Lethal photosensitization and guided - coimplante.odo.brcoimplante.odo.br/Biblioteca/Perimplantite/Lethal... · Lethal photosensitization and guided bone regeneration in treatment

Lethal photosensitization and guidedbone regeneration in treatment ofperi-implantitis: an experimentalstudy in dogs

Jamil Awad ShibliMarilia Compagnoni MartinsFernando Salimon RibeiroValdir Gouveia GarciaFrancisco Humberto Nociti JrElcio Marcantonio Jr

Authors’ affiliations:Jamil Awad Shibli, Department of Periodontology,Dental Research Division, Guarulhos University,Guarulhos, SP, BrazilMarilia Compagnoni Martins, Fernando SalimonRibeiro, Elcio Marcantonio Jr, Department ofPeriodontology, Dental School of Araraquara, StateUniversity of Sao Paulo (UNESP), Araraquara, SP,BrazilValdir Gouveia Garcia, Dental School of Marılia,University of Marılia (UNIMAR), Marılia, SP,Brazil; Dental School of Aracatuba, State Universityof Sao Paulo (UNESP), Aracatuba, SP, BrazilFrancisco Humberto Nociti Jr, Department ofPeriodontology, Dental School of Piracicaba,University of Campinas, (UNICAMP) SP, Brazil

Correspondence to:Elico Marcantonio JrDepartmento de PeriodontiaFaculdade de Odontologia de Araraquara-UNESPR. Humaita, 168014801-903 Araraquara, SPBrazilTel.:þ 55 16 3301 6369Fax: þ55 16 3301 6314e-mail: [email protected]

Key words: guided bone regeneration, histology, peri-implantitis, photodynamic therapy/

photosensitizers, re-osseointegration

Abstract: The purpose of this study was to evaluate the effect of lethal photosensitization

and guided bone regeneration (GBR) on the treatment of ligature-induced peri-implantitis

in different implant surfaces. The treatment outcome was evaluated by clinical and

histometric methods. A total of 40 dental implants with four different surface coatings (10

commercially pure titanium surface (cpTi); 10 titanium plasma-sprayed (TPS); 10 acid-etched

surface; 10 surface-oxide sandblasted) were inserted into five mongrel dogs. After 3

months, the animals with ligature-induced peri-implantitis were subjected to surgical

treatment using a split-mouth design. The controls were treated by debridment and GBR,

while the test side received an additional therapy with photosensitization, using a GaAlAs

diode laser, with a wavelength of 830 nm and a power output of 50 mW for 80 s (4 J/cm2),

and sensitized toluidine blue O (100 mg/ml). The animals were sacrificed 5 months after

therapy. The control sites presented an earlier exposition of the membranes on all coating

surfaces, while the test group presented a higher bone height gain. Re-osseointegration

ranged between 41.9% for the cpTi surface and 31.19% for the TPS surface in the test sites;

however differences were not achieved between the surfaces. The lethal photosensitization

associated with GBR allowed for better re-osseointegration at the area adjacent to the peri-

implant defect regardless of the implant surface.

Animal studies have shown that bacterial

biofilm accumulation around dental im-

plants promoted by ligature placement

may develop peri-implant tissue breakdown,

also known, as peri-implantitis (Lang et al.

1993; Schou et al. 1993; Ericsson et al.

1996). Plaque-induced peri-implantitis has

been indicated as one of the etiological

factors associated with long-term failure of

dental implants (Quirynen et al. 2002).

Different implant surfaces have been

proposed in order to improve clinical and

histological outcome of dental implants

and, therefore, the interaction between

different implant surfaces and peri-implan-

titis therapy may be of interest.

Recent studies (Persson et al. 2001b;

Kolonidis et al. 2003; Schou et al. 2003a;

Shibli et al. 2003b, 2003c) have investi-

gated the importance of implant surface

cleanliness after peri-implantitis treat-

ment. It has been hypothesized that surface

contaminants, released from contaminated

implant surfaces, enhance and perpetuate

the inflammatory response, thus altering

the healing process, and possibly leading to

the dissolution of titanium (Baier et al.

