Material Science & Engineering 6 82 2018) 12 Content lists available at ScienceDirect Materials Science & Engineering C ELSEVIER journal homepage: www.elsevier.com/locateimsec = Antibacterial activities and biocompatibilities of Ti-Ag alloys prepared by spark plasma sintering and acid etching fa Zeming Lei’, Hangzhou Zhang’, Erlin Zhang‘, Junhua You", Xiaoxue Ma‘, Xizhuang Bai" parm of Ocoee and Spore Metis and Jot Suge. Te Peis Hota of Cine Madea ner, Sega. Pepe Rp of Cine Deparment of Ores nd Spars Meine a Sey. Te Fo Atel Hat of Ca Meal Urey, Skene Pepe ep of Chine key Lary for Anny an Trav of Maes (ATA, hain Mey of Chia Schl of Maver Sane nd Pega areaton ne, Shey Prpe Rept of Cia “Sol of Maal Since angering Shenyang Vey of Teo Shee Fee ep of Chine Dogar of Medial Maly and Pratl, Clee of Bac Medal Sen, China Medel Unive, Shecag Peps Rep of Chins ARTICLE INFO ABSTRACT I ths work, T-Ag sintered alloys CTA) with ferent Ag contents were prepared y 2 spark plasma snterog (SPS) technique, which sa new, effieient and convenient method of power metallurgy. The T-Ag samples were then rated wath a mixture of 0 wis hyérelure aed (HE) and nie aeié (HNO,). The surace features, to release, antibacterial activites and bicompaslises ofthe acideehed Tg CT Ag(AB) samples were ye tematicallycharactrined. The srtace characterization rete revealed the fermation of miropts nd paler with igh Ag contents. Antibacterial experiments demonstrated that the antibacterial ratios ofthe Tg samples Inerezedsgnifcanay afer the ac etching treatment, and the pals with high Ag contents are thowght play a hey roe nthe antibacterial mechani. Bocempasbiliy experiments inicated tht the cl profes tion, cell morphology, and oteoblas ferentiation ei nat signfcamyeffer Bere the pore tani ep “T) and TrAg(A®) samples. The TeARAE) samples with 30s and Sues A not only parened sztained troduction ‘Tianium (1) and -based alloys are widely ulized as orthopaedic materials due to their excellent corrosion resistance, high mechanical steength and cytocompatbility [1]. Moreover, these materials also possess a superior fatigue strength, a high wear resistance, and a similar ‘Young’ modulus to that of natural human bone [1, 2]. However, im plant-associated infection (IAD) is one of most serious and common Postoperative complications that arises from the absence of ant ‘bacterial activity in Ti. In recent years, the occurrence of postoperative infection after arthroplasty oF fracture Fixation has ranged from 2% to 5 [3] IAT can in tur lead tothe removal of implants, radical surgical debridement and prolonged courses of antiblote therapy [2, 4}. These factions oflen result in exteme and unnecessary suffering and even, lsabilty (3), and the incurred treatment costs inthe United States has heen estimated tobe as high as $27 billion per annum (5]. Incomplete preoperative disinfection, non-standard protocols during surgical pro ‘cedures and the transfer of bacteria from infected tierue and blood ‘adjacent tothe implant surface ean result in IA [6]. Upon reaching the Implant surface, bacteria adhere to it and colonize, after which they synthesize a complex glycocalyx and form a biofilm, which can protect ‘the bacteria from the immune system of the host and antibiotle teat ments, leading (IAL [7]. To prevent the initial bacterial adherence and subsequent formation ofthe biofilm, iis necessary to endow Ti-based. implants with selfantibacterial properties to reduce the extensive burden of disease interme of both financial and human costs ‘One of the curren strategies for inhibiting IA ie modification ofthe Ti surface through ion implantation [5], plasma spraying [9], anodi- zation {10] and coating with antiboties [11 or antimicrobial polymers [12]. In addition to surface modification of Ti surfaces with anti- bacterial metal ions or nanoparticles [13, 14], another import strategy for reducing the occurrence of bacterial infections is the in= corporation of anubacterial