J Bagh College Dentistry              Vol. 28(4), December 2016          Physical and Histological  
  

 

Oral Diagnosis  89 
 

Physical and Histological Evaluation of Coated Implant 

with Nano ZrO2 after Creation Titania Nanotubes 
 

Mustafa Sh. Alhilfi, B.Sc. (a) 

Athraa Y. Alhijazi, B.D.S., M.Sc. Ph.D. (b) 
 

ABSTRACT 
Background: Contact between implant material and bones must be strong and fast creation, to fulfill these 

properties appropriate surface modifications must apply on used implants. In this contribution; double surface 

modifications are applied on Ti-6Al-4V alloy to accelerate osseointegration.  
Materials and methods: Anodic process is utilized to create titania nanotubes (TNTs) on the screws made from Ti-6Al-

4V alloy. These implants were coated with nano ZrO2 particles. Second modification was annealing anodized screws 

at 8000C, and implanted in tibiae of nine adult New Zealand white rabbits. 

Results: Physical and histological consequences of two surface modifications on Ti-6Al-4V alloy screws were studied. 

Scanning electron microscope (SEM) images shows inhomogeneous distribution of TNTs on screws surfaces. X-ray 

diffraction (XRD) patterns illustrate the covering of first group samples with ZrO2 and transformation of Ti to its oxide 

(Rutile phase) for second group. These pattern shows that TiO2 had higher crystallinity and larger grain size than ZrO2. 

Atomic Force Microscopy images (AFM) shows the increasing of roughness, grain size and internal diameter of TNTs 

after annealing process. Coated implant with ZrO2 at 4 month duration shows threads with newly Haversian canal 

feature. Annealed implant at same duration shows well developed threads, base of implant illustrates bone 

trabeculae filled the base of implant bed with active osteoblast cells.  

Conclusion: Modification of implant's surface produced an improvement of osseointegration in comparison to 

untreated one. 

Keywords: ZrO2, titanium nanotubes, annealing, osseointegration and electrophoretic deposition. (J Bagh Coll 

Dentistry 2016; 28(4):89-95) 

 

INTRODUCTION 
Surface modification (within the field of 

biomaterials) is of special interest since the 

interaction between the living tissues (including 

blood and arteries) and implanted devices are 

mediated by reactions at the surface of the 

implant; biocompatibility is widely be considered 

as a surface property (1). 

Difference surface modifications have been 

experienced to Ti and its alloys to enhance bone 

differentiation and consolidate direct contact 

between implant material and bones.  However 

for long periods,  none  of  these modifications  

have  produced  a  durable  interface  strong  

enough  to  support functional loading. Hence, 

there is specific necessity to develop implants 

with surface coatings designed to improve bone 

anchorage through enhanced osseointegration (2).  

Attempts to improve the osteoconductivity of 

medical alloys can be divided into two techniques:  

coating metallic  implants with bioactive  

materials  to  accelerate  bone  formation  , and  

forming  rough  surface  at  the  macro-level  on  

these  implants  and  the  ingrowth  of  bone  

results  in anchorage of the implants (3). 

 

 

 

 
(a)Department of Physics, College of Education, Al-Mustansiriya 

University 
(b)Professor. Department of Oral Diagnosis, College of Dentistry, 

Baghdad University 

The  integration  of  the  implant  into  bone  

takes  place  largely  at  the  tissue-implant  

interface. Development of this interface is 

complex and involves numerous factors.  These  

include  not  only implant  related  factors,  such  

as  material,  shape,  topography  and  surface  

chemistry,  but  mechanical loading, surgical 

technique and patient variables, such as bone 

quantity and quality, as well (4). 

Because of their good combination of 

mechanical properties and excellent 

biocompatibility, zircona ceramics are recognized 

as one of the best biomaterials for dental 

prostheses (5). This material is chosen in form of 

nanoparticles to coat Ti-6Al-4V alloy after 

creation TNTs on its surface.    

Annealing process (the second surface 

modification) has many merits in improving the 

performance of TNTs as active biomaterial. This 

process can reduce contamination of species from 

the electrolyte or organic solvents (6), improved 

the stability of TNTs samples (7), activated the 

anodic oxidation layer and the surface becomes 

bioactive (8). 

The aim of this work is investigation the effect 

of covering TNTs with nano ZrO2 and annealing 

these tubes for getting best osseointegration. 

