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Nanobiomaterials inClinical Dentistry

Second Edition

Edited by

Karthikeyan SubramaniAdvanced Education in Orthodontics & Dentofacial Orthopedics,

College of Dental Medicine, Roseman University of Health Sciences,Henderson, NV, United States

Waqar AhmedSchool of Mathematics and Physics, University of Lincoln,

Lincoln, United Kingdom

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CHAPTER

17Bioactive inorganic-ion-doped titania nanotubecoatings on bone implantswith enhanced osteogenicactivity and antibacterialproperties

Kaifu Huo1,3, Na Xu2, Jijiang Fu1 and Paul K. Chu31The State Key Laboratory of Refractories and Metallurgy, Institute of Advanced Materials and

Nanotechnology, Wuhan University of Science and Technology, Wuhan, P.R. China 2College of

Life Sciences and Health, Wuhan University of Science and Technology, Wuhan, P.R. China3Department of Physics and Materials Science, City University of Hong Kong, Hong Kong,

P.R. China

17.1 INTRODUCTIONOsteoporosis and bone fracture have become a worldwide problem with an aging

global population and the increase in average life expectancy. New artificial

implantable devices with enhanced mechanical and biological performance are

required in clinical surgery. Titanium (Ti) and its alloys are widely used as bone

implant materials and are expected to be the next generation of artificial orthope-

dic biomaterials due to their high strength, low modulus close to bone

(4�30 GPa), good biocompatibility, and high corrosion resistance [1]. However,

the natural titanium oxide film formed on the titanium surface makes it bio-inert

and difficult to bond with bone tissues. Furthermore, bacterial infection occurs

occasionally even in sterile circumstances. Implant failures with undesirable bone

integration, poor bone quality, or infection have been observed in clinics.

To improve the osseointegration of Ti implants, much attention has been

focused on the modification of implant surfaces. Considering that bone tissue is a

complex system with nano-/microcollagen fibers and nutrient chemical ions, it is

an effective way to develop nano-/microtopographical features doped with inor-

ganic ions on Ti surfaces for enhancing the bioactivity of Ti implants. Hence, tita-

nia nanotubes (NTs) with controllable nanoscale properties have been developed

on the Ti surface by anodization. The nanotubular topography not only mimics

the dimension of collagen fibril in bones to some extent [2,3], but also is an

Nanobiomaterials in Clinical Dentistry. DOI: https://doi.org/10.1016/B978-0-12-815886-9.00017-6

© 2019 Elsevier Inc. All rights reserved.401

https://doi.org/10.1016/B978-0-12-815886-9.00017-6

excellent drug-delivery platform, especially for inorganic ions [4]. Firstly, inor-

ganic ions are much smaller molecules than growth factors and antibiotics and

function at very low doses. Long-lasting activity can be achieved by increasing

the loaded amounts and controlling the release rate appropriately. Secondly, these

agents are stable due to their inorganic nature, thereby facilitating the use of load-

ing processes and loading methods that tend to have harsh conditions. Thirdly,

the stable properties of the agents may also permit relatively long storage after

fabrication of the implants and it is important for commercial adoption.

Many inorganic ions involved in bone metabolism, such as Sr, Zn, Mg, and

Ca, have been doped into NTs. The coatings of inorganic-ion-doped NTs have

been found to foster the growth of nano-structured hydroxyapatite in simulated

body fluids [5,6], enhance extracellular matrix secretion, mineralization, and other

functions of osteoblasts, and even induce the commitment of mesenchymal stem

cells (MSCs) toward bone lineage in the absence of extraosteogenic supplements

(OS) [7,8]. Emerging in vivo evidence also suggests the ability of NT coatings to

enhance osseointegration [9�12].

Implant-associated infection, which is another issue impairing the normal func-

tion of bone implants, is usually difficult to treat and sometimes requires implant

removal and repeated revision surgeries [13]. Various means, such as thorough

sterilization and stringent aseptic surgical protocols, have been proposed to miti-

gate bacterial contamination. However, bacterial invasion usually occurs after sur-

gery and complications can arise from infection of nearby tissues or a

hematogenous source at a later time. Infections associated with bone implants are

characterized by bacterial colonization and biofilm formation on the implanted

device and infection of the adjacent tissues (peri-implantitis). Bacteria in the bio-

film are far more resistant to antibiotics, resulting in persistent infection despite

aggressive antibiotic therapy [14]. As emerging antibiotic resistance becomes more

challenging, developing novel implants or surface modification methods with dual

functions of excellent bone bonding ability and long-lasting antibacterial ability

through a procedure ready for industrial production and clinical application is the

need of the hour in implant dentistry. Silver ions or nanoparticles are good antibac-

terial agents and have been doped into NTs. The antibacterial ability in vitro and

in vivo of silver-doped NT coatings has been thoroughly studied [15,16].

In this chapter, we summarize the latest progress on coatings of inorganic-ion-

doped TiO2 NTs and review the fabrication methods, the effects of the coatings

on bone cell functions in vitro, osseointegration in vivo, and antibacterial ability.

17.2 FABRICATION OF INORGANIC-ION-DOPED TIO2NANOTUBES

Inorganic-ion-doped TiO2 NTs are commonly fabricated through electronic anodi-

zation and following a hydrothermal treatment process. The fabrication methods

and mechanisms are detailed as follows.

402 CHAPTER 17 Bioactive inorganic-ion-doped titania nanotube coatings

17.2.1 ANODIZATION FOR DEVELOPING TIO2 NANOTUBES ON TI

Uniform and highly ordered NT arrays can be readily fabricated by anodization

of a Ti foil in F2-containing electrolytes. The as-anodized NTs, which grow in

situ on the Ti substrate, are highly oriented perpendicular to the Ti surface [17].

