Massimo DE SANCTIS1 Nicola BALDINI2 Fabio VIGNOLETTI3

1- PhD, Department of odontostomatological sciences, Sienna University, Italy 2- DDS, Department of odontostomatological sciences, Sienna University, Italy 3- Madrid Complutense University, Spain Accepted for publication: 30 April 2010

Biological width around implants

Histological evidence: a review on animal studies

Espace biologique pri-implantaire

Preuves histologiques : revue dtudes animales
RSUM ABSTRACT Soft tissue relationship to implant surface is one of the most challenging area for implant manufacturers as it is evident by different kind of connections, implant shoulders and platforms. The presence of a quite constant dimension of soft tissue attachment to dental implants, similar for many features to dentogingival junction, has been well documented in histological studies on animal models. Based on similar histological studies, the influence of different variables, like the surgical technique, the surgical protocol, implant loading, implant structure, titanium surfaces and abutment materials on peri-implant biological width has been evaluated. The aim of this study is to produce a review on these topics. KEY WORDS Biologic width, implant, soft tissues healing, junctional epithelium, connective tissue. Les relations entre les tissus mous et la surface implantaire reprsentent un dfi considrable pour les fabricants dimplants si lon considre les diffrents types de connexions, de cols implantaires et de plateformes qui existent. La prsence assez constante dune hauteur dattache conjonctive sur les implants dentaires, qui est semblable sous bien des aspects la jonction dento-gingivale, a t bien documente dans des tudes histologiques ralises sur des modles animaux. En se fondant sur des tudes histologiques similaires, on a pu valuer linfluence de diffrentes variables, telles que la technique chirurgicale, le protocole chirurgical, la mise en charge de limplant, la structure de limplant, les surfaces en titane et les matriaux composant les piliers, sur lespace biologique pri-implantaire. Le but de cette tude est de faire le point sur ces questions. MOTS CLS Espace biologique, implant, cicatrisation des tissus mous, pithlium de jonction, tissu conjonctif.

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Introduction
The large use of osteointegrated implants in modern dentistry and the increasing esthetic demands for implant rehabilitations has focused attention on soft tissue reactions to implant placement specially in the area of soft tissue relationship to implant surface. Probably this is, at the present time, the most challenging area for implant manufacturers as it is evident by different kind of connections, implant shoulders and platforms. The transmucosal area constitutes an effective barrier between the oral environment and the periimplant bone and in many features it is similar to dentogingival junction. The morphology of dentogingival junction has been studied since 1959 by Sicher, who found both an epithelial and a connective tissue attachment to the tooth (Sicher, 1959). In 1961, Gargiulo et al. measured the vertical dimension of this structure and named it biological width (Gargiulo et al., 1961). Biological width was composed by sulcus depth (SD), junctional epithelium (JE) and connective tissue attachment (CTA). The mean value was 2.73 mm and 2.04 mm for JE + CTA. These findings have been confirmed by Vacek in 1994: in this study, mean measurements were 1.34 0.84 mm for sulcus depth; 1.14 0.49 mm for epithelial attachment; 0.77 0.32 mm for connective tissue attachment (Vacek et al., 1994). Authors emphasized that connective tissue attachment has a variable width within a more narrow distribution and range than the epithelial attachment and sulcus depth. Peri-implant tissues have many similarities with periodontal tissues and

dentogingival junction, but there are also obvious anatomical differences like the lack of periodontal ligament and a different vascular distribution (Berglundh et al., 1991). Peri-implant biological width has been studied and measured in both histological animal studies and clinical human studies. The aim of the present study is to produce a review on the dimensions of the peri-implant biological width and to analyze the factors that may determine variations on biological width values.

Structure and biological dimensions

In the first animal studies of Berglundh et al. in 1991, it was demonstrated that the peri-implant mucosa established a cuff-like barrier which adhered to the surface of the titanium abutment (Berghlund et al., 1991). The peri-implant tissue is a scar tissue that repairs the injury of the implant insertion: the soft tissues of the edentulous crest, once repositioned and sutured, participate to the formation of a new tissue that protects the exposed bone and seals the emergence of the implant. As the gingiva, the peri-implant mucosa has a well-keratinized oral epithelium which is continuous with a junctional epithelium that faces the titanium surface. The structure of junctional epithelium is still matter of debate: a junctional epithelium similar to the junctional epithelium on teeth has been documented in a number of studies from different authors (Abrahamsson et al., 1996, 1999, 2002; Berglundh