metalic material, Ca and Ag, known and ‘sed at effective inorganic antibacterial agents, are frequently added to retalic materials to prevent bacterial infections. T-Cu alloys have ‘been reported to possess a strong (99%) antibacterial behaviour against Staphylococcus eureus(S. cures) and Escherichia coli (E.coli, but a copper content of atleast Swe was required [15]. Inorganic Ag may Shenyang 11001, Popes Repble of Ci eed september 2017, Received in eed far 12 May 2048; Accepted 13 June 2028 ‘niable one 1 Jane 2018 (0928:4981/ © 2018 Feever BY. Alrighe reservebea better choice Because ofits stronger antibacterial activity (7), and 4 small amount of Ag has been shown to significantly increase the Uibacterial properties of metalic materials, For example, an ant bacterial ratio of nearly 100%% was achieved against F. coli by adding (03 wits Ag to sailess steel (15). Moreover, Ag has been extensively used in antibacterial materials due to several unique advantages, including a broad antibacterial spectrum [17], 8 small possibility for developing resistant stains [18], low cytotoxicity [8] and satisfactory stability under physiological ‘oneitions [18]. Owing to these characteristics, Ag has been proposed for the prevention of infection in various medical products and in stuments, sich as urinary catheters (20), drainage catheters (211, megaprostheses [22], sealpels and needles (23). However, the anti: bacterial effects of most reported as-cast Ti-Ag alloys ae limited. Ka ‘ako Nakajo etal. [24] reported that an as-cast T-20 wt Ag and Ti 25 we. Ag alloy suppressed biofilm formation but was not bactericidal Furthermore, the bacteriostatic effec ofthe material was not satisfac tory, and the high Ag content would create high production costs. Y. F. ‘Zheng etal, (25] found thet @ TIN alloy exhibited an 86.2% anti bacterial ratio agsinst § aureus when 1.4 t% Ag was added, Further more, a powder sintering technology has been recently usd to fabricate ‘cAg alloys, which achieved good antibacterial properties with a small Amount of Ag, Mian Chen etal. 26] reported an antibacterial ratio of 198.2% for 4 Tis wi%s Ag alloy prepared by a conventional sintering ‘method. In addition to powder metallurgy, surface modification has also been used to improve the antibacterial activities of as-cast TiAg alloys. Kang et al [27] used surface modification by large-grit sand blasting and acid etching to obtain satisfactory antibacterial ratios (© 90%) for a5-ast Ti alloys with low Ag contents (4 wt) In the present study, T-Ag alloys were fabricated by a spark plasma sintering (SPS) technique instead of the conventional sintering method. the SPS technique is the mos efficient and convenient sintering ech nology available, and it has advantages of fast densifiation and minimal rain growth [28]. In contrast to the conventional sintering method, the heat created in the gape between particles by the joule effect from electri current pulses accelerates the densification kinetics and enhances the atom diffusion to more quickly complete the reaction (28, 29]. Im addition, biocompatibility should not be sacrificed when improving the antibacterial activity of biomaterials. Therefore, an acid ‘etching treatment, which hed been reported to form mieropits on the surface and promote cell adhesion and osseointegration, was used to modify the Ti-Ag samples (50, $1]. The antibacterial properties an Diocompatibiitis ofthe samples were then systematically investigated In vitro, the acid-etched ‘leAg (Il-Ag(AE)) sintered alloys were ex pected to have longterm antibacterial abilities and be non 90%, After culturing for 1 day, the samples were washed re- peatedly and thoroughly with PBS co collect both nonadherent and adherent bacteria. The viable bacteria in the PBS were quantified by standard serial lution and plate counting based on the National Standard of China (GB/T 4789.2-2010) [22]. Afterwards, the samples were utasonically cleaned, dried and