 

MATERIALS AND METHODS 
1- Samples preparation 

Ti-6Al-4V alloy discs shapes were prepared by 

using wire cut machine. These samples were 



J Bagh College Dentistry              Vol. 28(4), December 2016          Physical and Histological  
  

 

Oral Diagnosis  90 
 

utilized for characterization purposes. The same 

material was used to make medical screws with 

following dimensions: the head diameter was 

3.5mm while body was 3mm in diameter.  They  

have  a  slide  in  the  head  of  1.5mm  depth  and  

1mm  width. Ultrasonic  cleaning  bath 

(Sonomatic/170-2-T80,  Germany)  with  ethanol  

and  acetone  of  75:25  wt%  ratio was carried out 

to removing debris and contamination from the 

fabricated samples. 

  

2- Samples characterization  
The  crystalline  nature  of  the  materials was 

tested  by  X-ray  Diffractometer (XRD)  using  

Cu  Kα  radiation. Surface morphology of the 

modified surfaces was examined using Scanning 

Electron Microscopy (SEM), type (JEOL-JSM-

5600). 

 

3- Anodic and annealing processes 
To create TNTs on screws anodic process was 

used, the details are mentioned in reference (9). 

After anodic process; the thermal treatments on 

samples were performed in a tubular furnace for 

two hours at 800°C.  

 

 

 

4- Coating with ZrO2  
 EFD method was used to deposit nano-ZrO2 

material on screws after creation TNTs on their 

surfaces. Amer et al. (10) mentioned deposition 

details of this material on Ti-6Al-7NB alloy. The 

deposition conditions were: Deposition time 3 

min, applied voltage 50 volt, collide temperature 

25 ºC and the distance between electrodes was 1 

cm. Sintering of the coated screws was carried out 

using Carbolite furnace type MTF, England. The 

treatment was done at 800 ºC under inert gas 

(argon), to prevent oxidation of the specimen. 

Figure 1 shows untreated, annealed and coated 

samples. 

 

5- Surgical implantations and specimens 
collection 

      Nine adult New Zealand white rabbits 

weighing 1.5-2 kg were used. For each rabbit, two 

screws were implanted in the right tibia (untreated 

and annealed), coated screw with ZrO2 was 

implanted in left tibia. The details of surgical 

implantation were mentioned in reference (11). 

Specimens of bone with different implant samples 

were subjected to decalcification process and the 

slides were stained by H&E stain and examine 

under light microscope. 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 1: From left: Untreated, Coated with ZrO2 and Annealed sample. 
 

RESULTS AND DISCUSSION 
     Figure 2 shows XRD pattern of untreated, 

annealed and coated Ti-6Al-4V alloy. Annealed 

sample has mixed peaks, first type belongs to 

titanium and the second one belongs to titanium 

oxide. Strong lines of XRD profile of annealed 

alloy with the following Miller indices (hkl): 

(110), (101), (111), and (211) belong to (Rutile 

phase) (JCPDS file no. 211276).This reflections 

indicating that the as-annealed nanotubes layers 

are crystallized. Inside this pattern there is strong 

lines with high intensity can be assigned to Alpha 

phase titanium with Miller indices (101) and (100) 

(JCPDS file no.441294). This refers to incomplete 

transformation to TiO2 by annealing process.  

      Strongest reflections peaks for coated sample 

are (-111) and (111) which could be indexed to 

ZrO2 monoclinic phase corresponding to JCPDS 

file 37-1484. Comparing the values of Full Width 

at Half Maximum (FWHM) between the 

dominant peak of annealed and coated samples 

shows that the grain sizes of the former larger 

than that of latter. Also, TiO2 consists of highly 

crystalline form, since narrower peaks represent 

higher levels of crystallinity. 

 

 

 



J Bagh College Dentistry              Vol. 28(4), December 2016          Physical and Histological  
  

 

Oral Diagnosis  91 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 2: XRD patterns of untreated, annealed and coated samples. 
      

Figure 3(A) shows the SEM image of TNTs 

which were created on medical screws fabricated 

from Ti-6Al-4V alloy. These nanotubes are not 

uniform or have the same length over entire 

surface; this is might due to the effect of the 

geometric design of machined screw. It’s well 

known that (12): on the binary α + β type Ti alloys, 

inhomogeneous nanotube formation takes place.    

On  the  surface  of  this  body  the  arrays  of  

nanotubes  formed  on  α  phase region while the 

selective dissolution of β phase occurs, leading an 

inhomogeneous oxide  nanotube  layer.  