et al., 1991, 2007; Moon, 1999). In a work on rats (Ikeda et al., 2000), the presence of a basal lamina and hemidesmosomes in peri-implant junctional epithelium was confirmed although it was stated that the finding of a basal lamina was less evident than on the control tooth sites, and it was well detectable only in the lower part of junctional epithelium. Results from a recent article by Shioya et al. differ greatly by the work of Ikeda (Shioya et al., 2009): one week after implant insertion, periimplant epithelium was observed, 8 weeks after implant insertion, the peri-implant epithelium receded, and the implant interface appeared to be sealed by aligned special cells with surrounding elongated fibroblasts and bundles of collagen fibers. No hemidesmoses and no basal lamina were found in this tissue. This finding is in contrast with the previous scientific literature and opens new phases for further research. The most important difference between the two tissues is represented by the collagen fibers departing from the bone crest that are not inserted on the titanium surface and run parallel to implant surface, following the implant orientation (fig. 1 and 2). Furthermore according to Berglundh et al., experimental model, the collagen content of the peri-implant mucosa is higher, while the fibroblast density is much lower when compared to the gingival tissue (Berglundh et al., 1991). Another important observation was that all gingival and peri-implant units examined were free from infiltrates of inflammatory cells. It was suggested that under the conditions of that study, both types of soft tissues,

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Fig. 1. a. Immediate implant (3i Osseotite Certain, Biomet 3i, USA) and surrounding tissues after 8 weeks of healing. The keratinized oral epithelium continues with the junctional epithelium which is in intimate contact with the titanium surface of the abutment. Toluidine blue staining. Original augmentation 5X. b. Detail of (a). The connective tissue located immediately below the epithelium is rich in collagen fibers and poor in cells. Fig. 1. a. Implant mise en charge immdiate (3i Osseotite Certain, Biomet 3i, tats-Unis) et tissus environnants aprs 8 semaines de cicatrisation. Lpithlium oral kratinis est en continuit avec lpithlium de jonction qui est en contact intime avec la surface en titane du pilier. Coloration au bleu de toluidine. (grossissement 5) b. Dtail. Le tissu conjonctif situ immdiatement en dessous de lpithlium est riche en fibres de collagne et pauvre en cellules.

Fig. 2. Immediate implant (3i Osseotite Certain, Biomet 3i, USA) and surrounding tissues after 8 weeks of healing. The supracrestal connective tissue is cell rich in the area located close to the implant. Collagen fibers extend from periosteum of the bone crest and run lateral to this area with a direction that is parallel to the implantabutment surface. Toluidine blue staining. Original augmentation 2.5X. Fig. 2. Implant mise en charge immdiate (3i Osseotite Certain, Biomet 3i, tats-Unis) et tissus environnants aprs 8 semaines de cicatrisation. Le tissu conjonctif supracrestal est riche en cellules dans la zone situe proximit de limplant. Les fibres de collagne stendent du prioste de la crte osseuse et se dirigent latralement par rapport cette zone dans une direction qui est parallle la surface implant/pilier. Coloration au bleu de toluidine. Grossissement 2,5.

the gingiva and the peri-implant mucosa, present a proper potential to prevent subgingival plaque formation. Buser et al. investigated the soft tissue dimensions around three different titanium surfaces, a rough surface, a sandplasted surface and a

polished surface (Buser et al., 1992). No significant difference concerning soft tissue reactions were found between the three implant surfaces. The soft tissue barrier was composed by a sulcus with a non keratinized sulcular epithelium, a junctional epithelium, and a supracrestal connec-

tive tissue with an area of dense circular fibers near to the implant surface. Circular fibers were found in the inner zone of connective tissue, next to the titanium surface; in the outer layer, horizontal and vertical fibers were found: these fibers were