1984, 1988; Olefjord & Hansson 1993;

Esposito et al. 1999; Shibli et al. 2004). In

addition, the alterations of the titanium

oxide layer may not allow re-osseointegra-

tion. In earlier studies, Shibli et al. (2003b,Copyright r Blackwell Munksgaard 2006

Date:Accepted 15 January 2005

To cite this article:Shibli JA, Martins MC, Ribeiro FS, Garcia VG, Nociti JrFH, Marcantonio Jr E. Lethal photosensitization andguided bone regeneration in treatment of peri-implantitis: an experimental study in dogs.Clin. Oral Impl. Res. 17, 2006; 273–281doi: 10.1111/j.1600-0501.2005.01167.x

273

2003c) demonstrated the potential use

of photodynamic therapy associated with

guided bone regeneration (GBR) in the

treatment of experimental peri-implant le-

sions. The average re-osseointegration ob-

tained in this study was 25.15%, similar to

previous studies that have used mechanical

treatment associated with systemic antibio-

tics (Persson et al. 1999; Wetzel et al. 1999;

Nociti et al. 2001a; Schou et al. 2003a,

2003c). Photodynamic therapy uses a low-

power laser, following the application of a

photosensitizing substance, such as tolui-

dine blue O (TBO). The mechanism by

which TBO kills microorganisms, such as

Porphyromonas gingivalis, Prevotella inter-

media, Actinobacillus actinomycetemco-

mitans, and Fusobacterium nucleatum, is

currently under research. However, it is

believed that lethal photosensitization of

these microorganisms may involve changes

in their membranes, on the plasma mem-

brane proteins, and DNA damage mediated

by singlet oxygen (Dobson et al. 1992;

Sarkar & Wilson 1993; Henry et al. 1995;

Bhatti et al. 1997, 1998; Wainwright 1998;

Bhatti et al. 2000).

The objective of this study was to eval-

uate the efficacy of lethal photosensitiza-

tion associated with GBR, for the

treatment of ligature-induced peri-implan-

titis in dogs, using different dental implant-

coated surfaces.

Material and methods

Animals and anesthesia

The outline of the experiment is presented

in Fig. 1. Briefly, five healthy male mon-

grel dogs, with an average age of 2 years and

an average weight of 18 kg, were used in

the study. Animal selection, management,

and surgical protocol followed routines

approved for this study by the local In-

stitutional Animal Care and Ethics

Committee.

All surgical and clinical procedures, as

well as laser irradiation, were performed

under general anesthesia accomplished

with 0.05 mg/kg of subcutaneous prea-

nesthesia sedation (Atropine sulfate –

0.5 mg, Ariston Inds. Quımica e Farms.

LTDA, Sao Paulo, Brazil), intravenous in-

jection of chlorpromazine (Amplictil

25 mg, Rhodia Farma LTDA, Sao Paulo,

Brazil), and thiopental (Tiopental – AB-

BOTT Laboratorios do Brasil Ltda, Sao

Paulo, Brazil).

Tooth extraction

An edentulous ridge was created by the

extraction of all mandibular premolars and

first molars. The alveoli were allowed to

heal for a period of 3 months. The upper

premolars were also extracted to prevent

occlusion trauma interference during pla-

que-induced peri-implantitis. During the

healing period, bacterial biofilm control

was performed by scrubbing with 0.12%

chlorhexidine daily, in addition to scaling

and root planning once a month, until

cotton ligatures were placed.

Implant design, surface, and surgery

Forty dental implants of four different

surfaces were used: 10 commercially pure

titanium implants (cpTi; Sterngold, Im-

plamed, Attleboro, MA, USA); 10 titanium

plasma-sprayed (TPS; Sterngold, Im-

plamed); 10 hybrid surfaces – machined

titanium in the three first screws and acid

etched in other screws – acid (Osseotites

-

3is

Implants Innovations, Palm Beach

Gardens, FL, USA), and 10 sandblasted

with titaniun oxide – oxide (Porous, Con-

exao Implants, Sao Paulo, SP, Brazil).

All implants were 10 mm in length and

3.75 mm in diameter.

The implants were randomly distributed

among the dogs, so that each dental im-

plant surface could be represented on

each mandibular side. Dental implants

were placed after full thickness flap surgery

under aseptic conditions. The recipient

sites were prepared according to the surgi-

cal techniques indicated by each implant

manufacturer. The flaps were sutured with

single interrupted sutures, submerging all

implants. Potassic and sodic benzilpenici-

lin (Fort Dodge Saude Animal LTDA,

Campinas, SP, Brazil) was given once a

week for 2 weeks, in order to prevent post-

surgical infection, pain was controlled

with paracetamol (ABBOTT Laboratorios

do Brasil Ltda), and the sutures were re-

moved after 10 days.