immersed in 2m of PBS at 37°C for 30 8 um). With increasing Ag content in the TLAGCAF) samples, che were determined to be 1 5:26 By EDS,Table 1 DS rent for TARA. Stelaceroughnes ofthe ep, Teg, THAR, and THAS(AR) samples, Semple Reiom 5) km angia®) as 8 gon ast = ood Fig. 8 shows a igh magnfieation image of THSAR(AE), and Pig. tb-d shows the element mapping images ofthe particles (Atea A). The Ag. ‘ontent, which was analysed by EDS (spot mode), was found to vary within the particles, as shown in Fig. 3b-d. Within the same particle, approximately 67.25 8 Tl and 32.75 a9 Ag were detected a Point 1A whereas approximately 93.03 9% Ti and 6,97 3% Ag were ée- tected at Point B (Table 1), The atomic rato of T to Ag at Point A eas approximately 2:1, which indicates that this portion of the particle corresponds tothe Ti,Ag phase observed in the XRD pattern. Point Cin another particle contained 59.07 at 9% Ti and 40.93 at Aa, indicating that cifferene particles had different Ag contents. From the EDS and ‘XRD results, ¢ was deduced that Points B and C ere located on a T-Ag intermetallic phase, which doesnot form a stoichiometric composition. In general, the above results demonstrated that the particles on the surfaces of the TLAQ(AE) samples contained a significantly higher Ag Surface roughness (Ra) (um) Samples Fig. 5, Surface roughness (Ra) of the ep, TAG, sT(AB, and THAg(AB) samples. ""p = 001, content than the substrate and consisted of Tishg and a Teg inter- setalic phase The values of the average roughness (Ra), root mean square de- vation (Rq) and peak-to-valey roughness (Rx) for the samples are presented in Table 2 and 115. The surface of the -TI(AR) sample was the roughest among all samples (p < 0.01). "The sutface roughness ofthe T-Ag(AE) samples was markedly higher than that of {the ep-T and TieAg samples (p < 0.01). The surface roughness of the cp-Tl and Ti-Ag samples were nearly the same due to thei teatment by the same grining method (p > 0.05). Among the T-Ag(AE) samples, the surface roughness followed the order T-IAg(AE) > T3Ag (AE) > T-SAR(AE), However, there war no significant difference in Sere Fig. 4. CLSM images ofthe surfaces ofthe ph, Th, eTIAB), and TEARIAE) samples a) «pT, B) TAA ©) TAA € T-5Ag, 6 THAR, 0 THIAGIN 2AgAE),b) TSAR IT‘Water contact angle () Samples ig 6, Water contact angles ofthe «pl, ThAg, ICAI, and TARA sa ples tp = 003, ""p = 00) -e@ THAg = THHIAg(AE) “= Ti3Ag + Ti3Ag(AB) i THSAg -O THSAS(AE) Ae! concentration (ppb) 1D 4D Immersion time (D) Fig.7. Coeentaion ofA one leased from he TL.Ag and TLARAR) sample ™» the roughness between the @ > 0.05) ig 6 shows the results of the water contact angle measurements of the ep, T-Ag, STAB), and TAQ(AE) samples. The water contact angles ofthe samples not treated by acid etching were higher than those ofthe acid-etched samples except inthe case of T-SAR(AE) (P< 0.05). ‘Among the acid-etched samples, the contact angle increased with in ‘ressing Ag concentration. Moreover, the water contact angles of the and Ti-1Ag(AS) samples were smaller than those ofthe other samples (p = 0.05), However, the cfference in the water contact angle be tween sTI(AR) and. T-IAG(AR) was not statistically significant TILAR(AE) and T-3Ag(AE) samples Ma Sie & Engng € 82 208) 121-181 Log(gurrenty/A —S-THAE) THIAQ(AB) oa 2-10 08 0.6 Voltage’ 4 02 00 Fig. 8. Tafel curves of the ep, Tha, THAD, and T-AQAR) spl. ( > 005). Thus, among the TAS(AE) samples, the hydrophilicity followed the order T-IAR(AE) > Ti-2Ag(AE) > T-SAR(AR) Fig. 7 shows the concentration of Ag ions released from the TAg samples upon immersion in a 0.9% NaCl solution on days 1, 4 and 7. For both the Ti-Ag and TT-Ag(AE) samples, the amount of Ag ions re- leased increased with increasing Ag content, and the amounts released, from the T-Ag(AR) samples were higher than those released from the TIhAg samples, The concentration of Ag ions released from the TAg samples was steady from day 1 (07. In conteat, the