The NTs are generally fabricated in the aqueous hydrofluoric acid electrolyte in a

two-electrode electrochemical cell at a constant potential between 5 and 35 V

[5,18,19]. At a low anodization voltage of 5 V, the morphology of the anodized

film is sponge-like, with a typical pore size of 25 nm. At 20 V, hollow and cylin-

drical tube-like features with an inner diameter of 80 nm form (Fig. 17.1).

Besides the HF/H2O electrolyte, many other aqueous electrolytes have also been

developed to fabricate NTs, for example, H2SO4/NaF/H2O [3], NaH2PO4/HF [6],

and Na2SO4/HF [6]. The diameter of the NTs can be regulated to vary from 15 to

140 nm and the length can range from 200 to 1000 nm by adjusting the electrolyte

content and anodization voltage [17]. When using polar organic electrolytes,

much longer NTs of hundreds of micrometers can be fabricated [20]. Ethylene

glycol (EG) is the most commonly used organic solvent to fabricate NTs. The

typical NTs formed by anodization of a titanium foil in an EG solution with

0.5 wt.% NH4F, 5 vol.% CH3OH, and 5 vol.% H2O at 10 V for 1 hour and 40 V

FIGURE 17.1

(A) and (B) NTs formed at 5 and 20 V in 0.5 wt.% aqueous hydrofluoric acid solution with

tube diameters of 25 and 80 nm, respectively. (C) and (D), NTs formed at 10 and 40 V in

an EG solution with 0.5 wt.% NH4F, 5 vol.% CH3OH, and 5 vol.% H2O with tube diameters

of 30 and 80 nm, respectively. NT, Nanotube; EG, ethylene glycol.

(A) and (B) Reprinted with permission from L.Z. Zhao, S.L. Mei, W. Wang, P.K. Chu, Z.F. Wu, Y.M. Zhang, The

role of sterilization in the cytocompatibility of titania nanotubes, Biomaterials 31 (2010) 2055�2063 [18].

40317.2 Fabrication of Inorganic-Ion-Doped TiO2 Nanotubes

for 40 minutes are shown in Fig. 17.1C and D, respectively. The two NTs with

diameters of 30 and 80 nm have been used to incorporate various inorganic ions

[21,22].

17.2.2 HYDROTHERMAL TREATMENT FOR DOPING INORGANIC IONS

Many divalent inorganic ions (such as Sr, Zn, Ba, etc.) are incorporated into crys-

talline/amorphous TiO2 NTs through a hydrothermal treatment method [9,11,23].

The composites exist eventually in the form of perovskite-type titanates MTiO3

(M5 Sr, Zn, Ba, etc.). In the hydrothermal process, pH in the solution is demon-

strated to play important roles in the morphology and composition of the hydro-

thermal products [24]. Fig. 17.2 shows the morphology changes of amorphous

TiO2 NTs after hydrothermal treatment at 200�C for 6 hours in solutions with dif-

ferent pH. In HCl solution (pH5 3), nanorods are composed of compact nanopar-

ticles with a large diameter the same as previously formed NTs. In DI water

(pH5 6.5), some small NPs with a diameter of 40 nm are formed in nanorods. By

further increasing the pH to 11, smaller NPs of 20�30 nm in diameter are

FIGURE 17.2

Top and cross-sectional FE-SEM micrographs of amorphous TiO2 NTs after hydrothermal

treatment at 200 oC for 6 h in water with different pH values: (A) pH5 3, (B) pH5 6.5,

and (C) pH5 11. (D) The corresponding XRD pattern of (B). (E) and (F) TEM and HR-

TEM images of an NR and NP in (B). The pH value is adjusted with 1 M HCl and 1 M

NaOH and the inset scale bar is 500 nm. NT, nanotube.

Reprinted with permission from X.M. Zhang, B. Gao, L.S. Hu, L.M. Li, W.H. Jin, K.F. Huo, et al.,

Hydrothermal synthesis of perovskite-type MTiO3 (M5Zn, Co, Ni)/TiO2 nanotube arrays from an amorphous

TiO2 template, CrystEngComm 16 (2014) 10280�10285.


observed on the surface. At higher pH (pH5 12.2, 0.02 M NaOH), the nanorods

are dissolved and nanosheets begin to from.

The principle of perovskite-type titanates MTiO3 formation with the hydro-

thermal reaction involves a hydroxy radical-induced dissolution and recrystalliza-

tion with a combination of metal ions. The amorphous TiO2 NTs are not stable in

water and the unstable TiO622 absorb water molecules via the surface hydroxyl

groups to form soluble species of Ti(OH)622, which can be further dehydrated

and precipitated to form crystal anatase TiO2 nanoparticles or MTiO3 nanoparti-

cles when M21 exists. The overall reaction is described as follows [25]:

TiOx 1 4H2O1 12x

2

� �O2-Ti OHð Þ226 1 2H1ð1, x, 2Þ (17.1)

Ti OHð Þ226 1 2H1-TiO2k1H2O (17.2)

or

Ti OHð Þ226 1M21-MTiO3k1 3H2O (17.3)

where M is the metal source. In order to form MTiO3 precipitates, process (17.2)

should be controlled, as reactions (17.2) and (17.3) are competitive processes.

