This document discusses the immune response to dental implants and potential causes of implant failure. It begins by outlining the topics to be covered, including immuno-pathological explanations, immuno-allergological testing, and solutions. It then details the innate immune response to titanium debris, including macrophage activation and proinflammatory cytokine production. The specific adaptive immune response is also described, where titanium acts as a hapten and activates memory T-lymphocytes, perpetuating inflammation through cytokine release. Various in vivo and in vitro tests for exploring metal allergy are discussed, including patch testing, lymphocyte transformation testing (LTT), and lymphocyte activation testing (LAT). Failure rates are noted to be higher in patients with a history of metal

2. IMMUNE RESPONSE TO DENTAL IMPLANT
Toward an optimally functionalized Ti implant
Kheir Eddine KERBOUA, Pharm.D
BJKA Consulting
Tel.
Innate Immunity and Repruductive Immunology, Unit of Immunology, HMRUO, Oran, Algeria.
Email : K.K.Eddine@gmail.com
December 18th, 2014
The First Military Congress of Implantology
Military University Hospital of Constantine
HMRUC| 5 RM

3. II. Explication immuno-pathologique
III. Exploration immuno-allergologique
IV. Solutions
I. Le pourquoi du Comment
OUTLINE
Dr. Alphonse Laveran
FIRST NOBEL PRIZE
to a Military Doctor 1907
Military University Hospital
of Constantine

4. 1 century
?
MACROPHAGES

5. The multi or interdisciplinary aspects of these topics are obvious,
and without the results of basic science the field of alloplastic
materials and biological surface science COULD NOT HAVE
DEVELOPED SO EXTENSIVELY. Nevertheless, without
the experience and observations of clinical scientists, these
studies would be PURPOSELESS.
Kinga Turzo
University of Szeged, Faculty of Dentistry,
Hungary

6. Biological Surface Science
Bio – Osteo integration
Evidence Based Medecine
Immuno-
PharmacologyIndustry
Physical methods Biochemical methods
Carrier Systems for osteogenic drugs
DNA
TGF
Prot Career
Energy Protein adsorption
Charge Cellular metabolic activity
Composition Mechanical activity
Morphology Cell activity
Hypersensitivity IV
Cancer
Auto-immunity
DENTAL IMPLANTOLOGY
Bengt Kasemo 2002
Göteborg University, Sweden
R&D
Cluster Patients ????

7. THE FAILURE OF DENTAL IMPLANTS
inflammatory processes
The soft and hard tissues of THE ORAL CAVITY
(the alveolar bone, or the conjunctive and epithelial parts of the mucosa).
PERI-IMPLANT MUCOSITIS
defined as a reversible inflammatory change of the periimplant soft tissues without bone loss,
Norowski & Bumgardner, 2009
PERI-IMPLANTITIS
an inflammatory process resulting in a loss of supporting bones and associated with bleeding
and suppuration.

8. Le titane est-il capable de provoquer des
réactions allergiques?
Après pose d’implants
Après retrait des implants
D’apres Kohdera , allergologue à l’Université de Kyoto
Human oral mucosa that covered an implant closure
screw. Note the presence of free titanium particles or particles
phagocytosed by macrophagesat the epithelium-chorion
interface (→). H-E. Orig. Mag. X1000.
Le titane est-il capable de provoquer des
réactions Inflammatoires?
OR
AND

9. Ti-stimulated patient lymphocytes after rapid
differential haematology staining of cytospin
preparations of 5-day-old cell cultures.
MELISA® reactivity in a 54-year-old man before and after
removal of Ti-based dental implants and screws.
Does metal sensitivity affect patient function and failure rates?

10. Fawad Javed, 2011
?
the risk of an allergy to titanium is increased in patients who are allergic to other metals.
In these patients, an allergy evaluation is recommended, in order to exclude any problem with titanium
dental implants
The risk of an allergy to titanium
After 6 months !!