Experimental peri-implantitis

Three months after dental implant installa-

tion, healing abutments were installed,

according to the instructions of each dental

implant system. After 2 months of a pla-

que control program and the healing of the

soft tissue, cotton floss ligatures were

placed around the dental implants, as pre-

viously described (Shibli et al. 2003a). At

90 days, when approximately 40% of the

initial bone support was lost, the ligatures

were removed, and a 2-month plaque

control program was initiated by daily

scrubbing of the implants with 0.12%

chlorhexidine and scaling of the abutment

surface.

Randomization of the mandibular quad-

rants and a split-mouth design was estab-

lished as follows: control side – mechanical

debridment and GBR, and test side – me-

chanical debridment, lethal photosensitiza-

tion, and GBR to compare the effect of

lethal photosensitization (Table 1).

Clinical evaluation and treatment

A crestal incision was made through the

mucosa, and buccal and lingual full-thick-

ness flaps surgery was performed to remove

Extraction

Implant

Abutment

+Lig. –Lig.

Treatment

Biopsy

–8 –5 –2 0 3 5 9

0.12% chlorhexidine 0.12% chlorhexidine

Membrane removal

10 months

Fig. 1. Outline of the experiment. Animals n¼ 5, dental implants n¼ 40. Ligatures were placed at 0 month

(þLig.) and removed at 3 months (�Lig.).

Shibli et al . Lethal photosensitization in treatment of peri-implantitis

274 | Clin. Oral Impl. Res. 17, 2006 / 273–281

the abutments and the granulation tissue

present in bone craters around the dental

implants. A plastic scaller (Implacare–

IMPHDL6, Hu-friedy Mfg Co Inc., Chi-

cago, IL, USA) was used to prevent any

damage to exposed dental implant surface.

Pre- and post-treatment bone defects were

measured by a single trained examiner, i.e.,

the distance from the top of the cover screw

to the bottom of the peri-implant defect,

using a periodontal probe (PCPUNC 15

Hu-friedy Mfg Co Inc.), at four sites (me-

sial, buccal, distal, and lingual).

The implant surfaces of the test group

received 100mg/ml TBO (Sigma LTDA,

Poole, UK) injected into the peri-implant

defect, as far as the bony edge, on the

previously contaminated implant surface,

using a thin needle as previously described

(Shibli et al. 2003b, 2003c). TBO was

placed for 1 min, and then carefully re-

moved. The stained area was subsequently

irradiated with a GaAlAs diode laser

(Thera-Lase, DMC Equipamentos LTDA,

Sao Carlos, SP, Brazil) with a measured

power output of 50 mW, to emit radiation

in collimated beams (1 cm2) with a wave-

length of 830 nm, for 80 s, and a total

energy of 4 J (energy density of 4 J/cm2).

The diode laser was in contact with the

mesial, distal, buccal, and lingual surfaces

by a scanning method, for 20 s, on each

surface. Multiple perforations of the bone

surface were previously made to facilitate

the filling of the defect with the coagulum.

All the sites, including control sides,

received a PTFE membrane (TefGen-

PLUS, Lifecore Biomedical Inc., Chasca,

MN, USA), allowing implant penetration

through the site. The membrane was ad-

justed to extend circumferentially 3–5 mm

over the adjacent alveolar bone, avoiding

ingrowths of the soft connective tissue.

The membranes were stabilized at the

buccal and lingual sites not only by cpTi

tacks (INP Implantes Nacionais and Prot-

eses Comercio Ltda, Sao Paulo, SP, Brazil)

but also by cover screws. To allow flap

apposition and closure after placement,

incisions were made buccally and lingually

after membrane placement. Primary

wound closure was achieved with horizon-

tal mattress sutures alternated with

interrupted sutures. Anti-inflammatory

medication (2 mg betamethasone Celes-

tone, Schering-Plough S/A, Rio de Janeiro,

RJ, Brazil) was administered twice a day

and appropriate analgesia (paracetamol) for

3 days following surgery in order to reduce

postoperative swelling and pain. Antibio-

tics were administered neither before

nor after the surgical treatment of the

peri-implant defect. Two weeks after local

therapy, the sutures were removed and a

fluorochrome (Oxytetracycline 25 mg/kg

body weight; Pfizer do Brasil, Sao Paulo,

SP, Brazil) was injected intravenously (i.v.).