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running from the periosteum and the alveolar crest towards the oral epithelium. Authors reported that the orientation of the fibers differs in rough and smooth surfaces: smooth surfaces revealed an orientation of fibers parallel to the implant surface, while porous-coated surfaces promoted the formation of perpendicular fibers. The presence of this fibers has been confirmed in a recent study of Shioya et al. (Shioya et al., 2009). The overall tissue was described as an inflammation free scar tissue. The zone of dense collagen fibers was surrounded by a looser connective tissue with a 3dimensional network of collagen fibers running in different directions. Berghlund et al. compared the vascular system of the periodontal and peri-implant tissues in the beagle dog (Berglundh et al., 1994). In the tooth, blood vessels of the sub-epithelial oral plexus appeared as they were terminal branches of the larger supraperiosteal blood vessels. The density of the vascular units in this plexus was increased in the marginal portion of the free gingival tissue unit; there were capillary loops projected into the papillae with a diameter of 7-10 m. The vasculature of tissue portion lateral to the enamel surface was located a few microns from the basal cell layer of the junctional epithelium and formed a mesh of vascular units called creviclular plexus. The vessels of the supracrestal connective tissue, lateral to the root cementum, were found to originate mainly from the vasculature of the periodontal ligament with a minor contribution from the larger supraperiosteal vessels. The two systems showed an anastomotic system. In the peri-implant mu-

cosa thin capillary loops were found, they were terminal branches of supraperiosteal vessels with a diameter of 7-10 m, similar to those observed in the periodontal tissues. Lateral to the junctional epithelium at a distance of 50 m, a crevicular plexus could be observed. In the area of connective tissue two different portions could be found: a central part 300-500 m wide in intimate contact with the implant-abutment interface that was poor of blood vessels with only few capillaries present and a lateral part with larger vessels originating from the supraperiosteal arterioles. The existence of a central area poor of vessels have been well documented by Moon et al. in an histological animal experiment (Moon et al., 1999). Authors concluded that the junctional epithelium was 2 mm long and 40 m wide. Apical to the junctional epithelium the connective tissue portion (200 m wide) presented a 40 m wide zone in close contact to the titanium surface. This zone was characterized by the absence of blood vessels, abundance of fibroblasts oriented with their long axis parallel to the implant surface and thin collagen fibers that extended from the periosteum of the bone crest parallel to the implant surface (Astra Tech Implants) (fig. 3). Laterally, an area with less fibroblasts, greater collagen fibers and vascular structures could be evidenced. Connective attachment was composed by 80,61% collagen fibers, 12,98% fibroblasts, 3,42% vascular structures and 3,0% residual tissues. Authors concluded that due to the presence of a fibroblast rich layer next to the implant surface the peri-implant connective tissue was

a tissue with a high turnover. These data were in contrast with previous studies that described the periimplant tissue as scar-like tissue (Buser et al., 1992). Some authors have stressed the hypothesis of a connective tissue attachment to the implant surface. Few experiments and human biopsies have demonstrated collagen fiber bundles functionally orientated and running in different directions (Schwarz et al., 2007; Nevins et al., 2008). The precise tridimensional orientation of the collagen fibres in the periimplant mucosa have been described by Schierano et al. (Schierano et al., 2002) contrasting this hypothesis.

Fig. 3. Densely packed connective tissue with absence of blood vessels, abundance of fibroblasts with thin collagen fibers. Fibroblast are oriented with their long axis parallel to implant surface. Toluidine blue staining. Original augmentation 10X. Fig. 3. Tissu conjonctif tass de faon dense avec absence de vaisseaux sanguins, abondance de fibroblastes avec de fines fibres de collagne. Les fibroblastes sont orients selon leur grand axe paralllement la surface de limplant. Coloration au bleu de toluidine. Grossissement 10.

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In 1996, Berglundh and Lindhe evaluated in an animal study the biologic width around implants (Brnemark Implant System) at sites showing a surgically created thin mucosa compared to control sites demonstrating a normal mucosa (Berglundh and Lindhe, 1996). Results after 6 months of healing demonstrated that the biological width dimensions were rather similar: the junctional epithelium measured approximately 2.1 mm and 2.0 mm in the control and test groups, respectively; the corresponding values for the connective tissue were 1.8 and 1.3 mm. Interestingly the wound healing processes at the test sites included bone resorption and thus the formation of an angular bone defects. The authors indicated the need of a minimum dimension of the biological width in order to accommodate the soft tissue healing process. Recently, Berglundh et al. described in detail the morphogenesis of the peri-implant mucosa around titanium implants in dogs (ITI Implant System) (Berglundh et al., 2007). Immediately after surgery (day 0) a coagulum occupied the space between the mucosa and implant surface and between the mucosa and the alveolar process (fig. 4). At 4 days of healing, the blood clot was infiltrated by numerous granulocytes and an initial mucosal seal was established by the clustering of leukocytes in a dense fibrin network. At 1 week of healing, an area of leukocyte-infiltrated fibrin tissue was still present, but it was smaller and localized only close to the soft tissue margin. The central part of the tissue was occupied by fibroblasts and collagen fibers. At 2 weeks, the periimplant mucosa adhered to the