TLAR(AE) samples Initially released a lage amount of Ag tons, and the release then gre ually decreased, Hig. 8 shows the Tafel curves acquired fo the ep-T, Tig, 0.05). However, com- pared tothe T-Ag samples without acid etching, the acd-etched T-Ag, samples had significantly beter antibacterial ratios (P< 0.01), After Immersion ofthe samples in PBS for 30 days, the antibacterial ratios of| the TIIAg(AE), TI-3AR(AE) and T-SAg(AE) samples didnot obsiouslyBroa Ma Sie & Engng € 82 208) 121-181 Fig 9S ewe colonies on the ep, TA, UAE), and THAg(AE) sample after 20 dys of immersion in PRS) 0.05) between the ep-Ti and THAg(AE) samples throughout the culture time, although a proliferation trend of THLAS(AE) > Ti-3AQ(AE) > TESAG(AR) > epTi was detected. The Viabiliy% values of the TI-Ag(AE) samples were all above 100% (75%) theoughout the culture time, which meant that the TAG(AB) samples were non-ytotoxle according to 180 10893-5 [53} The morphologies ofthe cll onthe samples were observed on days 1 and 3 by FE-SEM, as shown in ig. 14. On day 1, the cells on ep-Ti ‘were well spread, while those on the acid-etched samples exhibited more filopodia However, aftr culturing for 3 days, the eels occxpied a larger area than thoce from day 1. The eell on all-day samples were well spread vell with many filopodia and lamellipodia, and che dif ference in the cell morphology was not obvious. ‘The MC3T3-E1 osteoblastic differentiation on the different samples was assessed based on ALP production, as ALP is regarded as an early smatker of osteogenic differentiation [18]. The ALP aetivtis of the MCSTS-EI cells after culturing on the diferent samples for 7 days arePrentn) Ma Sie & Engng € 82 208) 121-181 Fig. 11. CLSM images ofS: aueu cultured onthe eT Hg, TAB), and THAg(AE) samples er 1 day aller Guoreseent staining a) eb) T-LAg, TEAS 2) THAR, ©) eTWAH), D THAG(AB), ¢) TARE), B) THSAR(AB, ¥ Fe Fig. 12. SEM morphology ofS. eure cultured onthe cp, Teg, THAR), TASCA samples for I day) pt TAS €) TAG) THA ©) THAR), THIAG(AF, 2) T-3AQ(AP,b) T-SAg(AP) shown in Fig. 15, There was no significant difference (p > 0.05) inthe ALP production between the ep-T and acid-etched samples, suggesting, that the ace etching treatment had no influence on cell diferentition. ‘The above results indicated that the TI-Ag(AE) samples possessed ex cellent eytocompatibility without cytotoxicity Biomedical IAls are one of the most important challenges for oF ‘thopaedic surgeons [35, 36]. In addition to short-term antibacterial activity, long-term antibacterial activity is especially necessary for implants because of the constant risk of bacterial infection (37, 38] ‘Strategies that can endow biomaterials with long-lasting antibacterial effets without impairing the bioeompaubility are being atively pur ‘sed, Here, we wed a simple and economical method t fabricate Ag, alloys with long-term anubacteril activity and excellent biocompate ‘bility, Under equilibrium solidification conditions, a TioAg phase will form in Ti-Ag alloys when the concentration of Ag is < 20 wt hased on the binary phase diagram (39]. In our work, Ti and Ag powders veere sin tered at 900°C, which is below the Ti and Ag melting points, indicating, that only solid reactions occurred between the Tl and Ag powders ‘through physical diffusion. Mian Chen etal. [25] reported that a TA. phase and/or Tig intermetallic phase formed in T-Ag alloys sintered ft 900°C when the concentration of Ag was below § wide and thatthe Antibacterial mechanism of the TiaAg phase relied on direct contat between the Tig surface and the bacteria. According to the XRD re- salts, the T-Ag samples prepared by SPS also formed a TizAg phase Unfortunately, unlike the results of previous studies, the TI-1Ag. T-3Ag. and Ti-Stg samples in our work exhibited only alow anubacteral rao,mm pT TEE STAR) GED THAQ(AE) TrlAQ(AE) EEG Ti-SAg(AE) 04 (FL im 1D 3D cy ‘Time of culture (D) Fig, 13. Viblty and proliferation of MCSTS-E1 eels