Zhang et al. propose acetates of metals as reaction precursors and fabricated

ZnTiO3, CoTiO3, and NiTiO3 NTs by hydrothermal treatment [24]. In the reaction

process, weak acid radicals supplied in these solutions consume H1 and restricted

process (17.2). Therefore, process (17.3) is prompted for facilitating the produc-

tion of MTiO3 precipitates.

17.3 OSTEOINDUCTIVE ACTIVITY OF INORGANIC-ION-DOPEDTIO2 NTS

17.3.1 STRONTIUM-DOPED TIO2 NANOTUBES

Strontium (Sr), of which 98% in the human body can be found in the skeleton,

plays an important role in bone metabolism [26]. Strontium ranelate is a clinical

drug for treating and preventing osteoporosis in postmenopausal women [27].

Many experimental studies in vitro and in vivo have proved that strontium rane-

late can stimulate osteoblast differentiation, inhibit osteoclast formation, and

rebalance differentiation of bone marrow MSCs [28�31]. Therefore, strontium

has been widely incorporated into bone and dental implants, including biocera-

mics, cement, and metals, to improve the osteoinductive activity of scaffolds

[32�34].

As mentioned in the Section 17.1, TiO2 NTs are ideal carriers for inorganic

ions. Huo and his cooperators have fabricated a series of Sr-doped TiO2 NT sur-

faces and explored their bioactivity and osteointegration ability [12,22]. By con-

trolling voltage and anodization time in the anodization process and the

40517.3 Osteoinductive Activity of Inorganic-Ion-Doped TiO2 NTs

concentration of Sr(OH)2 in the hydrothermal process, NTs with diameters of

30 nm (NT10) and 80 nm (NT40) and different contents of Sr are fabricated

(Fig. 17.3) [22]. Initial adherent cell numbers and cell cytotoxicity characterized

by lactate dehydrogenase on different surfaces show no significant difference.

This demonstrates that Sr incorporation has no obvious influence on cell adhesion

and cell cytotoxicity. NT40 and NT40-Sr induce more protein deposition but the

proteins distribute unevenly compared to NT10 series. This is because the top

ends of the NT wall of NT10 and NT10-Sr are smoother than in the NT40 series.

Cell morphology and cell migration are closely related with surface topography.

It is demonstrated that a distance smaller than 50�70 nm between two neighbor-

ing integrins is favorable for integrin clustering and focal adhesion formation for

further cell spreading or migration [35�37]. Therefore, cell motility and cell

spreading on NT40-Sr are both better than NT40 due to the proper size of nanoto-

pography improved by protein distribution (Fig. 17.4). The influence of NT

FIGURE 17.3

FE-SEM images of NT and NT-Sr. NT, Nanotube.

Reprinted with permission from L. Zhao, H. Wang, K. Huo, X. Zhang, W. Wang, Y. Zhang, et al., The

osteogenic activity of strontium loaded titania nanotube arrays on titanium substrates, Biomaterials 34

(2013) 19�29.


surfaces on osteogenic differentiation of MSCs is a most important indicator to

evaluate the bioactivity of NTs. The mRNA expression levels of osteogenic-

related genes of MSCs on NT-Sr surfaces, including the runt-related transcription

factor 2 (RUNX2, a key transcript factor for bone formation), alkaline phospha-

tase (ALP, an early marker for osteogenic differentiation), osteocalcin (OCN, a

late marker for osteogenic differentiation), type I collagen (Col-I, main content of

bone ECM), and bone morphogenic protein 2 (BMP-2), are significantly upregu-

lated in the absence of osteogenic serum. The ALP staining and extracellular

matrix mineralization are also detected to evaluate the osteogenic differentiation

of MSCs (Fig. 17.5). Sr-doped NTs show more ALP staining and calcium nodules

indicating stronger osteogenic differentiation of MSCs than NTs.

17.3.2 ZINC-DOPED TIO2 NANOTUBES

Zinc is an essential trace element in the human body and its weight is about

1.4�2.3 g on the biochemical level [26]. The zinc content of bone is about

FIGURE 17.4

Fluorescence images showing: (A) morphology of GFP-labeled MSCs after 8 days of

culture and (B) cell migration on different samples. MSC, Mesenchymal stem cell.



(2013) 19�29.


0.015�0.025 wt.% of the total amount, which is relatively high compared to other

tissues. Zinc has been known to play important roles in many physiological beha-

viors, including normal growth, immune functions, and neurodevelopment. Zinc

is involved in various cellular signaling pathways through 1400 zinc-finger pro-

teins accounting for a large part of the transcription regulatory proteins [38]. In

bone, zinc participates in bone matrix calcification, stimulates bone apposition by

osteoblasts, and inhibits osteoclastic resorption [39�41]. In addition, zinc pos-

sesses effective antibacterial ability [42,43]. Considering the dual functions of

zinc, it has been incorporated into various bone implants to enhance the osteo-

genic activity and antibacterial ability of implants [44].

Zinc-doped TiO2 NTs with diameters of 30 nm (NT10-Zn) and 80 nm (NT40-

Zn) have been fabricated and their osteogenic and antibacterial function have

been detected by Huo et al. [21]. The loading amount of zinc on NTs in the

reports ranges from 1.2 to 60.2 μg/cm2, which is safe for the human body. As

excessive zinc uptake brings risks of anemia, leucopenia, and neutropenia [45],

and so the total loading amount is an important factor to be considered.

Antibacterial ability of NT-Zn samples toward adherent bacteria and planktonic

bacteria are shown in Fig. 17.6. The NT-Zn samples show higher antibacterial

FIGURE 17.5

(A) ALP product and (B) extracellular matrix mineralized nodules generated by MSCs

cultured on different samples. ALP, Alkaline phosphatase; MSC, mesenchymal stem cell.



