J Bagh College Dentistry Vol. 33(4), December 2021 Evaluation of Cytotoxicity

Evaluation of Cytotoxicity and Biocompatibility of

Ti2AlC in Rabbits

Luma M. Ibrahim (1), Raghdaa K. Jassim (2) Ahmed Al Gabban(3)

https://doi.org/10.26477/jbcd.v33i4.3015

ABSTRACT

Background: The Titanium and its alloys are suitable for dental implant and medical applications. Biocompatibility of the

materials is a major factor in determining the success of the implant and has a great impact on their rate of osseointegration.

The aim of this study was to evaluate the biocompatibility and cytotoxicity of Ti2AlC in comparison to CPTi & Ti6Al7Nb in

rabbits.
Materials and Methods: 10 male New Zealand White rabbits, weighing (2-2.5 kg), aged (10-12 months) were used in this

study. Cylindrical implants were prepared from the study materials (CPTi, Ti6Al7Nb and Ti2AlC) with (8mm) height and

(3mm) diameter for the evaluation of tissue response and disc specimens were prepared with (6 mm) diameter and (2 mm)

thickness for evaluation of cytotoxicity MTT test. A histological study was performed at 2 & 6 weeks post- surgical implant

insertion.

Results: Histological findings show that Ti2AlC has enhanced proliferation of osteo-progenitor cell and reported mature bone
formation at 6 weeks. Moreover, Ti2AlC has recorded a higher percentage for viable cells by MTT test in comparison to CPTi

and Ti6Al7Nb.

Conclusion: The new Ti2AlC dental implant is considered biocompatible and has showed a better bone formation than the

CPTi and Ti6Al7Nb materials at 2 & 6 weeks.

Keywords: Bone healing, CPTi, Ti6Al7Nb, Ti2AlC, Osseointegration, Dental implant, . (Received: 22/9/2021, Accepted:
13/10/2021)

INTRODUCTION
Titanium regards as a key factor for the

establishment of implant tissue interaction and for

the assessment of biocompatibility of its alloy [1].

Titanium is applicate in many studies in

prosthodontics, conservative and in orthodontics

due to their resistance to corrosion and their good

tolerance by tissue without causing harms or

damage. [2,3,4] Titanium and its alloys may release

ions in saliva that contact the oral mucosa and

may cause tissue reaction including toxicity or

allergy reaction [5,6].

Most researches record that titanium is the least

metal material that induces allergy; therefore, it is

regarded as material of choice for biological

application.

Moreover, Ti6Al7Nb alloy is light in weight, have

very high tensile strength and well tolerated by

bone tissue and reported to be used for biomedical

purposes [7,8,9]

(1) PhD Student, College of Dentistry, Al Farahidi University,

Baghdad, Iraq.
(2) Professor, Department of Prosthodontics, College of

Dentistry, University of Baghdad, Baghdad, Iraq

(3) Professor Ahmed Al Gabban, Department of Material
Engineering, University of Technology, Baghdad, Iraq

Corresponding email, dr_lumakurbasi@yahoo.com

The evaluation of cytotoxicity of implant

materials along with its osseointegration and bone

formation potential becomes important

concerning the clinical application of these

materials in service and their success in

implantation. The relationship between viability

of bone cell that contact implant surface and

tissue reaction have been recorded in several

studies [10,11,12].
The objective of this study was to evaluate the

cytotoxicity and bone tissue response in rabbit for

the new prepared Ti2AlC implant in comparison to

Commercially pure titanium CPTi and Ti6Al7Nb

alloy by using histological examination and Methyl

thiazolyl- tetrazolium MTT assay at different

periods.

MATERIALS AND METHODS

Animals

A total of 10 male New Zealand White rabbits,

weighing (2-2.5 kg) and aged (10-12 months)

were used in this study, and kept in the animal

department of (National Center of Drug Control

and Research /Iraq) at a constant humidity and

temperature of 23°C according to the National

https://doi.org/10.26477/jbcd.v33i4.3015

J Bagh College Dentistry Vol. 33(4), December 2021 Evaluation of Cytotoxicity

Council’s guide for the care of laboratory

animals.