Four months after treatment, the PTFE

membranes were surgically removed in

both groups and the implants were allowed

to heal for 1 month. Four days before the

euthanasia, a second fluorochrome (18 mg/

kg red alizarine body weight; Sigma

Chemical Co., St Louis, MO, USA) was

injected i.v. for observation of the

remodelation of peri-implant bone tissue

at 5 months after treatment. Five months

after treatment, the animals were sacri-

ficed by induction of deep anesthesia fol-

lowed by intravenous sodium pentobarbital

euthanasia.

Histological procedures

The mandibles were removed and block

biopsies of each implant site were dissected

and fixed in 4% neutral formalin for 48 h.

The biopsies were then prepared for ground

sectioning according to the methods pre-

viously described by Donath & Breuner

(1982). The specimens were cut in a me-

sio-distal plane using a cutting–grinding

unit (Exacts

Cutting, System, Apparatebau

Gmbh, Hamburg, Germany). One central

section of each biopsy was prepared and

reduced to a final thickness of 50–70mm,

by micro-grinding and polishing using a

micro-grinding unit (Exacts

Cutting, Sys-

tem, Apparatebau Gmbh). Before staining,

each section was evaluated with respect to

the location of the fluorochrome marker

by microscopy (Leitz DM-RBE micro-

scopy, Leica, Bensheim, Germany)

equipped with an image system (Qwin,

Leica). In the unstained sections, fluores-

cence light and a filter cube compatible

with the fluorochrome were used to assess

the bone defect border, as well as the

remodeling of the new bone.

The sections were stained in toluidine

blue in order to assess the histometric para-

meters: (1) Distance from the original

bottom of the defect – identified by the

difference in coloration after staining and

by the fluorochrome (a) to the most coronal

point of the newly formed bone with in-

timate contact with the implant surface, (b)

(¼ re-osseointegation); (2) area of (a) to the

most apical border of the newly formed

bone, (c) to implant shoulder, (d) (¼bone fill); (3) percentage of osseointegration

(mineralized bone contact with the im-

plant surface); and (4) bone area within the

limits of the implant threads at the portion

of the implant, apical of the peri-implant

defect, where peri-implantitis did not

occur (Fig. 2).

Data analysis

Data were obtained in pixels and pixels2

and transformed into percentage to prevent

the possible influence of the different

macrostructure of the different implant

Table 1. Distribution of dental implants with different surfaces in five dogs

Animal Test side Control side

PM2 PM3 PM4 M PM2 PM3 PM4 M

1 cpTi TPS Oxide Acid Acid Oxide TPS cpTi2 TPS Oxide Acid cpTi Oxide TPS cpTi Acid3 Oxide cpTi TPS Acid TPS cpTi Acid Oxide4 cpTi Acid Oxide TPS Acid TPS Oxide cpTi5 Acid TPS CpTi Oxide cpTi Acid Oxide TPS

PM2, PM3, PM4, and M, mandibular premolars and molar; cpTi, commercially pure titanium; TPS, titanium plasma-sprayed; Oxide, surface sandblasted with

titanium oxide; Acid, acid-etched surface.



systems evaluated in this study. The data

relative to defect depth, percentage of os-

seointegration, and bone area within the

limits of the threads were obtained from all

implants, independent of group.

Paired t-test and parametric analysis of

variance were performed to assess the dif-

ferences between test and control groups

and among the several implant surfaces,

respectively. The unit of analysis was the

animal (n¼5), and the level of significance

was 0.05.

Results

Clinical observations

One cpTi (test group) and one oxide surface

(control group) were lost during the experi-

mental peri-implantitis phase in two ani-

mals. All the other remaining implants

were included in the evaluation. The den-

tal implants of the test group successfully

integrated to the bone and survived the

subsequent periods of treatment. However,

89.47% of the control sites depicted earlier

exposition of the membranes when com-

pared with the 15.79% of the test sites

(Tables 2 and 3). Implants from the control

group showed several exposed threads,

while some dental implants from the test

group appeared to be covered by new bone

(Figs 3a–c). When the effect of the treat-

ment was compared between the test and

the control group independent of the im-

plant surface, the test group presented a

higher bone gain (Table 6).