11. Biocompatibilité (TiO2)
Ostéo-intégration (ME/Os)
Les titanes
The cost constraints(--)
1. osseointegration
2. mucosal seal
3. biomechanical forces
Exceptional corrosion resistance
The Metal strengthens

12. Hanawa T. Reconstruction and regeneration of surface oxide film
on metallic materials in biological environments. Corrosion Rev 2003;
21: pp. 2-3
In dental implantology:
1. CP Ti
2. an alloy (grade V titanium): Ti-6Al-4V, Nitinol (Ti-Ni), Ti-Co (Geetha et al, 2009)
3. or titanium-zirconium, especially in cases where narrow implants are recommended
(Evrard and Atash, 2009).
International Agency for Research on Cancer (IARC) has recently classified TiO2
as a possible human carcinogen
?
Chemical dissolution of titanium dioxide
21 ppm Ti, 10.5 ppm Al, 1 ppm V (Puleo & Nanci, 1999).
NON TOXIC
Titanium has a high affinity for proteins
Forte et al, 2008Immuno-compatibility

13. The outermost atomic layers
0.1-1 nm
BIORECOGNITION
Puleo & Nanci, 1999 and Kasemo, 2002
at the INTERFACE of the implant and host tissue
IMMUNO-RECOGNITION
Biomimetic
FAR FROM the INTERFACE of the
implant and host tissue
Biocompatibility : the acceptance of an artificial implant by the surrounding tissues
the body as a whole (????)
(Park, 2000).

14. Relargage des métaux dans
Une solution electrolyte La salive
Ivana Dimić, Metall. Mater. Eng. Vol 19 (2) 2013 p. 167-176
Diffusion et accumulation tissulaire du Titane
Réaction inflammatoire Gx200 Pigmentation marron noire dorée Gx400
Réaction de la muqueuse a l’implant en titane
Nilüfer Bölükbaşı, Int J Dent Case Reports 2013; 3(1): 83-88
Echec
Aseptic lymphocyte
dominated vasculitis-
associated lesion
(ALVAL)
100-300 ppm titanium can be
observed in tissues surrounding
implants, as well as in regional
LYMPH NODES and
pulmonary tissue
(Wennerberg et al, 2004).

15. La corrosion
et la biocorrosion
Gelin et Niot
l’Institut Universitaire de Chimie, Besançon
Oral cavity represents a multivariate external environment with a wide range of circumstances,
like foods, abrasion, acidic pH, temperatures from 5 to 55C, high masticator forces, bacteria, etc.
(Lemons, 1996).
Association#Causality
F¯ The rate of corrosion
titanium-aluminium-
vanadium implants
prevalence of allergic reactions
ph
?

16. oxidative processes can thicken and condense the TiO2 layer on the surface, improving the
corrosion stability of the underlying Ti
Reductive agents, such as fluoride (F¯)
accelerated at low pH
Reclaru & Meyer, 1998; Schiff, et al., 2002
oral care products containing F¯
toothpastes,
rinsing solutions,
or prophylactic gels
Stress corrosion cracking
Könönen et al., 1995
Galvanic corrosion
between orthodontic wires and brackets
NiTi and CuNiTi
Schiff et al., 2006
Hydrofluoric acid (HF)
?
> 30 ppm
Nakagawa et al., 1999
pH: ranges from 3.5 up to neutral
F¯: between 1000 and 10,000 ppm
Hexafluorotitanate complex
Messer and Wahata, 2002
Electric Potential Difference
Na2TiF6

17. Failed human dental implant showing
tissue in contact with the metallic surface
and tissue fragments obtained by curettage
of the surgical bed.
corrosion products in the peri-implant
environment, especially surrounding the blood
vessels, to be drained by them
Rat liver. Note the abundant presence of
titanium deposits in the parenchyma of the
organ of an animal injected with TiO2.
Grenacher carmine stain. Orig. Mag. X400
Rat blood smear. Titanium particles are
evident in a peripheral blood monocyte.
Safranin stain. Orig. Mag. X1000.

18. eczema, angular cheilitis,
cheilitis
erythema of the oral mucosa,
hyperplastic gingivitis
lichenoid reactions of the
oral mucosa
perioral dermatitis or a loss of
lingual papillae that can mimic
“geographical tongue”
Waroquier et al, 2009, Vamnes et al., 2004, Leigh et al., 2001, Alanko et al., 1996
Localized inflammatory response in a 17
year old adult as a result of ionic leaching
from an implant in 12 region.
Malignancy formation as a response to
adjacent implant leach (corrosion)
(Gawkrodger, 2005)
l’Hypersensibilite type IV
L’inflammation

