Abstract

Crestal bone loss has been reported to occur around dental implants. Even if the causes of this bone loss are not completely understood, the presence of a microgap between implant and abutment with a possible contamination of the internal portion of the implants has been suggested. The aim of this study was to see if there were differences in the vascular endothelial growth factor (VEGF) expression, microvessel density (MVD), proliferative activity (MIB-1), and inflammatory infiltrate in the soft tissues around implants with screwed and cemented abutments. Sandblasted and acid-etched implants were inserted in the mandibles of 6 Beagle dogs. Ten 3.5- × 10-mm root-form implants were inserted in each mandible. A total of 60 implants (30 with screwed abutments and 30 with cemented abutments) were used. After 12 months, all the bridges were removed and all abutments were checked for mobility. A total of 8 loosened screws (27%) were found in the screwed abutments, whereas no loosening was observed in cemented abutments. A gingival biopsy was performed in 8 implants with cemented abutments, in 8 implants with screwed abutments, and in 8 implants with unscrewed abutments. No statistically significant differences were found in the inflammatory infiltrate and in the MIB-1 among the different groups. No statistically significant difference was found in the MVD between screwed and cemented abutments (P = .2111), whereas there was a statistically significant difference in MVD between screwed and unscrewed abutments (P = .0277) and between cemented and unscrewed abutments (P = .0431). A low intensity of VEGF was prevalent in screwed and in cemented abutments, whereas a high intensity of VEGF was prevalent in unscrewed abutments. These facts could be explained by the effects induced, in the abutments that underwent a screw loosening, by the presence of bacteria inside the hollow portion of the implants or by enhanced reparative processes.

Introduction

Peri-implantitis is a site-specific, plaque-induced infection with progressive loss of the bone supporting a functioning implant.1 In peri-implantitis, it is possible to observe pocket formation, radiographic bone destruction, suppuration, swelling, color changes, and bleeding upon gentle probing.2–4 Angiogenesis consists of the formation of new capillaries by the budding of endothelial cells5 and is a complex process that involves epithelial cell division, selective degradation of vascular basement membranes and of surrounding extracellular matrix, and endothelial cell migration.6–12 It is physiologically important in developing organs, inflammation, and wound healing, but its role in the development and progression or in the healing of periodontal lesions has not been elucidated.12–16 In periodontal tissues, angiogenesis plays a role in the maintenance of tissue health and in chronic inflammatory periodontal disease.13 Moreover, angiogenesis takes part in the development and maintenance of chronic inflammatory diseases such as diabetic retinopathy, rheumatoid arthritis, psoriasis, age-related macular degeneration, and tumors.13,16,17 Changes in the relative balance of inducers and inhibitors of angiogenesis probably contribute to the angiogenic process.13 The major angiogenic activators are basic fibroblast growth factor–2 and vascular endothelial growth factor (VEGF), and the most important inhibitors are angiostatin and endostatin.11,13 

Vascular endothelial growth factor is a glycoprotein that shares homology with platelet-derived growth factor and is a potent inducer of microvascular permeability.11,12,17,18 It has been shown to have a remarkable potency in inducing specific endothelial cell proliferation in vitro and also to have an angiogenic activity in vivo.5,13,19 It induces microvascular permeability with a potency of about 50 000 times that of histamine.18 Induced angiogenesis and vasculogenesis are impaired in mice with mutated and nonfunctional VEGF gene.19 It is regulated by hypoxia-mediated control of gene transcription, alternative messenger-RNA splicing, and proteolytic processing.17 Inflamed tissues enhance the expression of inflammatory mediators, which, in turn, may promote angiogenesis.17 Vascular endothelial growth factor has been detected in vascular endothelial cells; inflammatory cells; and in the junctional, sulcular, and gingival epithelium17,20 and is expressed in a variety of highly vascularized tissues.11,21 A correlation between VEGF expression and neovascularization assessed by factor-VIII immunostaining and evaluated by microvessel density (MVD) has been found.10 Moreover, MVD is correlated to poor clinical outcome in several malignant tumors.10 

The aim of the present study in Beagle dogs was to conduct a comparative immunohistochemical evaluation of VEGF, inflammatory infiltrate, proliferative activity (MIB-1) expression, and MVD in the peri-implant soft tissues surrounding dental implants with cemented and screwed abutments.