The following materials were used in this study:

-CPTi rods and Ti6Al7Nb rods, 6 mm in diameter

from Straumann Company, Switzerland.

-Ti2AlC powder ASTM E8M03 (Famouschem

Technology Shanghai) was used to prepare implant,

by using (0.5g) of powder of Ti2AlC that was

condensed by dental condenser of (0.5mm) size.

The punch was allowed to seat over the solid steal

rod and when the mold was filled with a condensed

powder, compaction was started by using a punch

guide. Pressing with hydraulic press started using

(100 Mpa) for (10min). The specimen was ejected

by using the long punch after that the base removed

and left for drying 24 hours at room temperature.
Cylindrical implants were prepared from the study

materials with (8mm) height and (3mm) diameter

for evaluation of tissue response and disc specimens

were prepared with (6 mm) diameter and (2 mm)

thickness for evaluation of cytotoxicity assessment

by MTT test. [13]

In Vivo study
Three implants were implanted in the proximal

third of the lateral aspect of the femoral bone, the

Ti2AlC and Ti6Al7Nb implant were applicate in the

right femur while CPTi was implanted in the left

femur. According to the healing interval, the

experimental rabbits were divided into two groups

(2, 6 weeks), each group consists of 5 animals

sacrificed for histological study.

In Vitro Study (Cytotoxicity Test)
Cultured for fibroblast cell line (murine NIH 3T3

Cell Line 93061524 – Sigma) in Dulbecco's

Modified Eagle medium. Seed the cells in a 96-well

microplate at a density of (1 x 104 with 100 µl) per

well. Cultures were incubated at 37°C in a

humidified atmosphere of 5% CO2 in air. In the

present study, 6 cut samples from each rod of CPTi,

Ti6Al7Nb and Ti2AlC were used for cytotoxicity

evaluation with fibroblast cells. Cells were treated

with different doses of examined materials. Then,

these cells were estimated for their proliferation and

viability by Methyl thiazolyl- tetrazolium MTT

colorimetric assay, using spectrophotometer record

the absorbance at 570 nm as described by Wang et

al. [14]. Percentage viability was calculated as
follows:

Statistical Analysis
All records were entered into Excel spread sheets

for evaluation with the Statistical package deal for

social studies (SPSS) (Chicago, IL, united states

of America). The data were analyzed using one-

way ANOVA test.

RESULTS
1.Histological findings: microscopic features for

all specimens of implant for CPTi group at 2

weeks post-operative duration, show a sparse of

bone trabeculae surrounding by osteoblast with

basal bone around implant bed. At 6 weeks post-

operative duration, the specimens show basal

bone coalesce with newly formed thin bone

trabeculae at the bed implant region, with

presence of fibrous tissue surrounding implant

figure 1 (A&B).

Microscopic evaluation for all specimens of

implant for Ti6Al7Nb group at 2 weeks post-
operative duration shows bone marrow with a

sparse of bone trabeculae coalesce with basal

bone, while at 6 weeks post-operative duration,

the specimens show a thin rim of fibrous tissue

surrounding the implant with bone trabeculae full

most of implant bed, figure 1 (C&D).

Implant for Ti2AlC group at 2 weeks post-

operative duration shows basal bone with

attached newly formed bone trabeculae

surrounded by active proliferating osteogenic

cells. At 6 weeks all specimens show mature bone

surrounding the implant, figure1 (E&F).

2.MTT Results

The results of cytotoxicity of CPTi, Ti6Al7Nb and

Ti2AlC by detection and estimation of viable cells

for the whole concentration that used for MTT

test after 72 h are illustrated in figure (2) and table

(1). The material (Ti2AlC) showed a higher

percentage of cell viability (89.6461 ±7.6468)

followed by Ti6Al7Nb (80.6306 ±5.6362). A

significant P value (.001) is recorded for cell

viability within and between the examined

materials by using ANOVA test, table (2).