(2013) 19�29.


efficiency compared to NTs with the general trend of NT40-Zn3.NT10-

Zn3.NT40Zn1.NT10-Zn1. The antibacterial ability of NT-Zn samples is

related to the loading and release contents of Zn. The antibacterial ability of NT-

Zn samples decreases gradually with time and Zn loading content, which is the

same as the trend of Zn release kinetics. The antibacterial mechanism of Zn is

considered to be related to the production of reactive oxygen species [44].

Besides antibacterial ability, the osteogenic abilities of NT-Zn samples have

also been investigated. In the absence of exogenous osteoinductive serum, most

NT-Zn samples enhance the osteogenic differentiation of MSCs, characterized

with more dense nodular ALP areas and higher ECM mineralized nodule forma-

tion [21]. MC3T3-E1 mouse preosteoblasts are also used to detect the osteogenic

ability of NT-Zn samples [9]. Except the above characterization, the expression

levels of osteogenesis-related genes, including ALP, OCN, OPG, and Col-1, were

estimated by qRT-PCR assay. NT-Zn samples exhibit higher mRNA levels for all

osteogenic-related genes (Fig. 17.7).

ERK1/2 signaling is believed to play an important role in the osteogenic dif-

ferentiation of MSCs and found to be modulated by different biomaterials [46].

NT-Zn samples show a higher ERK expression degree compared to NT samples

(Fig. 17.8). The biological effects of NT-Zn samples are ascribed to the combined

effects of the topographical cues and Zn release. As discussed in the above sec-

tion on NT-Sr, the NTs within the proper range of diameters facilitate the forma-

tion of focal adhesions. Evidence collected from other tissues and cells indicates

that Zn can initiate ERK1/2 signaling [47], even though there is still no evidence

from MSCs.

In vivo experiment for evaluating the osteointegration of NT-Zn samples fab-

ricated by anodization at 40 V for 40 minutes and hydrothermal treatment for 1 or

FIGURE 17.6

(A) Antibacterial rates versus adherent bacteria on the specimen (Ra) and (B)

Antibacterial rates against planktonic bacteria in the medium (Rp). �, ��P,.05 and .01 vs

NT10; #, ##P,.05 and .01 vs NT40; 1 ,11 P,.05 and .01 vs NT10-Zn1; %, %%

P,.05 and .01 vs NT10-Zn3; $, $$ P,.05 and .01 vs NT40-Zn1.


3 hours has been performed by Li et al. [9]. Ti samples are inserted into the tibiae

of Sprague�Dawley rats aged 1 month. Four weeks later, the rats are sacrificed

for micro-CT evaluation. The transverse 3D images shown in Fig. 17.9 illustrate

that more trabeculae are observed around the NT implants, especially around the

zinc-loaded materials. The quantitative analysis of micro-CT shows that all the

zinc-loaded implants resulted in increased BV/TV, Tb. N, and Conn.D, and

decreased Tb. Sp compared with TiO2-NTs (Table 17.1). Unlike in vitro cellular

experiments, the differences between various Zn loading capacities are not statis-

tically significant. The authors test the biomechanical ability through a push-out

test. NT-Zn implants exhibit significantly increased strength of fixation compared

to Ti and TiO2-NT (Table 17.2), indicating better osteointegration ability than Ti

and TiO2-NT.

0.0

0.1

0.2

0.3

0.4

0.5(A) (B)

(D)(C)3.5

3.0

0

2

0

1

2

3

4

5

6

TiTi TiO2-NTsTiO2-NTs NT-Zn1hNT-Zn1h NT-Zn3hNT-Zn3h

Ti

**

**

**/##

**/##

**/##

**/##

**/#

**/#**/#

**/#

*

TiO2-NTs NT-Zn1h NT-Zn3h Ti TiO2-NTs NT-Zn1h

mR

NA

CO

L-1/

AC

TIN

mR

NA

OP

G/A

CT

IN

mR

NA

OC

N/A

CT

IN

mR

NA

ALP

/AC

TIN

NT-Zn3h

4

6

8

10

12

2.5

2.0

1.5

1.0

0.5

0.0

&

FIGURE 17.7

Relative expressions of (a) ALP, (b) OCN, (c) OPG, and (d) Col-1 by MC3T3-E1 cells

cultured on different substrates for 2 weeks, all values normalized to β-ACTIN. �P,.05,��P,.01 compared with Ti, #P,.05, ##P,.01 compared with TiO2-NTs, &P,.05

compared with TiO2-Zn1h. The data are expressed as mean6 SD (n5 4). ALP, Alkaline

phosphatase, OCN, osteocalcin.

Reprinted with permission from K. Huo, X. Zhang, H. Wang, L. Zhao, X. Liu, P.K. Chu, Osteogenic activity and

antibacterial effects on titanium surfaces modified with Zn-incorporated nanotube arrays, Biomaterials 34

(2013) 3467�3478.


FIGURE 17.8

Western blot of pERK1/2 and ERK1/2 levels in the MSCs cultured on the different samples

for 48 h. β-Actin is used as a control for equal loading. MSC, Mesenchymal stem cell.

Reprinted with permission from K. Huo, X. Zhang, H. Wang, L. Zhao, X. Liu, P.K. Chu, Osteogenic activity and

antibacterial effects on titanium surfaces modified with Zn-incorporated nanotube arrays, Biomaterials 34

(2013) 3467�3478.

FIGURE 17.9

Micro-CT transverse 3D images of the proximal tibiae with implants after 4 weeks.

