.	袁宁, 刘运德, 李雪, 等. 庆大霉素和O-羧甲基壳聚糖对硫酸钙骨水泥改性研究. 中国修复重建外科杂志, 2017, 31(3): 306-312.
21.	刘重, 郭永明, 马宁, 等. 扩髓灌洗联合抗生素骨水泥涂层髓内钉内固定治疗胫骨骨折髓内钉内固定术后感染. 中国骨与关节损伤杂志, 2021, 36(2): 139-142.
22.	张利兴, 田昂, 李锡, 等. TiO₂纳米管/羟基磷灰石载万古霉素涂层的释药性及生物毒性. 中国组织工程研究, 2021, 25(10): 1500-1506.
23.	Sun J, Liu X, Lyu C, et al. Synergistic antibacterial effect of graphene-coated titanium loaded with levofloxacin. Colloids Surf B Biointerfaces, 2021, 208: 112090. doi: 10.1016/j.colsurfb.2021.112090.
24.	Barbour ME, Gandhi N, el-Turki A, et al. Differential adhesion of Streptococcus gordonii to anatase and rutile titanium dioxide surfaces with and without functionalization with chlorhexidine. J Biomed Mater Res A, 2009, 90(4): 993-998.
25.	Franco CF, Pataro AL, E Souza LC, et al. In vitro effects of a chlorhexidine controlled delivery system. Artif Organs, 2003, 27(5): 486-491.
26.	杨园梦, 王爽, 李娇娇, 等. 氯己定接枝改善多孔钛抗菌性能初探. 中华口腔医学杂志, 2020, 55(2): 104-110.
27.	Tallury P, Alimohammadi N, Kalachandra S. Poly (ethylene-co-vinyl acetate) copolymer matrix for delivery of chlorhexidine and acyclovir drugs for use in the oral environment: effect of drug combination, copolymer composition and coating on the drug release rate. Dent Mater, 2007, 23(4): 404-409.
28.	Tambunlertchai S, Srisang S, Nasongkla N. Development of antimicrobial coating by layer-by-layer [corrected] dip coating of chlorhexidine-loaded micelles. J Mater Sci Mater Med, 2017, 28(6): 90. doi: 10.1007/s10856-017-5899-2.
29.	王小红, 曹阳, 张利, 等. 碱热处理对钛表面生物活性的影响. 功能材料, 2013, 44(2): 275-280.
30.	Zhao C, Zhu X, Liang K, et al. Osteoinduction of porous titanium: a comparative study between acid-alkali and chemical-thermal treatments. J Biomed Mater Res B Appl Biomater, 2010, 95(2): 387-396.
31.	杨帮成, 周学东, 于海洋, 等. 钛种植体表面改性方法. 华西口腔医学杂志, 2019, 37(2): 124-129.
32.	Pettygrove BA, Kratofil RM, Alhede M, et al. Delayed neutrophil recruitment allows nascent Staphylococcus aureus biofilm formation and immune evasion. Biomaterials, 2021, 275: 120775. doi: 10.1016/j.biomaterials.2021.120775.
33.	Lehnfeld J, Gruening M, Kronseder M, et al. Comparison of protein-repellent behavior of linear versus dendrimer-structured surface-immobilized polymers. Langmuir, 2020, 36(21): 5880-5890.
34.	Tao C, Jin M, Yao H, et al. Dopamine based adhesive nano-coatings on extracellular matrix (ECM) based grafts for enhanced host-graft interfacing affinity. Nanoscale, 2021, 13(43): 18148-18159.
35.	Fan Q, Bai J, Shan H, et al. Implantable blood clot loaded with BMP-2 for regulation of osteoimmunology and enhancement of bone repair. Bioact Mater, 2021, 6(11): 4014-4026.

1. Jang TS, Kim D, Han G, et al. Powder based additive manufacturing for biomedical application of titanium and its alloys: a review. Biomed Eng Lett, 2020, 10(4): 505-516.