J Bagh College Dentistry Vol. 33(4), December 2021 Evaluation of Cytotoxicity

Figure (1) Microscopic view for different examined materials at

(2 & 6 weeks) where basal bone (BB), few scattered bone

trabeculae (BT), Osteoid tissue (OST), Osteoblast (arrows).

A. CpTi implant at 2-week duration

B. CpTi implant at 6-week duration

C. Ti6Al7Nb implant at 2-week duration

D. Ti6Al7Nb implant at 6week duration

E. Ti2AlC implant at 2-week duration

F. Ti2AlC implant at 6- week duration

Figure (2) Cell viability of CPT(Ti), Ti6Al7Nb (Ti6) and

Ti2AlC(max) after 72 h.

Table (1) Descriptive statistic for MTT assay

Table (2) ANOVA Test for the all studied

groups for MTT assay

Material N Mean Std. 95% Confidence

Interval for Mean

Lower

Bound

Upper

Bound

CpTi 6 64.19 12.95 50.596 77.796

Nb7Al6Ti 6 80.63 5.63 74.715 86.545

AlC2Ti 6 89.64 7.46 81.621 97.670

Test of homoginity
Levene

Statistic
df1 df2 Sig.

3.364 2 15 .062

Sum of
Squares

Df Mean
Square

F Sig.

Between
Groups

1998.141 2 999.070 11.609 .001

Within
Groups

1290.920 15 86.061

Total 3289.060 17

H&E X20 H&E X10

H&E X20
H&E X10

H&E X10 H&E X10

J Bagh College Dentistry Vol. 33(4), December 2021 Evaluation of Cytotoxicity

DISCUSSION
Titanium and their alloys implant have been widely

used in various branches of dentistry. As implant

materials have direct contact with the bone tissue

and may interact with cells of the body, therefore,

their success not only require an acceptable physical

and chemical properties but also must have good

biocompatibility [15,16] .In vivo study hasbeen done

by implantation of different materials (CpTi,

Ti6Al7Nb and Ti2AlC) to investigate their ability in

enhancement of osseo-integration and bone

formation .Our results for Ti2AlC implant report an

obvious proliferation of osteo- progenitor cells at 2

weeks and a well mature bone formation at 6 weeks

in comparison to CpTi, Ti6Al7Nb which recorded a

rim of fibrous tissue around the implant with bone

trabeculae filled more than half of implant bed,

Although Ti6Al7Nb alloy showed more bone

formation than CpTi, immature bone was detected

in most of their examined specimens . Many studies

revealed that titanium and Ti6Al7Nb alloy were used

in dental implant due to their excellent

compatibility with surrounding tissues [17,18]. On the

other hand, the present results focus on excellent

findings related to tissue response by newly Ti2AlC

implant material.

In vitro studies have been performed by using of

cytotoxicity test to evaluate the biological effects of

the examined materials on growth and viability of

fibroblast cell which is derived from the

mesenchymal layer as having the same origin of the

osteoblast cells. The cell viability was recorded by

MTT test thatwas based on mitochondrial enzyme

which reduced the yellow MTT dye into insoluble

Formazan, and the number of viable cells were

calculated [19,20,21]. The results indicated that Ti2AlC

material showed a higher percentage of viable cells

in whole recorded concentration that coincided and

supported the histological findings in better bone

formation and maturation in comparison to CPTi

and Ti6Al7Nb materials.

CONCLUSION
The present study concludes that the new Ti2AlC

implant material is considered a biocompatible and

less toxic to cells by recording high percentage of

cell viability and showing a better bone formation

than the CpTi and Ti6Al7Nb materials at 2and 6-

week period.

Financial Support and Sponsorship: Nil.