Reprinted with permission from Y. Li, W. Xiong, C. Zhang, B. Gao, H. Guan, H. Cheng, et al., Enhanced

osseointegration and antibacterial action of zinc-loaded titania-nanotube-coated titanium substrates: in vitro

and in vivo studies, J. Biomed. Mater. Res. A 102 (2014) 3939�3950.


17.4 ANTIBACTERIAL ACTIVITY OF SILVER-DOPED TIO2NANOTUBES

Silver (Ag) has attracted a lot of attention as one of the strongest bactericides.

Silver shows many advantages compared to traditional antibiotics, such as broad

antibacterial spectrum, effective antibacterial activity to most bacteria including

antibiotic-resistant bacteria, noncytotoxicity at low doses, and low risk of produc-

ing resistant strains [48�50].

Silver nanoparticles have been controllably incorporated into NTs and their

antibacterial capability has been investigated [51�53]. The NT-Ag surfaces show

excellent antibacterial ability for both planktonic and adhesive Staphylococcus

aureus bacteria throughout 30 days, which effectively prevents biofilm formation

(Figs. 17.10 and 17.11) [51].

Table 17.1 Quantitative Results of the Femora With Implants by Micro-CTWithin Volume of Interest 4 Weeks After Implantation

Groups

Parameters Ti TiO2-NTs NT-Zn1h NT-Zn3h

BV/TV (%) 13.316 2.74 19.546 2.52� 27.366 4.53��,# 28.786 5.32��,#Tb. N (mm21) 1.636 0.21 2.256 0.24� 2.816 0.51��,# 2.926 0.34��,#Tb. Th ( μm) 70.346 6.04 68.326 7.03 72.296 8.03 75.326 6.05Tb. Sp (mm) 0.936 0.13 0.666 0.06�� 0.506 0.08��,# 0.466 0.08��,#Conn.D.(mm23)

23.716 2.20 29.686 2.37� 35.906 2.45��,# 37.566 3.58��,#

BV/TV, Ratio of bone tissue volume to total tissue volume; Tb. N, the mean trabecular number; Tb.Th, the mean trabecular thickness; Tb. Sp, the mean trabecular separation; Conn.D, the meanconnectivity density.�P, .05 and ��P, .01 compared with Ti, #P, .05 compared with TiO2-NTs. The data are expressedas mean6SD (n5 16).Reprinted with permission from Y. Li, W. Xiong, C. Zhang, B. Gao, H. Guan, H. Cheng, et al.,Enhanced osseointegration and antibacterial action of zinc-loaded titania-nanotube-coated titaniumsubstrates: in vitro and in vivo studies, J. Biomed. Mater. Res. A 102 (2014) 3939�3950.

Table 17.2 Results of the Biomechanical Push-Out Test 12 Weeks AfterImplantation

Groups

Parameter Ti TiO2-NTs NT-Zn1h NT-Zn3h

Maximal push-out force (N) 21.56 3.2 32.36 5.9� 50.16 9.1��,# 53.66 7.5��,#

�P, .05 and ��P, .01 compared with Ti, #P, .05 compared with TiO2�NTs. The data areexpressed as mean6SD (n516).Reprinted with permission from Y. Li, W. Xiong, C. Zhang, B. Gao, H. Guan, H. Cheng, et al.,Enhanced osseointegration and antibacterial action of zinc-loaded titania-nanotube-coated titaniumsubstrates: in vitro and in vivo studies, J. Biomed. Mater. Res. A 102 (2014) 3939�3950.


In addition, Ag2O nanoparticles have also been doped into NTs through TiAg

magnetron sputtering and anodization [54]. The surfaces exhibit long-term anti-

bacterial activity against both S. aureus and Escherichia coli up to 28 days and

show no significant difference in osteogenic induction for MC3T3-E1 cells after

14 days coculture (Fig. 17.12).

The antibacterial activity of NT-Ag surfaces has also been detected in vivo

through an infected rat model by Cheng et al. [16]. Bacteria are injected into rats

and NT-Ag-coated Ti implants are inserted simultaneously. X-ray examination,

histological analysis, and immunohistochemistry (Fig. 17.13) are used to analyze

the infection degree in rats. Histological analysis (H&E staining) shows that Ti

and NT implants present acute pyogenic infection with an abundant neutrophilic

exudate (white arrow) at 2 weeks, chronic inflammatory cell infiltration (black

FIGURE 17.10

(A) Noncumulative silver release profiles from NT-Ag into PBS, (B) antibacterial rates

against planktonic bacteria in the medium (Rp), and (C) antibacterial rates against

adherent bacteria on the specimen (Ra). The antibacterial assays data are expressed as

means6 standard deviations (n5 3). One-way ANOVA followed by SNK post hoc test is

utilized to determine the level of significance. �P,.05 and ��P,.01.

Reprinted with permission from L. Zhao, H. Wang, K. Huo, L. Cui, W. Zhang, H. Ni, et al., Antibacterial nano-

structured titania coating incorporated with silver nanoparticles, Biomaterials 32 (2011) 5706�5716.

41317.4 Antibacterial Activity of Silver-Doped TiO2 Nanotubes

arrow) at 3 weeks, and intramedullary necrosis including abscess and osteonecro-

sis (red arrow) at 4 weeks postsurgery. In contrast, an NT-Ag implant at 2 weeks

displays a small amount of neutrophilic exudate, which is a normal inflammatory

response because of implantation of the metal rod, and no neutrophilic exudate

can be observed at 3 and 4 weeks. Immunohistochemical analysis proves the exis-

tence of bacteria in Ti and NTs at 2, 3, and 4 weeks, respectively, but no bacterial

survival in NT-Ag group.