2. Arciola CR, Campoccia D, Ehrlich GD, et al. Biofilm-based implant infections in orthopaedics. Adv Exp Med Biol, 2015, 830: 29-46.
3. Arciola CR, Campoccia D, Montanaro L. Implant infections: adhesion, biofilm formation and immune evasion. Nat Rev Microbiol, 2018, 16(7): 397-409.
4. Vuong C, Kocianova S, Voyich JM, et al. A crucial role for exopolysaccharide modification in bacterial biofilm formation, immune evasion, and virulence. J Biol Chem, 2004, 279(52): 54881-54886.
5. Drago L, Clerici P, Morelli I, et al. The world association against infection in orthopaedics and trauma (WAIOT) procedures for microbiological sampling and processing for periprosthetic joint infections (PJIs) and other implant-related infections. J Clin Med, 2019, 8(7): 933. doi: 10.3390/jcm8070933.
6. Alves DF, Magalhães AP, Neubauer D, et al. Unveiling the fate of adhering bacteria to antimicrobial surfaces: expression of resistance-associated genes and macrophage-mediated phagocytosis. Acta Biomater, 2018, 78: 189-197.
7. Borse V, Pawar V, Shetty G, et al. Nanobiotechnology perspectives on prevention and treatment of ortho-paedic implant associated infection. Curr Drug Deliv, 2016, 13(2): 175-185.
8. Stanton IC, Murray AK, Zhang L, et al. Evolution of antibiotic resistance at low antibiotic concentrations including selection below the minimal selective concentration. Commun Biol, 2020, 3(1): 467. doi: 10.1038/s42003-020-01176-w.
9. Riool M, Dirks AJ, Jaspers V, et al. A chlorhexidine-releasing epoxy-based coating on titanium implants prevents Staphylococcus aureus experimental biomaterial-associated infection. Eur Cell Mater, 2017, 33: 143-157.
10. Hickok NJ, Shapiro IM, Chen AF. The impact of incorporating antimicrobials into implant surfaces. J Dent Res, 2018, 97(1): 14-22.
11. Xiang L, Zhang J, Gong L, et al. Probing the interaction forces of phenol/amine deposition in wet adhesion: Impact of phenol/amine mass ratio and surface properties. Langmuir, 2019, 35(48): 15639-15650.
12. Wiegand C, Abel M, Ruth P, et al. Analysis of the adaptation capacity of Staphylococcus aureus to commonly used antiseptics by microplate laser nephelometry. Skin Pharmacol Physiol, 2012, 25(6): 288-297.
13. 张一, 张宪高, 胡中岭, 等. 多孔医用植入材料抗菌性能研究进展. 中国修复重建外科杂志, 2020, 34(11): 1478-1485.
14. Villegas M, Alonso-Cantu C, Rahmani S, et al. Antibiotic-impregnated liquid-infused coatings suppress the formation of methicillin-resistant Staphylococcus aureus biofilms. ACS Appl Mater Interfaces, 2021, 13(24): 27774-27783.
15. Jennings JA, Carpenter DP, Troxel KS, et al. Novel antibiotic-loaded point-of-care implant coating inhibits biofilm. Clin Orthop Relat Res, 2015, 473(7): 2270-2282.
16. 陆肖璇, 张露露, 阳曦, 等. 钛植入物促骨整合及抗感染涂层的研究进展. 中国生物医学工程学报, 2021, 40(5): 620-627.
17. Cui J, Shao Y, Zhang H, et al. Development of a novel silver ions-nanosilver complementary composite as antimicrobial additive for powder coating. Chem Eng J, 2021, 420: 127633. doi: 10.1016/j.cej.2020.127633.
18. Wang S, Yang Y, Li W, et al. Study of the relationship between chlorhexidine-grafted amount and biological performances of micro/nanoporous titanium surfaces. ACS Omega, 2019, 4(19): 18370-18380.
19. Zhou L, Li QL, Wong HM. A novel strategy for caries management: Constructing an antibiofouling and mineralizing dual-bioactive tooth surface. ACS Appl Mater Interfaces, 2021, 13(26): 31140-31152.