Materials and Methods

Sandblasted and acid-etched implants (Bone System, Milano, Italy) were placed in the mandibles of 6 male Beagle dogs of at least 2 years of age. The Ethics Committee of the University of Madrid, Spain, approved the protocol. The 2 premolars and the first molars had been extracted 3 months beforehand. Each dog received ten 3.5- × 10-mm root-form implants in the mandible (5 on the right side and 5 on the left side). All surgical procedures were performed under general anesthesia (premedication with acepromazine 0.5 mg/kg subcutaneously, with nembutal 15 mg/kg intravenously) and antibiotic prophylaxis. The implant sites were prepared with drills under generously chilled, saline irrigation. The implants were then inserted with a tapping instrument. All implants were placed by a submerged approach, and the top of each implant was located clinically at the alveolar crest. The mucosal tissues were closed with 3-0 silk sutures. In the first 2 postsurgical weeks, the oral cavities were rinsed daily with chlorexidine-digluconate 0.12% (Peridex, Procter & Gamble, Cincinnati, Ohio). In addition, the dogs were fed a soft diet. The sutures were removed after 1 week. Three months after implantation, second-stage surgery was performed for abutment connection. After a midcrestal incision, the peri-implant soft tissues were evaluated with exposure of the peri-implant bone crest, and abutments were inserted in all implants. The abutments were either screwed by applying a total strength of 30 Ncm or cemented with Panavia 21 (J. Morita USA Inc, Tustin, Calif). The cement was mixed according to the manufacturer's recommendations and applied on the axial surface of the internal portion of the implants to minimize hydrostatic pressure during seating. Abutments were cemented on the implants with a load of 5 kg maintained for 10 minutes. Excess cement was removed with a scaler. One investigator (B.A.) carried out mixing and cementing procedures at room temperature. After cementation of the implant-abutment connection, a bridge of 6 elements was cemented with a load of 5 kg maintained for 10 minutes. After cementation of the bridge, the excess cement was removed with a scaler. A total of 60 implants were inserted, 30 with cemented abutments and 30 with screwed abutments. No postoperative complications or deaths occurred.

After 12 months, all the bridges were removed and all screwed and cemented abutments were checked clinically for mobility, alternatively pressing the facial and lingual surfaces of the abutments with the nonworking ends of 2 instrument handles.

A total of 8 loosened screws (27%) were found in the screwed abutments after 12 months. No loosening was observed in cemented abutments. A statistically significant difference was present between the incidence of the loosening in the screwed and cemented abutments (P = .0001).

A gingival biospy was performed near the top of the implant in 8 of the implants with cemented abutments, in 8 of the implants with screwed abutments, and in 8 of the implants where the abutments were found to be unscrewed after 12 months. The dimensions of the gingival biopsy were 1 mm in thickness and 1 mm in length. The dogs were not killed in this study.

All specimens were immediately fixed in 10% neutral buffered formalin and then embedded in paraffin. Three-micrometer sections were then obtained with a Leitz 1512 microtome and stained with hematoxylin and eosin.

For the immunohistochemical staining of VEGF, factor VIII, and MIB-1, 3-μm sections were cut and preincubated in phosphate-buffered saline (PBS) for 5 minutes and then with rabbit polyclonal antibodies (Santa Cruz Biotech Inc, Santa Cruz, Calif), which were diluted 1:100 in PBS and applied for 30 minutes at room temperature. Slides were then washed twice in PBS for 5 minutes and in Tris-HCl buffer, pH 7.6, for 10 minutes. Sections were incubated in the presence of biotynilated-specific secondary antibody, and avidin biotin peroxidase was developed with diaminobenzidine chromogen (Biomeda Corp, Foster City, Calif) and the nuclei were counterstained with hematoxylin. The secondary antibody was an alkaline phosphatase–labelled monoclonal calf antibody, and the detection antibody was a monoclonal anti–calf-mouse antibody. After incubation with a chromogen alkaline phosphatase substrate (Fast Red, Dako Cytomation, Hamburg, Germany), specimens were counterstained with Mayer hematoxylin and were cover slipped.