Silver ion release is believed to be a key factor for controlling the duration

time of antibacterial activity. Polymers are good adhesives for holding Ag nano-

particles and mitigate the release rate of silver ion. Polydopamine, known as a

reductive agent for noble metals and self-polymerization ability, has been applied

to fabricate a composite film of PDA/Ag [55,56]. The film shows a relatively

slower release rate and a longer-term antibacterial ability towards E. coli and S.

aureus than NT-Ag surfaces. In addition, the antibacterial activity of PDA/Ag

under visible light is higher than that in the dark [56].

The in vitro cell culture assay and in vivo assay have tested the bioactivity of

NT-Ag surfaces [57]. NT-Ag samples are incubated with epithelial cells and

fibroblasts to assess the degree of fibrosis. Cells on NT-Ag surfaces show spheri-

cal shape, exhibit dense filopodia, and secrete abundant extracellular matrix,

FIGURE 17.11

Representative images showing viability of the bacteria on samples after 7 days of

incubation displayed by acridine orange and ethidium bromide fluorescence staining. The

live bacteria appear green, while dead bacteria are orange.

Reprinted with permission from L. Zhao, H. Wang, K. Huo, L. Cui, W. Zhang, H. Ni, et al., Antibacterial nano-

structured titania coating incorporated with silver nanoparticles, Biomaterials 32 (2011) 5706�5716.


indicating that NT-Ag surfaces are inclined to fibrosis formation (Fig. 17.14). The

in vivo assays are performed to evaluate foreign-body reactions. As shown in

Fig. 17.15A, the hematoxylin and eosin staining results show that the reactive

capsule thickness of different samples accord with the trend of PT.NT-Ag-

0.5.NT�NT-Ag-1.0. Meanwhile, the inflammation reaction indicated

by cd68-positive macrophage cells shows the inflammatory trend of

NT-Ag-0.5. PT.NT�NT-Ag-1.0 (Fig. 17.15B). The results imply that

the nanotubular sample with high-dose superficial Ag is more inflammatory than

PT in vivo.

FIGURE 17.12

(A) Antibacterial assay against Staphylococcus aureus and Escherichia coli; ��P,.01

compared to the NT-Ag2O4.67, ## P,.01 compared to the NT-Ag2O7.53, &&P,.01

compared to the NT-Ag2O14.63. (B) Quantitative results of collagen secreted by MC3T3-E1

cells and extracellular matrix mineralization after osteogenic induction for 7 and 14 days.�P,.05 and ��P,.01 compared to the TiO2-NT, #P,.05 and ##P,.01 compared to the

NT-Ag2O1.27, &&P,.01 compared to the NT-Ag2O4.67.

Reprinted with permission from A. Gao, R.Q. Hang, X.B. Huang, L.Z. Zhao, X.Y. Zhang, L. Wang, et al., The

effects of titania nanotubes with embedded silver oxide nanoparticles on bacteria and osteoblasts,

Biomaterials 35 (2014) 4223�4235.


FIGURE 17.13

Histological and IHC analysis of three group of rats contaminating different implants at 2,

3, and 4 weeks postsurgery. Yellow scale bar5 1000 μm.

Reprinted with permission from H. Cheng, Y. Li, K.F. Huo, B. Gao, W. Xiong, Long-lasting in vivo and in vitro

antibacterial ability of nanostructured titania coating incorporated with silver nanoparticles, J. Biomed. Mater.

Res. A 102 (2014) 3488�3499.


A common view for the antibacterial effect of Ag nanoparticles to planktonic

bacteria is the released Ag1 into medium. Silver ions can act as a bridging agent

between thiols forming chains that lead to irreversible aggregation of thiol-

bearing molecules and following denaturation of DNA or peptides [58]. ROS is

another important pathway for the antibacterial activity of silver. Ag1 ions will

disrupt the balance of ROS system, increase intracellular ROS concentration, and

eventually cause cellular death [59]. This is the reason for enhanced antibacterial

effect of PDA/Ag under visible light because more ROS are produced under visi-

ble light irradiation [56]. A recent research reveals that electron transfer between

Ag nanoparticles and metallic substrate can increase intracellular oxidative stress,

FIGURE 17.14

Cell culture on the samples. (A, B) Time-dependent viabilities of (A) human epithelial cells

and (B) fibroblasts on the samples up to 7 days. One-way ANOVA followed by SNK post

hoc test is utilized to determine the level of significance, ��P,.01. (C�J) SEM

observations of human epithelial cells (C�F) and fibroblasts (G�J) after culturing on PT

(C, G), NT (D, H), NT-Ag-0.5 (E, I), and NT-Ag-1.0 (F, J) for 1 day noting the difference in

cellular morphologies between NT-Ag-0.5 and the other samples.

Reprinted with permission from S.L. Mei, H.Y. Wang, W. Wang, L.P. Tong, H.B. Pan, C.S. Ruan, et al.,

Antibacterial effects and biocompatibility of titanium surfaces with graded silver incorporation in titania

nanotubes, Biomaterials 35 (2014) 4255�4265.


FIGURE 17.15

In vivo biocompatibility of the samples: (A) Light micrographs of hematoxylin and eosin-

stained soft tissues previously in contact with the samples at 12 days postimplantation

noting the difference in thickness for the fibrous capsules elicited by various samples.