implant surface through a connective tissue that was rich in cells and vascular structures. First signs of proliferation of junctional epithelium could be observed. At 4 weeks, the junctional epithelium was completely formed and in a more apical position a mature connective tissue could be observed. In later specimens (6-8 weeks), maturation of the connective tissue with a dense layer of elongated fibroblasts at titanium interface was evident. Fibroblasts were situated between the collagen fibers which were oriented mainly parallel to the titanium surface. From

a dimensional point of view, the biologic width increased during the healing period (mainly between 1 and 2 weeks) from 3.1 to 3.5 mm. Barrier epithelium extended to a position about 0.5 mm apical to the mucosal margin, while at 4 weeks the distance was 1.42 mm. At the end of the study (6-12 weeks), the barrier epithelium varied between 1.7 and 2.1 mm. These findings corroborated previous clinical results from a longitudinal study (Bengazi et al., 1996). Authors evaluated the alterations in the position of the peri-implant soft

Fig. 4. a. Immediate implant (3i Osseotite Certain, Biomet 3i, USA) and surrounding tissues after 4 hours of healing. A coagulum occupies the void between the implant surface and the socket wall. Toluidine blue staining. Original augmentation 2.5X. b. Detail of (a). Remnants of a junctional epithelium can be recognized. Toluidine blue staining. Original augmentation 5X. Fig. 4. a. Implant mise en charge immdiate (3i Osseotite Certain, Biomet 3i, tats-Unis) et tissus environnants aprs 4 heures de cicatrisation. Un coagulum occupe lespace entre la surface implantaire et la paroi de lalvole. Coloration au bleu de toluidine. Grossissement 2,5. b. Dtail. On peut identifier les restes dun pithlium de jonction. Coloration au bleu de toluidine. Grossissement 5.

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tissue margin, during a 2-year period follow-up. One hundred and sixty-three Brnemark implants were inserted into 41 patients that were periodonatlly evaluated and re-examined after 6 months, 1 and 2 years. The results indicated an apical displacement of the soft tissue margin that mainly occurred during the first 6 months of observation. Lingual sites in the mandible showed the most pronounced soft tissue recession, decrease of probing depth, and decrease of width of masticatory mucosa. Authors suggested that the recession of the periimplant soft tissue margin could be the result of the re-modelling of the soft tissue in order to establish the appropriate biological dimensions of the peri-implant soft tissue barrier.

Factors that may influence peri-implant biologic width

Surgical technique
Abrahamsson et al. evaluated the influence of the surgical protocol (i.e. one stage versus two stage) on the soft tissue healing around 3 different implant systems (Astra Tech Implants, Brnemark and Bonefit-ITI) (Abrahamsson et al., 1996). The histological results demonstrated similar dimension and composition of the epithelial and connective tissue components. Ericsson et al. found similar soft tissue adaptation and proper osseointegration in Brnemark implants installed according to a 1-stage or to a 2-stage surgical procedure (Ericsson et al., 1996). These findings were further confirmed by

Hermann et al. who compared nonloaded implants with loaded implants (ITI Implant System) at different time intervals (3-12 months) and according to a submerged or non-submerged healing (Hermann et al., 2000). The study demonstrated that the dimension of the biological width around non-submerged, one-piece titanium dental implants was not different whether the implants were unloaded or loaded for a period of 1 year. Nevertheless differences were observed in the dimensions of the components of the biological width (sulcus depth, epithelial junction and connective tissue) at the three different healing intervals. Histometric measurements demonstrated that although the biologic width dimension remained constant over the 15-month healing period, a decrease of the sulcus depth and connective tissue contact were observed whereas an increase of the junctional epithelium occurred. Thereafter data from different authors support the conclusion that a similar soft tissue dimensions is established regardless the use of a submerged or a nonsubmerged installation technique.