Vascular endothelial growth factor has been evaluated in the vessels, in the cells of the inflammatory infiltrate (mainly lymphocytes, plasma cells, and neutrophils), and in the stromal cells (fibroblasts). Inflammatory infiltrate, factor VIII, and MIB-1 expressions were evaluated by a semiquantitative analysis: low (+), intermediate (++), and high (+++). The value was considered low (+) when fewer than 10% of the cells were positive for these factors, intermediate (++) when the percentage of positive cells was between 10% and 50%, and high (+++) when the percentage of positive cells was more than 50%. The antibody against human factor VIII–related antigen was used to highlight the blood microvessels; all the morphologic structures with a lumen surrounded by factor VIII–positive endothelial cells were considered as blood microvessels. Two pathologists (L.A. and C.R.) performed the microvessel count in a blinded and independent fashion with an IBAS-AT image analyzer (Kontron, Munich, Germany). For the evaluation, a ×400 magnification was used and the individual microvessel profiles were circled to prevent the duplication or omission of microvessel count. For each case, 10 high-power fields corresponding to 1.1 mm2 were measured. The values were expressed as the number of microvessels per square millimeter of peri-implant soft tissues surrounding screwed, cemented, and unscrewed abutments (MVD).

Quantitative analysis was performed for VEGF. A light microscope (Laborlux S, Leitz, Wetzlar, Germany) connected to a high-resolution video camera and interfaced to a monitor and personal computer was used to evaluate VEGF in the vessels of screwed, cemented, and unscrewed abutments. This optical system was associated with a digitizing pad and a histometry software package with image-capturing capabilities (Image-Pro Plus 4.5, Media Cybernetics Inc, Immagine & Computer, Milano, Italy). Five random fields were chosen for each specimen. An evaluation was performed in each field after 3 different intensities of the expression of VEGF were distinguished, which the personal computer software recognized as red (low), yellow (intermediate), and green (high) (Figures 1 through 3).

Figures 1–9. Figure 1. High intensity of vascular endothelial growth (VEGF) expression. Figure 2. Intermediate intensity of VEGF expression. Figure 3. Low intensity of VEGF expression. Figure 4. VEGF positivity of the endothelial cells lining the blood vessels (arrow) (magnification ×30). Figure 5. VEGF positivity of the stromal cells (arrow) (magnification ×30). Figure 6. VEGF positivity of the cells of the inflammatory infiltrate (arrow) (magnification ×30). Figure 7. Implant with a cemented abutment: low intensity of VEGF positivity of the endothelial cells lining the blood vessels (arrow) (magnification ×30). Figure 8. Implant with an unscrewed abutment: high intensity of the VEGF positivity of the endothelial cells lining the blood vessels (arrow) (magnification ×30). Figure 9. Implant with a screwed abutment: intermediate intensity of VEGF positivity of the endothelial cells lining the blood vessels (arrow) (magnification ×40)

Figures 1–9. Figure 1. High intensity of vascular endothelial growth (VEGF) expression. Figure 2. Intermediate intensity of VEGF expression. Figure 3. Low intensity of VEGF expression. Figure 4. VEGF positivity of the endothelial cells lining the blood vessels (arrow) (magnification ×30). Figure 5. VEGF positivity of the stromal cells (arrow) (magnification ×30). Figure 6. VEGF positivity of the cells of the inflammatory infiltrate (arrow) (magnification ×30). Figure 7. Implant with a cemented abutment: low intensity of VEGF positivity of the endothelial cells lining the blood vessels (arrow) (magnification ×30). Figure 8. Implant with an unscrewed abutment: high intensity of the VEGF positivity of the endothelial cells lining the blood vessels (arrow) (magnification ×30). Figure 9. Implant with a screwed abutment: intermediate intensity of VEGF positivity of the endothelial cells lining the blood vessels (arrow) (magnification ×40)