on the ep, THA, and THAg(AB staples ater cltrig for 1,2 and 7 days Which could be caused by the use of a diferent sintering method ‘Compared to the traditional sintering method, the SPS system drama tically shortened the sintering time from 3 to 10 min, which led to an incomplete resetion. In addition, during the sintering proces, the Va ‘coum inthe SPS chamber was approximately 7Pa weaker than that in the traditional sintering method, which likely resulted in slight oxida- tion of the material and influenced the antibacterial activity LEP has ability to dissolve the oxidation layer generated during the simering process, and the mixture of HP and HINO, created micro features on the sample surface (30,40). As shown in Fig. 10, after acid ‘etching, the TH-Ag sample shoved high antibacterial ratios. Based on ‘the XRD diffraction pattern shown in Fig. 1 andthe surface morphology shown in Fig. 2, the acid etching treatment did not change the phase ‘composition and le tothe formation ofa porous layer ané particles on the surface, From the EDS investigation, the particles generated by acid fetching. contained a high conceatration of Ag. Many studies have proven that Ag-containing particles exhibit excellent antibacterial ac tivities (9, 27, 41-43]. Mian Chen eal. (26] found that only 2 physical lfsion reaction occurred for the Ti powder and Ag when the sintering, temperature was below the Ti and Ag melting points, and the Th Ag phase was formed at the regions with high Ag concentration. Ad 0.08). Thus, it ean be concluded that the TARCAE) Ma Sie & Engng € 82 208) 121-181 samples did not impair the cell structure and fanetion and therefore bhave potential for use in vivo, Moreover, as shown in Figs. 13 andl 14, cell proliferation was highest on the s-TW(AE) sample, and the cel, spread was extensive. The STW(AE) and Ti-AgtAP) samples possessed {004 biocompatbilities, not only because ofthe low concentration of releazed Ag” but also because the acié etching treatment led to 2 porous structure onthe surface, which improved the surface roughness and hydrophilicity [53, 54]. A porous stueture, high roughness and high hydrophilicity have been reported to de beneficial for the re- sponses of eels in contact with the implant [55-57]. However, it should ‘be mentioned that the acid eiching treatment didnot significantly im prove the cell proliferation abilities and osteogenesis on the TI-Ag(AI samples as expected, Thus, a better acid etching method should be in- vestigated in subsequent studies. 5. Conclusion ‘ToAg samples were fabricated by an SPS system. The antibacterial satios of the Ti-Ag samples before acid etching were limited and un- Satisfactory. After the acid eithing treatment, mieropits and particles ‘ith high Ag contents were formed on the surfaces ofthe TI-Ag(AE) samples, and the TI-3AgIAE) and TL5Ag(AB) samples showed excellent Antibacterial activites, After immersion for up to 30days, the TI-3Ag. (AB) and TH-SAg(AF) samples still showed antibacterial rotor higher ‘than 96%, indicating that they possessed long-term and effective anti- bacterial activities. Moreover the T-3Ag(AB) and Ti-SAg(AE) samples also exhibited good biocompatbiltes, which is beneficial for pro longed clinical use. ‘Acknowledgements ‘This work was supported by the National Natural Seience Foundation of China (grant No. 81671811, 81501857), the Special Research Foundation ofthe Health and Family Planning Commission of Liaoning Province (LNCCC-A03-2014), the Science and Technology Project of Shenyang city (17-230-9-09, F15-139-9.23, F16.206-9-21, 2015020274) References 117. hen, GA Roan Stic pin Biomater, Mater. Sek Eng 8 Re. 97 (Coro dour, Daur apa, Nea, KR. Sy S aie 0 God) tas 1008, ° ie nee 9 (3 (2001) 8793, Sates Gu J. Geaier RA Sagel, T. Lib KM Mess i onie mo {esa eneale Bats 952) GOL 42h Sel tng later io Ape el (2016) 965.975 [init coo ¥. han Gin M Geng. 2 Vang Y-Sang 2 An X Pag. 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