Conflict of interest: There are no conflicts of

interests.

REFERENCES
1. Wang RR, Fenton A. Titanium for prosthodontic

applications: a review of the literature. Quintessence

Int. 1996 Jun; 27(6):401-408.
2. Abdel-Hady Gepreel M, Niinomi M.

Biocompatibility of Ti-alloys for long-term

implantation. J Mech Behav Biomed Mater. 2013

Apr; 20:407-415.
3. Hwang YJ, Choi YS, Hwang YH, et al.

Biocompatibility and Biological Corrosion

Resistance of Ti-39Nb-6Zr+0.45Al Implant Alloy.

J Funct. Biomater. 2020 Dec 29;12(1):2.
4. Okazaki Y, Rao S, Ito Y, et al. Corrosion resistance,

mechanical properties, corrosion fatigue strength

and cytocompatibility of new Ti alloys without Al

and V. Biomaterials. 1998 Jul; 19(13):1197-1215.

5. Chandar S, Kotian R, Madhyastha P, et al. In vitro
evaluation of cytotoxicity and corrosion behavior of

commercially pure titanium and Ti-6Al-4V alloy for

dental implants. J Indian Prosthodont Soc. 2017 Jan-

Mar;17(1):35-40.

6. Jorge JR, Barão VA, Delben JA, et al.Titanium in
dentistry: historical development, state of the art

and future perspectives. J Indian Prosthodont Soc.

2013 Jun; 13(2):71-79.

7. Kajzer A, Grzeszczuk O, Kajzer W, et al. Properties
of Ti-6Al-7Nb titanium alloy nitrocarburized under

glow discharge conditions. Acta Bioeng Biomech.

2017;19(4):181-188.

8. De Oliveira DP, Toniato TV, Ricci R, et al.
Biological response of chemically treated surface of

the ultrafine-grained Ti-6Al-7Nb alloy for

biomedical applications. Int J Nanomedicine. 2019

Mar 6;14: 1725-1736.

9. Okulov IV, Joo SH, Okulov AV, et al. Surface
Functionalization of Biomedical Ti-6Al-7Nb Alloy

by Liquid Metal Dealloying. Nanomaterials (Basel).

2020 Jul 28;10(8):1479.

10. Savadi RC, Goyal C. Study of biomechanics of
porous coated root form implant using overdenture

attachment: A 3D FEA. J Indian Prosthodont Soc.

2010;10: 168–175.

11. McMahon RE, Ma J, Verkhoturov SV, et al. A
comparative study of the cytotoxicity and corrosion

resistance of nickel-titanium and titanium-niobium

shape memory alloys. Acta Biomater. 2012;8:

2863–2870.

12. Soenen SJ, Manshian B, Montenegro JM, et al.
Cytotoxic effects of gold nanoparticles: A

multiparametric study. ACS Nano. 2012;6: 5767–

5783.

13. Luma M., Raghdaa K., Ahmed Al Gabban.
Manufacturing and characterizing of TiAlC (Max

phase alloy) in comparison with CPTi and Ti6Al7Nb

as a dental implant material. J Res Med Dent

Science. 2021 Jul; 9 (8) :2347-2367

14. Wang QY, Wang YB, Lin JP, et al. Development
and properties of Ti-In binary alloys as dental

https://www.ncbi.nlm.nih.gov/pubmed/24431713/
https://www.ncbi.nlm.nih.gov/pubmed/24431713/
https://www.ncbi.nlm.nih.gov/pubmed/24431713/

J Bagh College Dentistry Vol. 33(4), December 2021 Evaluation of Cytotoxicity

biomaterials. Mater Sci Eng C Mater Biol Appl.

2013; 33: 1601–1606.

15. Siddiqi A, Payne AGT, De Silva RK et al. Titanium
allergy: could it affect dental implant integration?

Clin Oral Implants Res. 2011 Jul;22(7):673-680.