EDS spectra of anodized screw are illustrated 

in figure 3 (B). The concentrations of the elements 

are listed in table (1); these concentrations did not 

seem to be directly related to the overall 

composition of Ti-6Al-4V alloy. Anodized alloy 

contains  69.3%  Ti  and  19.077%  O  which  is  

not  close  to  the stoichiometric  composition  of  

TiO2 .So  Ti does not  transforms  completely  to  

titania  phase after anodization. Wt% of vanadium 

element in table has  zero  value  because  of  the  

overlapping  between  Ti-Kβ  line  and  V-Kα 

line. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

  

 

 

 

 

 

 

                                                                                           Figure 3. For anodized Ti-6Al-4V. A-SEM  

                                                                                               image. B- EDS spectra. 
 

 

 

A 

 

B 



J Bagh College Dentistry              Vol. 28(4), December 2016          Physical and Histological  
  

 

Oral Diagnosis  92 
 

Table 1: Concentration of anodized Ti-6Al-4V alloy elements determined by EDS 

at% wt% 
Energy 

(KeV)of Kα1 lines 

Atomic 

number 
The Element 

19.077 7.822 0.523 8 O 

69.297 85.07 4.51 22 Ti 

8.948 6.185 1.487 13 Al 

0 0 4.951 23 V 

         

Figure (4-A and C) shows Atomic Force 

Microscopy (AFM) images for anodized and 

annealed alloy. This image illustrates the effect of 

annealing on TNTs. Figure (4-B and C) illustrates 

upper views of single Ti nanotube on anodize and 

annealed samples respectively.  

Table 2 shows the data of AFM images of 

anodized and annealed Ti-6Al-4Valloy. These 

data confirm the increasing of roughness, grain 

size and internal diameter of TNTs after annealing 

process. The increasing of roughness has good 

effect on accelerate osseointegration.            

 

Table 2: AFM images parameters of anodized and annealed samples 

Sample's type 
Roughness Average  

(nm) 

Grain Size 

(nm) 

Internal diameter of TNTs 

 (nm) 

Anodized alloy 1.2 96.40 171 

Annealed alloy 2.7 171.57 572 

         

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
  

 

 

 

 

Figure 4: (A),(C):AFM images of anodized and annealed Ti-6Al-4V alloy respectively.  

(B),(D): Measurement of Ti–tube after anodic and annealing processes respectively. 
       

 

 

Ti nanotube diameter (μm) D 

 
A  Ti nanotube diameter (μm) 

 

B 

 

C 



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Oral Diagnosis  93 
 

Removal torque forces have been used as a 

biomechanical measure of anchorage or 

osseointegration in which the greater forces 

required to remove implants may be interpreted as 

an increase in the strength of osseointegration (13). 

Table 3 illustrates the effect of current surface 

modification to build strength bonding between 

implants and bone tissue. 

 

Table 3: Average values of removal torque 

values of untreated and treated samples after 

implantation for four months 

Treatment type 

Average values  

of removal   

torque  

values (N.cm) 

Untreated 40 

Anodized and  

coated with ZrO2 
55 

Anodized and  

annealed at 8000C 
65 

 

Histological findings 

    Untreated implant at 4 month duration shows 

threads with immature bone feature, base of 

implant illustrates fine and few trabeculae with 

active osteoblast cell rimming the bone, figure 

5(A,B,C). 

Coated implant with ZrO2 at 4 month duration 

shows threads with newly bone formation, base of 

implant illustrates fine, numerous trabeculae with 

active osteoblast  and osteocyte cells, figure 

6(A,B,C). 

The enhancement of bone formation at the 

bone-implant interface has been achieved through 

the modulation of osteoblasts adhesion and 

spreading, induced by structural modifications of 

the implant surface, particularly at the nanoscale 

level (14).  

In present results the improvement of the 

bone-forming activity at the bone-implant 

interface for coated implant with ZrO2 is 

committed to nanoscale features that have the 

ability to induce the differentiation of stem cells 

along the osteogenic pathway and because of their 

ability to differentiate into different types of 

functional cells, as they posses great potential to 

restore and regenerate native tissues (15). 

For annealed implant at 4 month duration, 

result shows well developed threads, and the base 

of implant illustrates bone trabeculae that filled 

the base of implant bed with active osteoblast 

cells, figure 7(A,B,C).The present result shows 

the great potential for the conversion of natural 

by-product into highly valuable compounds for 

bio- applications, using a simple and effective 

valorization process (16). In addition, the 

improvement in bone-implant interface shows to 

be more by use of natural biomaterial rather than 

the synthetic. 

     As a conclusion; due to its high roughness and 

histologic findings that revealed for annealed 

process, it had superiority in production of active 

osseointegration, in comparison to others. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 5: Untreated implant at 4 month: A- threads (arrows)x4. B-bone trabeculae (BT) at base 

of implant bedx10. C- bone trabeculae (BT) rimming by osteoblast(arrows)x10. 
 