Scale bars for insets5 50 mm. (B) Confocal micrographs of cd68-positive cells and

counterstained cells from soft tissues previously in contact with the samples at 12 days

postimplantation noting the abundance of cd68-positive cells from soft tissues previously

in contact with PT and NT-Ag-0.5. Scale bars for insets5 50 mm. (C) Thickness of fibrous

capsules elicited and the ratio of cd68-positive cells recruited by various samples. One-

way ANOVA followed by SNK post hoc test is utilized to determine the level of significance,�P,.05, ��P,.01.

Reprinted with permission from S.L. Mei, H.Y. Wang, W. Wang, L.P. Tong, H.B. Pan, C.S. Ruan, et al.,

Antibacterial effects and biocompatibility of titanium surfaces with graded silver incorporation in titania

nanotubes, Biomaterials 35 (2014) 4255�4265.


which enhances the antibacterial activity of Ag nanoparticles [60]. Electron trans-

fer take place when bacteria are in contact with electrically conductive materials

and ROS is believed to be controlled and consumed sequentially in the electron

transfer process. As shown in Fig. 17.16, in the Ag-NPs@Ti systems, the elec-

trons produced by Ti may go to the intracellular part of the bacteria through the

Ag-NPs to disturb the electron transfer process to induce abnormal intracellular

production of ROS [60]. Furthermore, NT-Ag surfaces have been found to

enhance the bactericidal efficiency of antibiotics against various bacteria, even

methicillin-resistant S. aureus [15]. The enhancement of bactericidal activity is

considered to derive from various synergistic effects of Ag and antibiotics

(Fig. 17.17). Ag and vancomycin can both lyze the bacteria cell wall for penetra-

tion of bactericidal agents into the bacterium. The cell wall destruction caused by

Ag effectively helps other antibiotics to enter the bacteria for further reaction

with DNA. Gentamicin and Ag can both denature the 30 S ribosomal subunit of

bacteria to stop protein translation for bacteria survival.

FIGURE 17.16

Generation of oxidative stress in the surrounding solution and within the bacteria.

Reprinted with permission from G.M. Wang, W.H. Jin, A.M. Qasim, A. Gao, X. Peng, W. Li, et al., Antibacterial

effects of titanium embedded with silver nanoparticles based on electron-transfer-induced reactive oxygen

species, Biomaterials 124 (2017) 25�34.


17.5 DUAL FUNCTIONS OF MULTIION-DOPED TIO2NANOTUBES

There are various inorganic ions in bone tissue, therefore effective strategies

incorporate dual or multi-ions to synergistically enhance osteointegration of Ti

implants. Many trials have been performed to prove this view. Huang et al. fabri-

cated Sr- and Si-loaded NTs (Si-Sr-NTs) through anodization and hydrothermal

treatment in the solution of SiO2 and Sr(OH)2 [11]. The ALP production of

MC3T3-E1 osteoblastic cells is higher on Si-Sr-NTs than NTs. It is worth noting

that the Si-Sr-NTs exhibit better corrosion resistance than NT samples. Si-Sr-NT

samples possess lower icorr values in the Tafel polarization curves and larger

capacitive loops in the Nyquist plots, suggesting that Si-Sr-NTs display superior

corrosion resistance ability (Fig. 17.18).

FIGURE 17.17

The potential mechanism of synergistic effect between antibiotic and Ag: (1) lyzing the cell

wall; (2) stimulating the penetration of nano-Ag into cell; (3) inactivating RNA polymerase;

(4) denaturing 30 S ribosomal subunit; (5) preventing DNA unwinding; and (6)

inactivating DNA gyrase.

Reprinted with permission from N. Xu, H. Cheng, J. Xu, F. Li, B. Gao, Z. Li, et al., Silver-loaded nanotubular

structures enhanced bactericidal effciency of antibiotics with synergistic effect in vitro and in vivo, Int. J.

Nanomed. 12 (2017) 731�743.


Dual-functional NT surfaces on Ti implants with both antibacterial activity

and osteoinductive ability also have been fabricated. Cheng et al. fabricated Sr-

and Ag-loaded NTs (NT-Ag/Sr) and detected the antibacterial activity and

osteoinductive ability in vitro and in vivo [10]. The SEM and ZOI test results

shown in Fig. 17.19 show that the antibacterial ability of NT-Ag/Sr towards

E. coli (ATCC25922) and S. aureus (ATCC25925 and ATCC43300) is better

than Ti and NTs and the antibacterial ability increased with an increase of Ag

loading content. The evaluation of bone regeneration has been performed in an

osteoporotic animal model, namely ovariectomized rats. The qualitative micro-CT

evaluation shown in Fig. 17.20 displayed the information on the peri-implant tra-

becular microstructure and the implant osteointegration. More trabecular micro-

structures are developed around NT implants, especially in Sr-loaded samples.

NT-Ag/Sr samples display higher Tb. N, Conn. D, and BV/TV, and lower Tb. Sp

than NTs, indicating better osteointegration of NT-Ag/Sr in vivo. Zhang et al.

fabricated Sr/ZnO-loaded NTs grafted with octadecylphosphonic acid (OPDA)-

toluene [61]. The surface exhibits multibiofunctions, including the osteoinductiv-

ity ascribing to Sr and self-antibacterial ability contributing to the synergistic

effects of ZnO and hydrophobic OPDA.

FIGURE 17.18

(A) Tafel polarization curves of the TNs and Si-Sr-TNs, (B) experimental and fitted Nyquist

plots of TNs and Si-Sr-TNs. The parameters Ecorr and icorr stand for the corrosion potential

and the corrosion current density, respectively.