19. Host tissue response to an implant material
The keystone: Ti DEBRIS
Explication immuno-pathologique
I. Innate Immunity Response: Macrophage Oxidative stress Damage
II. Specific Immunity Response: Macrophage + TH1 Clinical Manifestations

20. tissue damage
Titanium+++/Zirconium+
CD68 macrophages haptenes
inflammatory mediators
macrophage recruitment
activating osteoclasts
+suppress the osteoblast function
osteolysis
“In situ” degradation
Relargage des particules
Inflammation Hypersensitivity
150 nm to 10 micro-m
Immunité Innée Immunité adaptative
Ions Ti or <1000 D
Métal de transition
Forte affinité pour les protéines
Néo épitope
Auto-immunité
may disrupt normal cell physiology
Ti-bound intra-cellular proteins
Cancérogenese
De-implantation
DNA damage
Size of particles, Large particles may not bond to proteins and may not act as antigens
ROS
Perte de l’Implant
unfavourable conditions: acidic pH (peri-implantitis ), excessive
mechanical forces on the implant, contact of the implant with
another metal (amalgam, gold alloy)

21. I. Innate Immunity Response

22. Titanium has also been reported to activate macrophages, which may secrete
cytokines involved in various disease processes
Photomicrograph of macrophages near the surface of the implant. Note the presence of particles in their
cytoplasm. Ground section. Orig. Mag. X1000
Muller and Valentine-Thon, 2006
cytokines
MACROPHAGES

23. Phagocytosing
debris induce inflammasome
danger-signaling
(NALP3).
Potent proinflammatory
cytokine IL-1β is produced.
NFkβ pathway
induction of
TNF-α, IL-1β, IL-6, and
PGE2
2
Decreased osteoblast function
1
Osteoclast activity increases
Ti

24. l’hypersensibilité retardée Cell-mediated response (Type IV)
Concept de l’haptene
1. Antigens activate Anti Ti
Memory T-lymphocytes
2. T-Lymphocytes release cytokines
•INTERFERON
•Tumor necrosis factor TNFα
•Interleukin 1&2 IL1 et IL2
3. Cytokines recruit and activate
macrophages,
monocytes and neutrophils
Le cercle vicieux
D’apres Duché et Barré
Nickel
Cobalt
Chrome
Beryllium
Titanium
Tantellum
Vanadium
Macrophages provoquent l’activation des T-cells
T-cells activent macrophages
Macrophages provoquent l’activation des T-cells
T-cells activent macrophages
Métaux inducteurs d’allergie
++++
Ti
Ti
Ti
II. Specific Immunity Response
TH1

25. Titanium has a high affinity for proteins
Environmental sources
and not necessarily from previous contact with dental implants.
1. watches, jewellery and spectacle frames
2. E171 TiO2 :cosmetics such as
sunscreens, make-up and deodorants,
and in food, medicines or toothpaste
50 ppm of titanium
4,2%
Pranay D. Khare 2010
sensitization to titanium is it possible?
Forte et al, 2008
Anti Ti Memory T-lymphocytes???

26. Exploration immuno-allergologique
Les tests In-Vivo+/-
Les tests In-Vivo+++
Patch tests
Blood tests +++
Cluster patients!!!!!

27. Tests cutanés
D’apres Dr Guillot.
Les tests In-Vivo ?
• Les tests cutanés ne pourront pas etre appliqué pour
les tests de relargage des implants
– Temps d’exposition trop faible
– La réponse d’Hypersensibilité n’est pas contre
implant lui meme, mais contre les produits de
dissolution ou de corrosion product
– L’optimisation de l’agent inititaeur n’est pas
claire (eg, chlorides du metal, complexes
specifiques metal-protein).
– La peau sollicite une seule Cellule Présentratrice
d’antigene (Langerhans cells) qui sont 10 fois
moins nombreuses dans les tissues
perimplantaire.
– La vascularisation de muqueuse buccale
élimine les allergens plus rapidement que la
peau (Forte et al, 2008).
– M. orale less permeable to antigens
– Induction de l’ hypersensibilite
Seulement 3 /4 patients
diagnostic efficiency of 75% for metal allergy
lack of standardization in the patch tests, especially for allergens such as titanium
Fischer, 2008, Forte et al, 2008