20. 袁宁, 刘运德, 李雪, 等. 庆大霉素和O-羧甲基壳聚糖对硫酸钙骨水泥改性研究. 中国修复重建外科杂志, 2017, 31(3): 306-312.
21. 刘重, 郭永明, 马宁, 等. 扩髓灌洗联合抗生素骨水泥涂层髓内钉内固定治疗胫骨骨折髓内钉内固定术后感染. 中国骨与关节损伤杂志, 2021, 36(2): 139-142.
22. 张利兴, 田昂, 李锡, 等. TiO₂纳米管/羟基磷灰石载万古霉素涂层的释药性及生物毒性. 中国组织工程研究, 2021, 25(10): 1500-1506.
23. Sun J, Liu X, Lyu C, et al. Synergistic antibacterial effect of graphene-coated titanium loaded with levofloxacin. Colloids Surf B Biointerfaces, 2021, 208: 112090. doi: 10.1016/j.colsurfb.2021.112090.
24. Barbour ME, Gandhi N, el-Turki A, et al. Differential adhesion of Streptococcus gordonii to anatase and rutile titanium dioxide surfaces with and without functionalization with chlorhexidine. J Biomed Mater Res A, 2009, 90(4): 993-998.
25. Franco CF, Pataro AL, E Souza LC, et al. In vitro effects of a chlorhexidine controlled delivery system. Artif Organs, 2003, 27(5): 486-491.
26. 杨园梦, 王爽, 李娇娇, 等. 氯己定接枝改善多孔钛抗菌性能初探. 中华口腔医学杂志, 2020, 55(2): 104-110.
27. Tallury P, Alimohammadi N, Kalachandra S. Poly (ethylene-co-vinyl acetate) copolymer matrix for delivery of chlorhexidine and acyclovir drugs for use in the oral environment: effect of drug combination, copolymer composition and coating on the drug release rate. Dent Mater, 2007, 23(4): 404-409.
28. Tambunlertchai S, Srisang S, Nasongkla N. Development of antimicrobial coating by layer-by-layer [corrected] dip coating of chlorhexidine-loaded micelles. J Mater Sci Mater Med, 2017, 28(6): 90. doi: 10.1007/s10856-017-5899-2.
29. 王小红, 曹阳, 张利, 等. 碱热处理对钛表面生物活性的影响. 功能材料, 2013, 44(2): 275-280.
30. Zhao C, Zhu X, Liang K, et al. Osteoinduction of porous titanium: a comparative study between acid-alkali and chemical-thermal treatments. J Biomed Mater Res B Appl Biomater, 2010, 95(2): 387-396.
31. 杨帮成, 周学东, 于海洋, 等. 钛种植体表面改性方法. 华西口腔医学杂志, 2019, 37(2): 124-129.
32. Pettygrove BA, Kratofil RM, Alhede M, et al. Delayed neutrophil recruitment allows nascent Staphylococcus aureus biofilm formation and immune evasion. Biomaterials, 2021, 275: 120775. doi: 10.1016/j.biomaterials.2021.120775.
33. Lehnfeld J, Gruening M, Kronseder M, et al. Comparison of protein-repellent behavior of linear versus dendrimer-structured surface-immobilized polymers. Langmuir, 2020, 36(21): 5880-5890.
34. Tao C, Jin M, Yao H, et al. Dopamine based adhesive nano-coatings on extracellular matrix (ECM) based grafts for enhanced host-graft interfacing affinity. Nanoscale, 2021, 13(43): 18148-18159.
35. Fan Q, Bai J, Shan H, et al. Implantable blood clot loaded with BMP-2 for regulation of osteoimmunology and enhancement of bone repair. Bioact Mater, 2021, 6(11): 4014-4026.

Chinese Journal of Reparative and Reconstructive Surgery

Chlorhexidine-grafted phenolamine coating to improve antibacterial property of the titanium surface

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