Reprinted with permission from Y. Huang, X. Shen, H.X. Qiao, H. Yang, X.J. Zhang, Y.Y. Liu, et al.,

Biofunctional Sr- and Si-loaded titania nanotube coating of Ti surfaces by anodization-hydrothermal process,

Int. J. Nanomed. 13 (2018) 633�640.

42117.5 Dual Functions of Multiion-Doped TiO2 Nanotubes

FIGURE 17.19

Antiadhesive, antibacterial assay and zone of inhibition test. (A) The antiadhesive effect of

the sample surface was verified by SEM. Shrinkage of bacteria (black arrows) can be

detected on the four silver-containing samples; (B) the four silver-loaded samples present

a visible inhibition zone on the Mueller�Hinton plates spread with the three bacterial

strains. The TiO2-NT samples and pure Ti samples showed no evidence of an inhibition

zone. The diameter of the ZOI was around 1.8 cm for the NT40-Ag2.0Sr3, NT10-Ag2.0Sr3samples and approximately equal to 1.7 cm for the NT10-Ag1.5Sr3, NT40-Ag1.5Sr3samples on the Mueller�Hinton plates spread with ATCC25922. On the ATCC25923

Mueller�Hinton plates, the ZOI diameter was 1.9 cm for the NT40-Ag2.0Sr3, NT10-

Ag2.0Sr3 samples and 1.6 cm for the NT10-Ag1.5Sr3 and NT40-Ag1.5Sr3 samples.

Reprinted with permission from H. Cheng, W. Xiong, Z. Fang, H. Guan, W. Wu, Y. Li, et al., Strontium (Sr) and

silver (Ag) loaded nanotubular structures with combined osteoinductive and antimicrobial activities, Acta

Biomater. 31 (2016) 388�400.


0.00

Tb.Th (mm) Tb.N (mm–1) Tb.Sp (mm)

Conn.D (mm–3)

BV/TV (%)

2.00

3.00

4.00

5.00

0.00 0.00

0.00

0.000

0.010

Ti

TiO 2-NTs

NT10-Ag 1.5Sr 3

NT10-Ag 2.0Sr 3

NT40-Ag 1.5Sr 3

NT40-Ag 2.0Sr 3

Ti

TiO 2-NTs

NT10-Ag 1.5Sr 3

NT10-Ag 2.0Sr 3

NT40-Ag 1.5Sr 3

NT40-Ag 2.0Sr 3

Ti

TiO 2-NTs

NT10-Ag 1.5Sr 3

NT10-Ag 2.0Sr 3

NT40-Ag 1.5Sr 3

NT40-Ag 2.0Sr 3Ti

TiO 2-NTs

NT10-Ag 1.5Sr 3

NT10-Ag 2.0Sr 3

NT40-Ag 1.5Sr 3

NT40-Ag 2.0Sr 3Ti

TiO 2-NTs

NT10-Ag 1.5Sr 3

NT10-Ag 2.0Sr 3

NT40-Ag 1.5Sr 3

NT40-Ag 2.0Sr 3

0.020

0.030

0.040

0.050

10.00

20.00

30.00

40.00

50.00

0.20

0.40

0.60

0.80

1.00

1.20

1.0020.00

40.00

60.00

80.00

100.00

Ti TiO2-NTs NT10-Ag1.5Sr3

NT10-Ag2.0Sr3

2000 µm

NT40-Ag1.5Sr3 NT40-Ag2.0Sr3

(A) (B)a

b

edc

FIGURE 17.20

Micro-CT evaluation in a bone osteoporosis model (female rats, 6 weeks old). (A)

Transverse 3D images of the implant osseointegration and peri-implant trabecular

microstructure 5 mm below the tibial plateau; (B) compared with Ti, all the nanotube-

coated implants showed increased BV/TV (a, the bone volume per total volume), Conn.D

(b, the mean connective density), and Tb. N (d, the mean trabecular number), decreased

Tb. Sp (e, the mean trabecular separation), and no obvious change in Tb. Th (c, the

mean trabecular thickness). Compared with TiO2-NTs, the Sr-loaded implants resulted in

increased BV/TV, Tb. N, and Conn. D and decreased Tb. Sp. The largest BV/TV, Tb. N,

and Conn. D were observed for NT40-Ag1.5Sr3 and NT40-Ag2.0Sr3 rod samples, together

with the smallest Tb. Sp. In comparison, the NT10-Ag1.5Sr3 and NT10-Ag2.0Sr3 rod

samples showed a smaller increase in BV/TV, Tb. N, and Conn. D. �P,.05 vs Ti, #P,.05

vs TiO2-NTs, and ¢P,.05 vs NT10-Ag2.0Sr3.

Reprinted with permission from H. Cheng, W. Xiong, Z. Fang, H. Guan, W. Wu, Y. Li, et al., Strontium (Sr) and

silver (Ag) loaded nanotubular structures with combined osteoinductive and antimicrobial activities, Acta

Biomater. 31 (2016) 388�400.

42317.5 Dual Functions of Multiion-Doped TiO2 Nanotubes

17.6 CONCLUSIONSThe osteointegration ability and antibacterial ability of Ti implants are two major

problems in clinical application. The NT surfaces, especially inorganic ion-doped

NTs, have been demonstrated to effectively improve the bioactivity of Ti implants

by delivering bioactive or antibacterial inorganic ions. These biofunctionalized

surfaces with nanotopography have huge promise in fabricating orthopedic

implants with better clinical performance.

ACKNOWLEDGMENTThis work was jointly supported by National Natural Science Foundation of China (No.

31700824, 31500783, 81571816, and 81601611), and Hubei Provincial Natural Science

Foundation of China (2017CFB191).

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