Statistical evaluation

The Friedman test was used to evaluate the presence of statistically significant differences of VEGF within and among the 3 groups considered. The percentages have been expressed as a mean ± SD. Statistically significant differences were set at P < .05. Inflammation was evaluated by the χ2 test. Finally, the Wilcoxon test was used to evaluate MVD and MIB-1.

Results

In all cases in all 3 groups, there was an inflammatory infiltrate composed mainly of lymphocytes, plasma cells, and granulocytes. There was no statistically significant difference in the amount of the inflammatory infiltrate between screwed and cemented abutments (P = .548), between screwed and unscrewed abutments (P = .391), and between cemented and unscrewed abutments (P = .682) (Table 1). There were no statistically significant differences in MVD between screwed and cemented abutments (P = .2111). On the contrary, there was a statistically significant difference in MVD between screwed and unscrewed abutments (P = .0277) and between cemented and unscrewed abutments (P = .0431) (Table 2). There was no statistically significant difference in MIB-1 among the 3 groups considered (Table 3).

Table 1

Inflammatory infiltrate χ2 test

Inflammatory infiltrate χ2 test
Inflammatory infiltrate χ2 test
Table 2

Microvessel density Wilcoxon test

Microvessel density Wilcoxon test
Microvessel density Wilcoxon test
Table 3

Proliferative activity Wilcoxon test

Proliferative activity Wilcoxon test
Proliferative activity Wilcoxon test

Vascular endothelial growth factor was expressed in all groups but with different intensity in vascular endothelial cells. Vascular endothelial cells (Figure 4), stromal cells (fibroblasts) (Figure 5), and the cells of the inflammatory infiltrate (Figure 6) were positive for VEGF. A low intensity of VEGF expression was prevalent in screwed and cemented abutments (Figure 7). There was a statistically significant difference among the 3 degrees of intensity (P < .0001). However, a high intensity of VEGF expression was prevalent in unscrewed abutments (Figure 8). In this group, there was no statistically significant difference (P = .0805) because the 3 degrees of intensity were always expressed, even if it was possible to observe a tendency from low to high intensity (Figure 9, Table 4). Moreover, high and intermediate intensities were significantly different among the 3 groups (P < .0001), whereas low intensity was not statistically significant (Table 4). No quantitative analysis of VEGF expression was performed in the stromal cells and in the cells of the inflammatory infiltrate because of technical difficulties.

Table 4

Vascular endothelial growth factor Friedman test

Vascular endothelial growth factor Friedman test
Vascular endothelial growth factor Friedman test