Histological examinations andmeasurements

The peri-implant soft and hard tissues from

all treatment groups generally appeared

healthy, with the junctional epithelium

Table 2. Overview of clinical measurements (mm) for the test and the control group pre- and post-treatment as well as membraneexposure per animal

Animal cpTi TPS Acid Oxide

Test Control Test Control Test Control Test Control

Pre, post Pre, post Pre, post Pre, post Pre, post Pre, post Pre, post Pre, post

1 1, 0 5.5, 5w 3, 1.5 4.35, 3.75z 2.25, 0 5, 5z 2.25, 0 2.5, 3.25w2 Failed implant 3, 2.5 5.5, 0.75 5, 4z 1.25, 0.25 0.5, 0.5y 2.5, 0.75 1, 3.2n

3 2.75, 2.5 2.75, 2.5w 2.75, 2.5 2.5, 4.75n 2.5, 2 3.25, 3.75w 3.25, 3.25w Failed implant4 3, 0 3, 3.1w 4.5, 4 3, 0 3.75, 0 2.25, 2.25w 3, 0 2.25, 2.25w5 3, 1.75 2.75, 4n 7, 3y 1, 3n 3.25, 2z 3-3w 3, 1.15 6, 6wMean 2.43, 1.26 3.4, 3.42 4.55, 2.35 3.15, 3.1 2.60, 0.85 2.8, 2.9 2.8, 1.1 2.8, 3.79SD 0.96, 1.26 1.18, 1.07 1.77, 1.27 1.55, 1.84 0.96, 1.05 1.63, 1.68 0.41, 1.35 1.88, 1.42P 0.091NS 0.954NS 0.07NS 0.96NS 0.038S 0.37NS 0.026S 0.107NS

Calculated values are based on the mean value of four sites (mesial, buccal, distal, and lingual).nExposure of membrane before the first post-operative month.

wExposure of membrane between the first and second post-operative month.

zExposure of membrane between the second and third post-operative month.

yExposure of membrane after the third post-operative month. sStatistically significant.NS, not significant; cpTi, commercially pure titanium; TPS, titanium plasma-sprayed; Oxide, surface sandblasted with titanium oxide; Acid, acid etched

surface.

Table 3. Clinical height measurements (mm� SD) of the peri-implant bone defects for test and control groups

Test side Control side

cpTin TPS Oxide Acid cpTi TPS Oxiden Acid

Pre-treatment

2.43 � 0.96 4.55 � 1.77 2.80 � 0.41 2.60 � 0.96 3.40 � 1.18 3.15 � 1.55 2.93 � 2.14 2.80 � 1.63

Post-treatment

1.06 � 1.26 2.35 � 1.27 1.10 � 1.35 0.85 � 1.05 2.92 � 1.88 3.10 � 1.84 3.67 � 1.61 2.90 � 1.68

Bone heightgain (mm)k

1.81 � 0.89w,z 2 � 0.5w,z 1.7 � 1.11y,z 1.75 � 1.28y,z 0.48 � 1.56w 0.05 � 2.19w � 1.17 � 1.14w � 0.50 � 1.11w

Bonegain (%)k

67.06 � 42.8z 42.74 � 32.76z 66.33 � 40.93z 66.15 � 40.93z 14.20 � 53.06 � 31.24 � 11.98 � 28.17 � 36.45 0

Calculated values are based on the mean value for four sites (mesial, buccal, distal, and lingual).nOne implant was lost at ligature phase (n¼ 4 implants).

wNot significant between pre- and post-treatment (P40.05).

zNot significant between groups test and control (P40.05).

yStatistically significant between pre- and post-treatment (P�0.03).

zStatistically significant between groups test and control (P�0.02).

kNot significant among the different implant surfaces (P � 0.711).

cpTi, commercially pure titanium; TPS, titanium plasma-sprayed; Oxide, surface sandblasted with titanium oxide; Acid, acid etched surface.