Loading
The influence of loading on soft tissue healing around implants was one of the topics most frequently investigated. Cochran et al. evaluated the dimension of the implanto-gingival junction around non submerged loaded and not loaded implants testing two different surfaces (SLA and TPS) at 3 and 12 months after implant placement (Cochran et al., 1997). At 3 months, the dimension of the

constituents of the biological width in the unloaded group were 0.49 mm for the sulcus depth (SD), 1.16 mm for the junctional epithelium (JE), and 1.36 mm for the connective tissue component (CTC). The corresponding measurements in the loaded group were 0.50 mm for SD, 1.44 mm for JE, and 1.01 mm for CTC for the loaded group. Results were similar after 12 months of loading, confirming that the biological width around implants resembles the one present around teeth and that the dimension of its constituents are independent from the loading variable. Siar found similar results comparing immediate versus delayed implant loading (Siar et al., 2003). The overall mean value of the biologic width was 3.9 mm in the immediate group and 3.8 mm in the delayed group. They concluded that no statistical differences were observed in the dimensions and compositions between the two groups. In a review of Glauser, it was concluded that, on the basis of the few available data, once immediately loaded or loaded and restored implants integrate successfully, they appear to show a soft-tissue reaction with regard to periodontal as well as morphologic aspects comparable with those of conventionally loaded implants (Glauser et al., 2006).

Titanium surfaces and abutment materials

The reaction of cells and tissues to implanted foreign bodies depends on the materials properties and its behavior upon contact with the body fluids. Abrahamsson et al.

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demonstrated that surface characteristics (smooth vs rough titanium surfaces) do not influence the biological width dimension (3i Implant System) (Abrahamsson et al., 2002). More recently, new titanium surfaces have been indicated to determine a better quality of peri-implant soft tissues relationship (Rossi et al., 2008) (ITI Implant System). Glauser et al. demonstrated in a histological study that the dimension of the biologic width (range 4-4.5 mm) in humans are similar to values found on animal models and that the soft tissue formed to oxidized and acid etched implants showed a minor epithelial down-growth and longer connective tissue seal when compared to machined implants (Glauser et al., 2005). In two different works, it has been demonstrated that abutment materials influenced the histological outcome on biologic width dimensions and in particular, titanium and zirconia abutments seemed to produce better histological results than gold and platinum abutments (Welander et al., 2008; Abrahamsson et al., 1998). Nevertheless, these findings were not consistent with a later study from the same group (Abrahamsson et Cardaropoli, 2007) that reported that the peri-implant soft tissue dimensions were not influenced by the use of titanium or gold alloy in the marginal zone of the implant. Kohal et al. further investigated the influence of zirconia and titanium abutments on soft tissue healing and demonstrated no significant differences (Kohal et al., 2004). In a review article by Rompen et al., it was concluded that titanium was the only material that demonstrated soft tissue biocompatibility; zirco-

nium and aluminum oxide showed favorable histological outcomes whereas dental porcelain and gold were less biocompatible and it was suggested to avoid them (Rompen et al., 2006).

Implant structure and position

Implant structure may differ between various implant systems: one piece implants present a transmucosal part in continuity with the endosseous part, whereas two piece implants present an interface between the implant (endosseous component) and the abutment (transmucosal component), resulting in a microgap between the two components (fig. 5 to 7). Abrahamsson et al. evaluated the influence of three different implant systems on the biological width (Astra Tech Implants, Brnemark and Bonefit-ITI) in beagle dogs (Abrahamsson et al., 1996). The authors compared a one-piece implant (Bonefit) versus two two-piece implants (Astra Techand Brnemark). The histological results demonstrated similar dimension and composition of the epithelial and connective tissue components at the end of the study. Abrahamsson et al. further investigated the influence of the abutment dis/reconnection on the marginal peri-implant tissues (Brnemark System) (Abrahamsson et al., 1997). The authors observed that abutment manipulation compromised the mucosal barrier and induced an apical migration of the connective tissue. Thus, while normal proportions and dimensions of the hard and soft tissues were observed in the control

group, at test sites the abutment manipulation resulted in a mechanical injury to the soft tissue barrier that had to reestablish more apically, causing a marginal bone resorption (1.5 mm). In contrast, a single abutment reconnection proved to induce no marginal bone remodeling (Astra Tech Implant System) resulting in a transmucosal attachment of adequate quality and dimensions (Abrahamsson et al., 2003). Hermann et al. further tested the hypothesis of a different biologic width between 1-piece and 2-piece implants (Hermann et al., 2001). Authors reported that dimensions of the peri-implant soft tissues, as evaluated by histometric measurements, were significantly influenced by the presence/absence of a microgap (interface) between the implant and the abutment, and the location of microgap (interface) in relation to the crest of the bone. Hermann et al. evaluated in an animal study (5 dogs, 59 ITI Implants) six different clinical situations: nonsubmerged one-piece implants with rough-smooth border placed at bone crest (group A), non-submerged onepiece implants with rough-smooth border placed 1 mm below crest (group B), non-submerged two-piece implants with a microgap placed at bone crest (group C), submerged two-piece implants with microgap placed at bone crest (group D), twopiece implants with the microgap placed above the bone crest (group E), two-piece implants with the microgap placed below the bone crest (group F) (Hermann et al., 1997). Authors concluded that one-piece, non submerged implants with a rough/smooth border placed at the