Discussion

The majority of studies in implant dentistry have focused more on the bone integration of dental implants and much less on the dimensions and relationship of the peri-implant soft tissues.22 Crestal bone loss has been reported to occur in the first year after implant insertion. This initial bone loss forms a V- or U-shaped pattern.23 The precise causes of this bone loss are unknown, but several hypotheses have been suggested, such as surgical trauma, overload, peri-implantitis, microgap, reformation of the biologic width, and implant crestal module.24 The determination of the etiology of this crestal bone loss is important to obtain an improved environment for long-term peri-implant health.23 Occlusal overload and peri-implantitis are 2 of the main causative factors for implant failure in later stages.24 Moreover, a correlation between plaque accumulation and progressive bone loss around implants has been reported in experimental and clinical studies.24 In most 2-stage implants, after abutment connection, a microgap is produced at or below the alveolar crest.24 This microgap represents a site of infection and the host reacts with an inflammatory response.25–29 Several studies have demonstrated a second discrete inflammatory cell infiltrate, in addition to that located in the marginal portion of the soft tissues, in the connective tissue lateral to the abutment fixture junction.30–34 This infiltrated connective tissue (abutment ICT) is believed to arise as a result of a microbial contamination of the internal portion of the implant.30–34 In nonsubmerged implants, however, the implant itself extends above the alveolar crest level, and the microgap does not exist at the level of the bone.23,24 Minimal or no resorption was found in these implants.25–29 One-piece implant designs seem to determine the least inflammation and the smallest resultant tissue changes.25–29 The degree of inflammation has been reported to be less around 1-piece implants as compared with 2-piece implants.25–29 In 2-piece implants, the crestal bone levels appeared dependent on the location of the microgap.25–29,35 The least bone resorption and peri-implant inflammation was reported to occur if the microgap was located 1 mm above the alveolar crest.28 From the literature, it can be speculated that though the microgap may not be considered as the only cause of early implant bone loss, it might cause implant crestal bone loss during the healing phase if it is placed at or below the alveolar crest.23,24 In a previous in vitro study from our laboratory, we demonstrated that a bacterial penetration in implants with screwed abutments was observed at the implant-abutment interface, whereas no bacteria were observed in the hollow portion of the implants with cemented abutments.36 This bacterial reservoir could interfere with the peri-implant tissue health and produce an inflammation of the peri-implant tissues, which could interfere with the long-term success of the implants.37–40 

Angiogenesis is a process in which new blood vessels are produced by sprouting from established vessels.15 An enhanced expression of inflammatory mediators, many of which can promote angiogenesis, can be found in inflamed tissues.15 Angiogenesis can also contribute to the severity of the inflammation as the result of the ability of the new blood vessels to transport proinflammatory cells to the lesion and supply nutrients and oxygen to the inflamed tissues.15,19 

Vascular endothelial growth factor is probably involved in the etiology of gingivitis and its progression to periodontitis by an expansion of the vascular network, an increase in tissue edema, and a decrease in the rate of the blood flow.15,17 Aberrant angiogenesis and persistent granulation have been reported in periodontitis, and these phenomena may contribute to its progression (ie, the aberrant angiogenesis enhances leukocytes infiltration into the lesion and formation of persistent granulation tissue).16 

The aim of the present study was to see if there were differences in the MVD, inflammatory infiltrate, MIB-1, and VEGF expression in the soft tissues around implants with screwed and cemented abutments. No statistically significant difference was found in the MVD, inflammatory infiltrate, and MIB-1 between screwed and cemented abutments, and the significance was prevalently low in the VEGF intensity between screwed and cemented abutments. However, it was interesting to note that the MVD was statistically significantly higher compared with the screwed and cemented abutments in the peri-implant soft tissues around implants whose abutments had become unscrewed after a loading period of 12 months. In the unscrewed specimens, it was possible to find a prevalence of a high intensity of VEGF. These results could point to the fact that though screw loosening in the screwed abutments did not occur, no differences in the soft tissue vasculature between the screwed and the cemented abutments exist. However, when there was a loosening of the screws, which is frequently reported in the literature,41 we observed a significant increase in the MVD and in the intensity of VEGF expression in the peri-implant tissues. These facts could be explained by the effects induced by the presence of bacteria inside the hollow portion of the implants or by the presence of enhanced reparative processes in which VEGF is implicated.13,15 

Further studies are certainly needed to try to understand the significance of the possible relationship between bacterial contamination of the internal portion of the implants and long-term peri-implant soft tissue health.

Acknowledgments

This work was partially supported by the National Research Council (C.N.R.), Rome, Italy; by the Ministry of Education, University, Research (M.I.U.R.), Rome, Italy; and by AROD (Research Association for Dentistry and Dermatology), Chieti, Italy.