Fig. 2. Schematic drawing illustrating the land-

marks used for the histometric measurements.



extending a short distance apical to the

implant shoulder. The connective tissue

seemed to be compromised primarily by

dense collagenous fibers, which ran parallel

to the previously ‘contaminated’ implant

surface. The old bone was mostly lamellar

and compact, and numerous osteocytes

were presented in their lacunae, while

the newly formed bone exhibited different

stages of maturation and remodelation,

mainly in the test group.

The biopsies from the sites where the

membrane had been exposed showed very

few spicules of bone at the base of the

connective tissue cap. In some cases, os-

teoblasts were connected to newly formed

bone, indicating ongoing bone formation,

and minor apposition of new bone could be

found, specifically at the apical regions of

the originally induced defects; however,

the bone tissue appeared immature. The

bone marker projected in a lateral–coronal

direction and separated a triangular-shaped

portion of newly formed regenerated bone

from the ‘old’ bone (Figs 4a–c).

Only the TPS implants presented surface

debris or particle inclusions in the sur-

rounding tissue at the bone area, within

the limits of the implant threads at the

apical portion of the peri-implant defect,

where peri-implantitis did not occur (Figs

5a and b).

The defect depth, the percentage of

mineralized bone contact with the dental

implant surface, and the bone area within

the limits of the threads are presented in

Fig. 6. The highest percentage of osseointe-

gration was 73.58% of the TPS surface and

the lowest was 61.48% of the cpTi surface

(P¼ 0.029).

Bone filling was statistically significant

between the test and control groups

(P�0.036) (Tables 4 and 6). In some speci-

mens, the lateral aspect of the coronal part

of the dental implant was covered by a

dense connective tissue capsule separating

the newly formed bone from the dental

implant surface (Fig. 5b).

The mean percentage of re-osseointegra-

tion was 31–41% for the test group and 0–

14% for the control group (Tables 5 and 6).

In some specimens in the control group,

there was no contact between new bone

and the previously contaminated implant

surface (Figs 7a and b).

Discussion

The difficulties in obtaining re-osseointe-

gration after treatment of peri-implantitis

have been documented in several animal

studies (Grunder et al. 1993; Ericsson et al.

1996; Persson et al. 1996; Hanisch et al.

1997; Wetzel et al. 1999; Shibli et al.

2003b). Recently, Kolonidis et al. (2003)

showed that re-osseointegration occurred

on a surface previously contaminated

with bacterial biofilm. However, the ani-

mal model used by Kolonidis et al. (2003) is

not comparable, either with the animal

model used in our study, or with the pre-

viously cited studies (Grunder et al. 1993;

Ericsson et al. 1996; Persson et al. 1996,

1999; Wetzel et al. 1999; Nociti et al.

2001a, 2001b; Persson et al. 2001a; Shibli

et al. 2003b) because of lack of important

factors such as peri-implant soft and hard

tissue breakdown induced by ligatures, the

presence of bone defect, and presence of

bone debris at the implant surface.

The amount of bone regeneration was

significantly influenced by the photosensi-

tization treatment. The earlier exposition

of the membranes, as well as the mainte-

nance of these barriers can jeopardize tissue

regeneration (Lekholm et al. 1993;

Machado et al. 1999; Haas et al. 2000).

The complications because of membrane

exposition in the control group may sup-

port the findings that the presence of perio-

dontal pathogens at the dental implant

fixture altered the cell responses, together

with enhanced protease activities and for-

eign body reactions, decreasing the efficacy

of GBR (Wakabayashi et al. 1996; Slots et

al. 1999). The control group presented 5.6

times more earlier membrane exposition

when compared with the test group. In the

present investigation, the clinical evalua-

tion pre- and post-treatment revealed a

variable degree of appreciable hard-tissue

fill of the peri-implant defects, mainly for

the test group.

The histometric analysis depicts a statis-

tically higher amount of new bone forma-

tion for the test group. Although the

percentages of bone fill observed in studies

such as those by Wetzel et al. (1999) and

Persson et al. (1999) were lower, our

results ranged between 41% for the TPS

surface and 60.87% for the acid surface, in

agreement with Persson et al. (1996, 1999),

although the last two studies cited utilized

smooth surfaces.

Re-osseointegration was achieved on all

dental implant surfaces of the test group,

principally at the base of the angular bony

defect, in agreement with previous studies

(Jovanovic et al. 1993; Singh et al. 1993;

Persson et al. 1996; Shibli et al. 2003b).