16. Lapin J, Kamyshnykova K, Klimova A.
Comparative Study of Microstructure and

Mechanical Properties of Two TiAl-Based Alloys

Reinforced with Carbide Particles Molecules. 2020

Jul 28;25(15):3423.

17. Comino-Garayoa R, Cortés-Bretón Brinkmann J,
Peláez J, et al. Allergies to titanium dental implants:

what do we really know about them? A scoping

review. Biology (Basel). 2020 Nov 18;9(11):404.

18. Hoornaert A, Vidal L, Besnier R, et al.
Biocompatibility and osseointegration of

nanostructured titanium dental implants in minipigs.

Clin Oral Implants Res. 2020 Jun;31(6):526-535.

19. Brunet G, Cabioch Th, Chartier P et al. Reaction
Synthesis of Layered Ternary Ti2AlC Ceramic, J

European Ceramic Society 2009; 29(1):187-194.

20. Jin J, Zhang L, Shi M, et al. Ti-GO-Ag
nanocomposite: the effect of content level on the

antimicrobial activity and cytotoxicity. Int J

Nanomedicine. 2017 Jun 7; 12:4209-4224.

21. Zhenhuan W, Yu D, Junsi L et al. Physio-chemical
and biological evaluation of SLM-manufactured Ti-

10Ta-2Nb-2Zr alloy for biomedical implant

applications. Biomed Mater. 2020 Jun 23;15 (4)

5004-5017.

الخالصة
ئيسياً في تحديد ر: يَُعدُّ التيتانيوم وسبائكه مناسباً لزراعة األسنان والتطبيقات الطبية، إذ يمثّل التوافق الحيوي )البيولوجي( للمواد عامالً الخلفية

ةنجاح عملية الزرع وله تأثير كبير على معدل اندماجها العظمي. كان الهدف من هذه الدراسة هو تقييم التوافق الحيوي )البيولوجي( والسمي

( في األرانب.Nb7Al6Ti)( وسبائك التيتانيوم الطبية CPTi( مقارنة بـالتيتانيوم النقي تجارياً )AlC2Tiالخلوية لكربيد األلومنيوم التيتانيوم )

كغم( لكل منها، وتتراوح أعمارها 2.2-2من ذكور األرانب النيوزيلندية البيضاء في هذه الدراسة، بوزن ) 01مواد البحث وطرقه: تم استخدام

ملم( 3قطر )ملم( وب 8( بارتفاع )AlC2Tiو CPTi ،Nb7Al6Tiشهراً(. وتم تحضير زرعات أسطوانية من مواد الدراسة ) 02-01بين )

. وأجريت الدراسة MTTملم( لتقييم السمية الخلوية عن طريق اختبار 2ملم( وسمك ) 6لتقييم استجابة األنسجة، وإعداد عينات قرصية بقطر )

أسابيع من وضع الزرعة بعد الجراحة. 6النسيجية بعد أسبوعين و

6لخاليا السلفية )األولية( العظمية، والحظ تكوين عظام ناضجة في غضون عّزز من تكاثر ا AlC2Tiالنتائج: تظهر النتائج النسيجية أن الـ

.Nb7Al6Tiوالـ CPTiمقارنةً بـالـ MTTنسبة مئوية أعلى للخاليا الحيوية عن طريق اختبار AlC2Tiأسابيع. عالوة على ذلك، سجل الـ

ة متوافقة حيوياً، وأظهرت تكوين أفضل للعظام مقارنة بمواد الـ الجديد AlC2Tiاالستنتاجات: تعتبر زرعات األسنان المحضرة من مادة الـ

CPTi والـNb7Al6Ti أسابيع. 6خالل أسبوعين و

، االندماج العظمي، زراعة األسنانiCPT ،Nb7Al6Ti ،AlC2Ti الكلمات الرئيسة: شفاء العظام ،

Articles Published by Journal of Baghdad College of Dentistry is licensed under a Creative
Commons Attribution 4.0 International License.