References

References
1
Esposito
,
M.
,
J. M.
Hirsch
,
U.
Lekholm
, and
P.
Thomsen
.
Biological factors contributing to failures of osseointegrated oral implants. II) Etiopathogenesis.
Eur J Oral Sci
1998
.
106
:
721
764
.
2
Mombelli
,
A.
Etiology, diagnosis, and treatment considerations in peri-implantitis.
Curr Opin Periodontol
1997
.
4
:
127
136
.
3
Mombelli
,
A.
and
N. P.
Lang
.
Clinical parameters for the evaluation of dental implants.
Periodontol 2000
1994
.
4
:
81
86
.
4
Mombelli
,
A.
and
N. P.
Lang
.
The diagnosis and treatment of peri-implantitis.
Periodontol 2000
1998
.
17
:
63
76
.
5
Unlu
,
F.
,
P. G.
Guneri
,
M.
Hekimgil
,
B.
Yesilbek
, and
H.
Boyaciaglu
.
Expression of vascular endothelial growth factor in human periodontal tissues: comparison of healthy and diabetic patients.
J Periodontol
2003
.
74
:
181
187
.
6
Folkman
,
J.
Angiogenesis in cancer, vascular, rheumatoid and other diseases.
Nat Med
1995
.
1
:
27
31
.
7
Folkman
,
J.
,
K.
Watson
,
D.
Ingber
, and
D.
Hanahan
.
Introduction of angiogenesis during the transition from hyperplasia to neoplasia.
Nature
1989
.
339
:
58
61
.
8
Hanahan
,
D.
and
J.
Folkman
.
Patterns and emerging mechanisms of the angiogenic switch during tumourigenesis.
Cell
1996
.
86
:
353
364
.
9
Senger
,
D. R.
,
S. J.
Galli
,
A. M.
Dvorak
,
C. A.
Peruzzi
,
V. S.
Harvey
, and
H. F.
Dvorak
.
Tumour cells secrete a vascular permeability factor that promotes accumulation of ascites fluid.
Science
1983
.
219
:
983
985
.
10
Artese
,
L.
,
C.
Rubini
,
G.
Ferrero
,
M.
Fioroni
,
A.
Santinelli
, and
A.
Piattelli
.
Microvessel density (MVD) and vascular endothelial growth factor expression (VEGF) in human oral squamous cell carcinoma.
Anticancer Res
2001
.
21
:
689
696
.
11
Kohno
,
S.
,
M.
Kaku
, and
K.
Tsutsui
.
et al
.
Expression of vascular endothelial growth factor and the effects on bone remodeling during experimental tooth movement.
J Dent Res
2003
.
82
:
177
182
.
12
Kumamoto
,
H.
,
K.
Ohki
, and
K.
Ooya
.
Association between vascular endothelial growth factor (VEGF) expression and tumor angiogenesis in ameloblastomas.
J Oral Pathol Med
2002
.
31
:
28
34
.
13
Yoshino
,
H.
,
I.
Morita
,
S. I.
Murota
, and
I.
Ishikawa
.
Mechanical stress induces production of angiogenic regulators in cultured human gingival and periodontal ligament fibroblasts.
J Periodontal Res
2003
.
38
:
405
410
.
14
Mamose
,
M.
,
M.
Murata
, and
Y.
Kato
.
et al
.
Vascular endothelial growth factor and transforming growth factor-α and β1 are released from human cultured gingival epithelial sheets.
J Periodontol
2002
.
73
:
748
753
.
15
Johnson
,
R. B.
and
F. G.
Serio
.
Leptin within healthy and diseased human gingival.
J Periodontol
2001
.
72
:
1254
1257
.
16
Oyama
,
T.
,
T.
Sakuda
,
K.
Matsushita
,
I.
Maruyama
,
S.
Nagaoka
, and
M.
Torii
.
Effects of roxythromycin on tumor necrosis factor-alpha-induced vascular endothelial growth factor expression in human periodontal ligament cells in culture.
J Periodontol
2000
.
41
:
1546
1553
.
17
Suthin
,
K.
,
K.
Matsushita
, and
M.
Machigashira
.
et al
.