 

 

 

 

 

 

 
A 

 

B 

 

C 



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Oral Diagnosis  94 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 6: Coated implant with ZrO2 at 4 month. A- Threads (arrows)x10. B-bone trabeculae 

(BT)at base of implant bedx10. C- Active osteoblast (arrow); osteocyte (arrow head)x20. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 7: Annealed implant at 4 month. A- Well developed threads (arrows) x20. B-bone 

trabeculae (BT) filled the base of implant bedx10. C- Bone trabeculae rimming by osteoblast 

(arrow) x40. 
 

REFERENCES 
1. Peter K. Sol-gel-derived Zirconia thin film coatings 

for Ti-6AI-4V and 316L Stainless steel implant 

applications. A master thesis, University of 

Toronto,1997. 

2. Zreiqat  H,  Valenzuela  S  M,  Nissan  B  B,  Roest  
R,  Knabe  C, Radlanski  R J,  Renz  H,  Peter  J.  

Evans PJ.  The effect of surface chemistry 

modification of titanium alloy on signalling pathways 

in human osteoblasts. Biomaterials 2005; 26: 7579–

86. 

3. Kuroda K, Okido M. Hydroxyapatite coating of 
titanium implants using hydroprocessing and 

evaluation of their osteoconductivity. Bioinorganic 

Chemist Appl 2012; Article ID: 730693, 1-7. 

4. Doblar M, Garc JM, Gomez MJ. Modelling bone 
tissue fracture and healing: a review. Engineering 

Fracture Mechanics 2004; 71: 1809–40. 

5. Chevalier J.  What future for zirconia as a biomaterial? 
Biomaterials 2006; 27: 535-43. 

6. Regonini D, Jaroenworaluck A, Stevens R, Bowen 
CR. Effect of heat treatment on the properties and 

structure of TiO2 nanotubes:  phase composition and 

chemical composition. Surf Interface Anal 2010; 42: 

139-44. 

7. Anca M, Georgeta V, Roxana T, Daniela I. Heat 
treatment of TiO2  nanotubes, a way to significantly 

change their behavior. U.P.B. Sci Bull Series B 2011; 

73(1): 97-108. 

8. Lukáčová H, Plešingerová B, Vojtko M. Bioactivity of 
chemical, Electrochemical and thermal treatment of 

Ti-6Al-4V. Acta Metallurgica Slovaca 2011; 17(1): 

11-7.  

9. Mustafa Sh A, Thair LA. Corrosion characterization of 
medical alloys modified by forming titanium 

nanotubes via anodic oxidation and annealing process. 

J Materials Technol 2013; 28(6): 297-304. 

10. Waheed AS. Mechanical and histological evaluation 
of nano zirconium oxide coating on titanium alloy               

Ti-6Al-7NB dental implants, Baghdad University, 

2013. 

11. Mustafa ShA , Thair LA, Al-Hijazi AY. Enhancement 
of early osseointgration by coating TiO2  nanotubes  

with annealed fishbone (In Vivo Study).  J Natural 

Science Res 2014; 4(2): 38-47. 

12. Emanuela S, Fedor F, Maria N, Margitta U, et al. Ti -6 
Al-7Nb Surface Modification by anodization in 

electrolytes containing HF. U.P.B. Sci Bull Series B 

2012; 74(2): 277-88. 

13. Jung-WK. Biomechanical evaluation of dental 
implants with different surfaces: Removal torque and 

 
A 

 
B 

 
C 

 
A 

 B  C 



J Bagh College Dentistry              Vol. 28(4), December 2016          Physical and Histological  
  

 

Oral Diagnosis  95 
 

resonance frequency analysis in rabbits. J Adv 

Prosthodont 2009; 1:107-12. 

14. Variola F, Yi JH, Richert L, Wuest JD, Rosei F, Nanci 
A. Tailoring the surface properties of Ti6 Al4V by 

controlled chemical oxidation. Biomaterials 2008; 29: 

1285–98. 

15. Atala A, Lanza R, Thomson JA, Nerem RM. 
Principles of Regenerative Medicine. Burlington: 

Elsevier; 2008. 

16. Piccirillo C, Silva MF, Pullar RC, Braga da Cruz I, 
Jorge R, Pintado MM, Castro PM. Extraction and 

characterization of apatite- and tricalcium phosphate-

based materials from cod fish bones. Mater Sci Eng C 

Mater Biol Appl 2013; 33(1): 103-10. 

 

 

 

http://www.ncbi.nlm.nih.gov/pubmed/25428050
http://www.ncbi.nlm.nih.gov/pubmed/25428050
http://www.ncbi.nlm.nih.gov/pubmed/25428050