The higher percentage of re-osseointegra-

tion observed in our investigation was

41.90%, of the cpTi surface, and the low-

est was on the TPS surface (31.19%),

which is in agreement with the results

described by Hanisch et al. (1997) and

higher when compared with the results

presented by Wetzel et al. (1999) and Shibli

et al. (2003b).

On the other hand, Persson et al. (2001a)

found 83.7� 8.6% of re-osseointegration

Fig. 3. (a) Peri-implant defect adjacent to commer-

cially pure titanium (left) and acid surface (right)

after mechanical debridment; (b) clinical view of the

same defect 4 months after lethal photosensitization

and guided bone regeneration. Note the contour of

the newly formed bone (arrows) on the peri-implant

defect; (c) clinical view of peri-implant defects from

the control group covered by fibrous tissue after

membrane removal. The membrane exposure oc-

curred after 1 month post-treatment.



in sandblasted large grit acid-etched sur-

faces (SLA) and 21.8� 16.7% for turned

surfaces. The authors speculated that the

SLA surface could provide a better condi-

tion for coagulum stability, facilitating the

bone regeneration process. However, the

different coated surfaces evaluated in our

study showed lower means of re-osseointe-

gration than for the cpTi surface. One may

speculate about the differences between the

present study and the study performed by

Persson et al. (2001a): (1) the treatment

using systemic antibiotics may decrease

the number of periodontal pathogens

present in the hard and soft peri-implant

tissue, and (2) the SLA surface may present

specific chemical properties that facilitate

re-osseointegration.

The healing of the dental implant is

initiated immediately after implant inser-

tion by initial blood clot formation in the

peri-implant gaps and the development of a

biofilm (D’Hoedt 1985; Meyer et al. 1988;

Zechner et al. 2003). The roughnesses of

the implant surface are an important factor

in this process. In our study, there were no

statistical differences for the amount of re-

osseointegration in different dental implant

surfaces. The adsorption of microorgan-

isms as well as the application of the

TBO on the implant surface may influence

the direct coagulum–implant contacts and,

consequently, re-osseointegration. In addi-

tion, a series of co-ordinated events, in-

cluding protein adsorption, proliferation,

and deposition of bone tissue, are probably

affected by experimental peri-implantitis.

In addition, a dense connective tissue

capsule that separated the newly formed

bone from the dental implant surface was

Fig. 4. (a) Ground section of commercially pure titanium implant from the test group. Fluorescence light (original magnification � 200). The fluorochrome marks show

the border of the bone defect (yellow) and the new bone formation (red); (b) mesio-distal ground section of the same area (toluidine blue staining, original magnification

� 200) showing the re-osseointegration with dental implant surface contamined previously; (c) the fluorochrome marks show the border of the bone defect (yellow) and

the new bone formation (red). on the oxide surface (original magnification � 200).

Fig. 5. (a) Surface debris and particle inclusions of titanium plasma-sprayed (TPS) surface in the surrounding

tissue at the bone area within the limits of the implant threads apical of the peri-implant defect (toluidine blue

staining, original magnification � 100); (b) ground section of one TPS surface from the test group (toluidine

blue staining, original magnification � 100) showing the borderline between the ‘old’ original bone and the

newly formed bone (arrows) in direct contact with the dental implant surface contamined previously (re-

osseointegration) (arrows heads). Note a dense connective tissue (CT) between the implant surface and newly

formed bone.



observed in some specimens, as previously

reported (Persson et al. 1996; Wetzel et al.

1999). Despite controversy regarding the

amount of re-osseointegration (Persson et

al. 1996; Nociti et al. 2001a, 2001b; Schou

et al. 2003a, 2003c), these different results

may be attributed to different experimental

designs and variables such as the ligature-

induced peri-implantitis period, material

used for ligatures (cotton, silk, orthodontic

elastics), microstructure utilized, cleaning

methods of the contaminated implant sur-

face and their efficiency, bony defect shape,

and combination of graft materials and

GBR.

In addition, the use of photodynamic

therapy to kill periodontal pathogens offers

some advantages over the use of conven-

tional antimicrobials: it prevents the devel-

opment of resistance among target

organisms to the photochemically gener-

ated free radicals thought to be responsible

for bacterial killing and, unlike antiseptics

and antibiotics, there would be no need

to maintain high concentrations of the

TBO in the peri-implant defects for long

periods.