Enhanced expression of vascular endothelial growth factor by periodontal pathogens in gingival fibroblasts.
J Periodontal Res
2003
.
38
:
90
96
.
18
Leonardi
,
R.
,
M.
Caltabiano
,
M.
Pagano
,
V.
Pezzuto
,
C.
Loreto
, and
G.
Palestro
.
Detection of vascular endothelial growth factor/vascular permeability factor in periapical lesions.
J Endod
2003
.
29
:
180
183
.
19
Yuan
,
K.
,
Y. T.
Jin
, and
M. T.
Lin
.
The detection and comparison of angiogenesis-associated factors in pyogenic granuloma by immunohistochemistry.
J Periodontol
2000
.
71
:
701
709
.
20
Chapple
,
C. C.
,
R. K.
Kumar
, and
N.
Hunter
.
Vascular remodelling in chronic inflammatory periodontal disease.
J Oral Pathol Med
2000
.
29
:
500
506
.
21
Uchida
,
S.
,
A.
Sakai
, and
H.
Kudo
.
et al
.
Vascular endothelial growth factor is expressed along with its receptors during the healing process of bone and bone marrow after drill-hole injury in rats.
Bone
2003
.
32
:
491
501
.
22
Cochran
,
D. L.
,
J. S.
Hermann
,
R. K.
Schenk
,
F. L.
Higginbottom
, and
D.
Buser
.
Biologic width around titanium implants. A histometric analysis of the implanto-gingival junction around unloaded and loaded nonsubmerged implants in the canine mandible.
J Periodontol
1997
.
68
:
186
198
.
23
Oh
,
T. J.
,
J.
Yoon
,
C. E.
Misch
, and
H. L.
Wang
.
The causes of early implant bone loss: myth or science.
J Periodontol
2002
.
73
:
322
333
.
24
Misch
,
C. E.
Early crestal bone loss etiology and its effect on treatment planning for implants.
Postgrad Dent
1995
.
2
:
3
16
.
25
Hermann
,
J. S.
,
D. L.
Cochran
,
P. V.
Nummikosky
, and
D.
Buser
.
Crestal bone changes around titanium implants. A radiographic evaluation of unloaded nonsubmerged and submerged implants in the canine mandible.
J Periodontol
1997
.
68
:
1117
1130
.
26
Hermann
,
J. S.
,
D.
Buser
,
R. K.
Schenk
,
F. L.
Higginbottom
, and
D. L.
Cochran
.
Biological width around titanium implants. A physiologically formed and stable dimension over time.
Clin Oral Implants Res
2000
.
11
:
1
11
.
27
Broggini
,
M.
,
L. M.
McManus
, and
J. S.
Hermann
.
et al
.
Persistent acute inflammation at the implant-abutment interface.
J Dent Res
2003
.
82
:
232
237
.
28
Hermann
,
J. S.
,
J. D.
Schofield
,
R. K.
Schenk
,
D.
Buser
, and
D. L.
Cochran
.
Influence of the size of the microgap on crestal bone changes around titanium implants. A histometric evaluation of unloaded non-submerged implants in the canine mandible.
J Periodontol
2001
.
72
:
1372
1383
.
29
Hermann
,
J. S.
,
D.
Buser
,
R. K.
Schenk
, and
D. L.
Cochran
.
Crestal bone changes around titanium implants. A histometric evaluation of unloaded non-submerged and submerged implants in the canine mandible.
J Periodontol
2000
.
71
:
1412
1424
.
30
Abrahamsson
,
I.
,
T.
Berglundh
,
J.
Wennstrom
, and
J.
Lindhe
.
The peri-implant hard and soft tissues at different implant systems. A comparative study in the dog.
Clin Oral Implants Res
1996
.
7
:
212
219
.
31
Abrahamsson
,
I.
,
T.
Berglundh
,
P. O.
Glantz
, and
J.
Lindhe
.
The mucosal attachment at different abutments. An experimental study in dogs.
J Clin Periodontol
1998
.
25
:
721
727
.
32