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Fig. 5. Implant into fresh extraction socket. Twopiece implant (Astra Micro Thread, Osseospeed, Astra Tech, Sweden) with surrounding tissues. Biological width after 6 weeks of healing. Note the inflammation free connective tissue in intimate contact with the implant-abutment interface. Levai-Laczko staining. Original augmentation 5X. Fig. 5. Implant dans une alvole dextraction frache. Implant en deux parties (Astra Micro Thread, Osseospeed, Astra Tech, Sude) avec les tissus environnants. Espace biologique aprs 6 semaines de cicatrisation. Noter labsence dinflammation du tissu conjonctif en contact intime avec linterface implant/pilier. Coloration de Levai-Laczko. Grossissement 5.

Fig. 6. Implant into fresh extraction socket. Two-piece implant (Thommen SPI Element, Thommen Medical AG, Switzerland) with surrounding tissues. Biological width after 6 weeks of healing. PM : marginal mucosa aJE : most apical junctional epithelium B : most coronal bone to implant contact Levai-Laczko staining. Original augmentation 2.5X. Fig. 6. mplant dans une alvole dextraction frache. Implant en deux parties (Thommen SPI Element, Thommen Medical AG, Suisse) avec les tissus environnants. Espace biologique aprs 6 semaines de cicatrisation. PM : muqueuse marginale. aJE : pithlium de jonction le plus apical. B : os le plus coronaire au contact de limplant. Coloration de Levai-Laczko. Grossissement 2,5.

Fig. 7. Implant into fresh extraction socket. One-piece implant (Straumann Standard Plus, AG, Switzerland) with surrounding tissues. Biological width after 6 weeks of healing. PM : marginal mucosa a.JE : most apical junctional epithelium B : most coronal bone to implant contact Levai-Laczko staining. Original augmentation 2.5X. Fig. 7. Implant dans une alvole dextraction frache. Implant monobloc (Straumann Standard Plus, AG, Suisse) avec les tissus environnants. Espace biologique aprs 6 semaines de cicatrisation. PM : muqueuse marginale aJE : pithlium de jonction le plus apical B : os le plus coronaire au contact de limplant Coloration de Levai-Laczko. Grossissement 2,5.

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alveolar crest (group A) presented the smallest value of soft tissues dimension at the end of the study. The apical displacement of the rough/smooth border of the implant (type B) resulted in a wider biologic width (average increase of 0.73 mm). This change occurred through both an increase of the junctional epithelium (average 0.41 mm) and of connective tissue dimension (average 0.34 mm). Furthermore, the authors observed the negative influence of the microgap on the histological outcome evidenced by higher soft tissues dimensions as well as an increase in bone resorption. Authors speculated that this finding was probably due to a bacterical colonization as indicated by previous reports (Persson et al., 1996; Quirynen et al., 1993), or to abutment micromovements (King et al., 2002). Todescan et al. investigated the influence of the implant shoulder position on the soft tissue healing (Todescan et al., 2002). The authors placed 24 implants (Brnemark System) in the mongrel dog and divided the implants into three groups: group 1, implants remained 1 mm above the bone crest, group 2, the implant shoulder was placed at the level of the bone crest and group 3, implants remained 1 mm below the bone crest. In group 2 and 3, a countersink bur was used. The junctional epithelium showed a mean value of 1.67 mm in group 1, 1.93 mm in group 2 and 2.78 mm in group 3. The corresponding values for the band of connective tissue were 1.13 mm, 0.92 mm and 1.60 mm, respectively. Differences between the groups were not statistically different, except for group 2 versus group 3. The authors demonstrated