The results of the present investigation

support the use of GBR associated with

antimicrobial local therapy for the treat-

ment of peri-implant defects. In addition,

the use of photodynamic therapy as local

treatment may be a useful alternative to

antibiotics for the treatment of local infec-

tion by eradication of target cells using

oxygen species produced by interaction

between a photosensitizing agent and light

of an appropriate wavelength (Dougherty et

al. 1998; Komerik & Wilson 2002).

In conclusion, data from the present

study showed that the treatment of liga-

ture-induced peri-implantitis using lethal

photosensitization, associated with GBR,

may achieve significant bone fill associated

with re-osseointegration. The different

coating surfaces evaluated in our study

presented the same healing responses to

peri-implantitis treatment. However, these

results should be considered with caution

and further investigations must be con-

ducted.

Table 4. Percentage of bone fill measured from the base of the defect to the implantshoulder

Implantsurfacen

Test Control P 95% CI

Mean � SD Range Mean � SD Range

cpTi 54.85 � 21.77 23.32–72.31 14.98 � 17.79 0–45.73 0.036 4.65–73.44TPS 41 � 29.2 0–68.62 15.3 � 19.74 0–47.21 0.142 � 13.57–64.91Oxide 49.29 � 7.1 43.83–61.54 12.06 � 14.49 0–31.6 0.027 14.22–76.48Acid 60.87 � 21.83 42.43–95.79 15.52 � 3.41 10.75–19.99 0.015 14.22–76.48

nNot significant among the surfaces P¼ 0.98.

cpTi, commercially pure titanium; TPS, titanium plasma-sprayed; Oxide, surface sandblasted with

titanium oxide; Acid, acid etched surface.

Fig. 6. Box-plots of histometric measurements of

(a) defect depth (mm) in an apical direction from

the implant shoulder to the level of the ‘old’ bone,

(b) percentage of mineralized bone contact with the

implant surface, and (c) bone area within the limits

of the implant threads.

Table 6. Clinic and histometric effect of the treatment by comparing the test and thecontrol group independent of the implant surface (n¼5 animals)

Variable Test Control P 95% CI


Bone heightgain (mm)n

1.79 � 1.37 0–4.75 � 0.09 � 1.22 � 2.22–3 0.002 1.81–2.59

Re-osseointegration(%)w

37.08 � 19 0–76.21 8.44 � 11.21 0–40.17 0.001 � 39.11 to 18.18

Bone fill(%)w

51.33 � 21.08 0–95.79 14.59 � 14.01 0–47.21 0.001 � 48.51 to � 24.95

nClinical variable.

wHistometrical variables.

Table 5. Percentage of implant–bone contact in the previously ‘contamined’ implant sur-face (re-osseointegration) measured from the base of the defect to the implant shoulder

Implantsurfacen

Test Control P 95% CI


cpTi 41.90 � 32.71 0–76.21 10.02 � 17.39 0–40.17 0.089 � 8.34 to 67.07TPS 31.19 � 17.55 0–42.14 0 0–0 0.016 9.39–52.98Oxide 34.69 � 9.49 20.97–45.15 9.79 � 11.82 0–23.9 0.033 3.78–7.57Acid 41.53 � 20.28 16.6–69.33 14.23 � 4.79 10–20.21 0.036 2.73–51.85

nNot significant among the surfaces P¼ 0.82.

cpTi, commercially pure titanium; TPS, titanium plasma-sprayed; Oxide, surface sandblasted with

titanium oxide; Acid, acid etched surface.



Acknowledgements: This research

was supported by FAPESP (Fundacao de

Amparo a Pesquisa do Estado de Sao

Paulo, SP, Brazil) grant no. 00/02433-1

and FUNDUNESP (Fundacao para o

Desenvolvimento da UNESP, Sao Paulo,

Brazil). The authors appreciate the

assistance of Dr Vagner Samy Lemo in

all the surgical phases. The authors are

also indebted to Conexao Implants,

Brazil, for providing the oxide dental

implants, INP – Brazil for the titanium

tacks, and Implamed Brazil for cpTi and

TPS implants.

Source of support: FAPESP (00/02433-1)

and FUNDUNESP.

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