Abrahamsson
,
I.
,
T.
Berglundh
, and
J.
Lindhe
.
Soft tissue response to plaque formation at different implant systems. A comparative study in the dog.
Clin Oral Implants Res
1998
.
9
:
73
79
.
33
Ericsson
,
I.
,
L. G.
Person
,
T.
Berglundh
,
C. P.
Marinello
,
J.
Lindhe
, and
B.
Klinge
.
Different types of inflammatory reactions in peri-implant soft tissues.
J Clin Periodontol
1995
.
22
:
255
261
.
34
Ericsson
,
I.
,
K.
Nilner
,
B.
Klinge
, and
P. O.
Glantz
.
Radiographical and histological characteristics of submerged and non-submerged titanium implants. An experimental study in the Labrador dog.
Clin Oral Implants Res
1996
.
7
:
20
26
.
35
Piattelli
,
A.
,
G.
Vrespa
,
G.
Petrome
,
G.
Iezzi
,
S.
Annibali
, and
A.
Scarano
.
Role of the microgap between implant and abutment: a retrospective histologic evaluation in monkeys.
J Periodontol
2003
.
74
:
346
352
.
36
Piattelli
,
A.
,
A.
Scarano
, and
M.
Paolantonio
.
et al
.
Fluids and microbial penetration in the internal part of cement-retained versus screw-retained implant-abutment connections.
J Periodontol
2001
.
72
:
1146
1150
.
37
Persson
,
L. G.
,
U.
Lekholm
,
A.
Leonhardt
,
G.
Dahlén
, and
J.
Lindhe
.
Bacterial colonization of internal surfaces of Branemark system implant components.
Clin Oral Implants Res
1996
.
7
:
90
95
.
38
Jansen
,
V. K.
,
G.
Conrads
, and
E. J.
Richter
.
Microbial leakage and marginal fit of the implant abutment interface.
Int J Oral Maxillofac Implants
1997
.
12
:
527
540
.
39
Quirynen
,
M.
and
Van
Steenberghe D
.
Bacterial colonization of the internal part of two stage implants. An in vivo study.
Clin Oral Implants Res
1993
.
4
:
158
161
.
40
Quirynen
,
M.
,
C. M. L.
Bollen
,
H.
Eyssen
, and
Van
Steenberghe D
.
Microbial penetration along the implant components of the Branemark system. An in vitro study.
Clin Oral Implants Res
1994
.
5
:
239
244
.
41
Goodacre
,
C. J.
,
G.
Bernal
,
K.
Rungcharassaeng
, and
J. Y. K.
Kan
.
Clinical complications with implants and implant prostheses.
J Prosthet Dent
2003
.
90
:
121
132
.

Author notes

Bartolomeo Assenza, MD, DDS, is a visiting professor, Dental School, University of Chieti-Pescara, Chieti, Italy, and is in private practice in Cologno Monzese (MI), Italy.

Antonio Scarano, DDS, MD, is a researcher, Vittoria Perrotti, DDS, is a research fellow, Maurizio Piattelli, MD, DDS, is an associate professor, Marcello Piccirilli, BSc, is a chief technician, and Adriano Piattelli, MD, DDS, is a professor of Oral Pathology and Medicine, Dental School, University of Chieti-Pescara, Chieti, Italy. Address correspondence to Prof Adriano Piattelli at Via F. Sciucchi 63, 66100 Chieti, Italy (apiattelli@unich.it).

Luciano Artese, MD, is a professor of Pathology, University of Chieti-Pescara, Chieti, Italy.

Corrado Rubini, MD, is a researcher in the Department of Pathologic Anatomy and Histopathology, University of Ancona, Ancona, Italy.

Ulf Thams, MD, DDS, is in private practice in Madrid, Spain.

Fidel San Roman, MD, is a professor of Surgery, Director of Veterinary Teaching Hospital, Complutense University, Madrid, Spain.