28. Les tests In-Vitro
1. Le test LTT (Lymphocyte transformation testing)
 Developé en 1960
 Measure la réponse proliferative des
lymphocytes aux antigens
 Lymphocytes sensibilisés par les allergens
se transforment en blasts et proliferent lors
du deuxieme exposition à cet antigene
 La réplication de l’ADN est utilisée comme
mesure de la prolifération
 Ratio de la prolifération lymphocytaire
apres la stimulation antigenique sur la
prolifération lymphocytaire en absence
d’antigene.
Ce ratio est applé the stimulation index (SI)
Avantages de la LTT
• Résultats Quantifiable
• Sensibilité importante
• Sang et pas la peau
• N’induit pas une hypersensibilité
• Permet l’analyse de plusieurs métaux à
différentes concentrations
• sometimes lack specificity(Faux positifs)
Fisher’s, 2008
Inconvenient de la LTT
Test MELISA; Stjekstal et al, 1994,
Muller and Valentine-Thon, 2006

29. Isolat des Lymphocytes
Co-culture avec les sels de métaux
Mesure:
Incorporation de la thymidine-H
Contrôle Qualité:
Verifier la Morphologie
10-40 ml Sang Total Avant la centrifugation
Apres la centrifugation0
Isolat des Lymphocytes
Séparation des CMN
6 jours
A stimulation index of is a 2 cut-off value
SI < 2: is considered negative
SI ≥ 2 but <3: a possible sensitization
SI ≥ 3: positive sensitization
SI 2–3 “weakly positive”
SI 3–5 “positive”
SI > 5 “strongly positive”
Accredited in Germany since 2001, should be considered for patients with
Suspicion of Ti allergy
!

30. Flow cytometry, for the purpose of detecting the activation of lymphocytes stimulated
by a metal, and measuring different mediators (cytokines, inflammatory mediators)
released in response to the metal.
2. LAT = lymphocyte activation test
Les tests In-Vitro
Measurement of CD69 up regulation on antigen-reactive T-cells and T-cell clones
from peripheral blood mononuclear cells (PBMC) cultures isolated from patients and
stimulated with antigen. Its require FACS, it distinguish CD4+ and CD8+ T cells, highly
quantitative.
Freshly isolated PBMC cultured for 48h in U-bottomed
tissue culture plate
metal concentrations
PHA (positive control)
flow cytometry
PE-CD69, PerCP-CD3,
FITC- CD4, APC-CD8
and PE-IgG1 as isotype control (BD).

31. The prevalence of metal sensitivity in
patients with FAILED IMPLANTS is
approximately six-times that of the
general population
and approximately two- to three-
times that of all patients.
Hallab Bulletin of the NYU Hospital for Joint Diseases 2009
(#) Pas de réactions granulomateuses !!!!!!!!!!!!!!!
25% vs 60%
Messer and Wataha, 2002, Nakashima et al, 1999, Muller and Valentine-Thon, 2006
Future !!!!!

32. oral care products containing F¯
Hexafluorotitanate complex (Na2TiF6)
> 0.2 % Pd
pH: ranges from 3.5 up to neutral
Ti-0.2Pd
Nakagawa et al., 2001
Stress corrosion cracking
Galvanic corrosion
between orthodontic wires and brackets
NiTi and CuNiTi
Patients allergic to other metals
MELISA
Pre-Implant Per-Implant Post-Implant
LAT

33. Le chirurgien-dentiste pourra prescrire un test d’activation lymphocytaire ou test MELISA pour vérifier
l’intolérance éventuelle aux métaux qu’il envisage d’utiliser chez son patient.
le diagnostic d’une allergie ou d’une intolérance aux métaux utilisés est important lorsqu’un patient est déjà
porteur d’une réhabilitation orale avec des implants et qu’il présente des symptômes allergiques apparus en
concomitance avec le placement des implants et/ou des prothèses
La décision quant à la dépose éventuelle des prothèses et implants ne doit pas se faire à la légère.
Une concertation pluri-disciplinaire est à mettre en œuvre.
Un dialogue constructif et une prise de décision en collaboration avec les allergologues
et/ou immuno-dermatologues seront alors nécessaires
L’ostéointégration d’un implant
N’EST PAS SYNONYME
de bio-intégration
Conclusion

34. CHOKRANE !!!!!
AS’ILA???