a tendency towards longer junctional epithelium and connective tissue component the deeper the implants were placed. Pontes reported in a study in the mongrel dogs on 2-piece implants (Conexao System) positioned at bone level, 1 and 2 mm below the bone crest (Pontes et al., 2008). Implants were further divided in two groups: immediate loading (24 hours, non occlusal contact) and conventional loading (30 days after second stage surgery). Animals were killed for histological analysis after 3 months of non-submerged healing. Findings from this study are partially consistent with Todescan et al. (Todescan et al., 2002) demonstrating a tendency towards a longer connective tissue component the deeper the position of the implant, whereas the length of the junctional epithelium resulted to be independent of the implant position. The effect of the loading did not demonstrate any influence on the soft tissue healing. Recently, Lazzara and Porter introduced the platform switching technique represented by a non matching implant/abutment interface (Lazzara and Porter, 2006). They elaborated the concept on the basis of a 13 years follow-up case series. Implants that presented non-matching abutments (i.e. the implant platform was wider than the abutment) showed limited marginal bone resorption when compared with normal implants covered by matching abutments. Authors speculated that the soft tissue healing around a non matching abutment might exploit a horizontal direction and utilize the space available between the implant shoulder and the abutment for the establishment of the biological wid-

th. This hypothesis would justify a less marginal bone loss apical to implant-abutment interface. Recently, Luongo et al. reported on the soft tissues response to the platform switching technique (Luongo et al., 2008). Authors observed in a human biopsy of one implant that the inflammatory connective tissue infiltrate measured about 0.35 mm apical and coronal to the implantabutment interface. This finding is not consistent with data reported by Ericsson et al. (Ericsson et al., 1996) who demonstrated in an experimental study in dogs the presence of an inflammatory connective tissue infiltrate that measured approximately 0.75 mm apical and coronal to the interface. The smaller inflammatory reaction observed with the platform switching technique (Luongo et al., 2008) may in part justify the limited marginal bone loss observed by Lazzara and Porter (Lazzara and Porter, 2006). In conclusion, the type of implant (i.e. one- or two-piece implants) and the surgical procedure (i.e. one- or twostage surgical protocol) do no influence the dimensions and composition of the biological width. Nevertheless, limited data are available on the influence of the position of the implant shoulder in relation to the bone crest. It may be suggested that the deeper the implant shoulder position the longer the biological width. The clinical consequences of such histological findings are still unknown.

Immediate post-extractive insertion

In the past two decades, the immediate implant placement protocol has been introduced into clinical

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practice, while most of the studies on biological width on animal models have been conducted on healed ridge models. It may be speculated that the surgical protocol of placing an implant immediately upon tooth extraction may influence the formation and maturation of the biological width. Arajo et al. investigated in the beagle dog the healing of implants (Straumann Implant System) placed into the distal sockets of third and fourth mandibular premolars (Arajo et al., 2005). Findings from this experiment demonstrated that the dimensions and composition of the mucosal seal around immediate implants were similar to those around standard implants (Berglundh et al., 1991). The histometrical analysis only showed a different dimension of the soft tissue barrier when comparing buccal and lingual sites. The overall dimensions of the biological width after 3 months of healing was 3.9 0.5 mm and 2.6 0.4 mm, at the buccal and lingual aspect, respectively. The difference between buccal and lingual dimensions was due to the connective tissue component that was 1.8 0.8 mm and 0.7 0.2 mm on the buccal and lingual aspects, respectively. This difference could be in part explained by a greater marginal bone loss observed on the buccal aspect of the implants that corresponded to 2.6 0.4 mm. These differences were confirmed by another study from the same laboratory (Arajo et al., 2006) that utilized a similar experimental model with different time intervals. At 1 month of healing the distance from the mucosal margin to the first boneimplant contact varied from 3.3 mm at the buccal sites and 3.5 mm at

the lingual sites whereas at 3 months the corresponding values were 4.2 0.8 mm and 2.7 0.2 mm respectively. As previously reported, the difference was due to the dimensions of the connective tissue that reached 1.9 0.6 mm and 0.6 0.2 mm at the buccal and lingual sites after 3 months of healing. Nevertheless, other experimental studies that have compared healing at implants placed in a healed ridge and implants immediately placed in fresh extraction sockets have reported, at 8 months, a larger dimension of the soft tissue barrier in implants placed immediately (Schultes

and Gaggl, 2001). Similar results were reported in a recent experimental study in the minipig model (Rimondini et al., 2005). They evaluated the epithelial dimensions after placing implants in fresh extraction sockets in minipigs and reported that the epithelial length was 3.02 mm 30 days after implant installation and then remained stable up to 60 days (fig. 8 to 10). In a recent study, Vignoletti described the differences in the healing of the soft tissue barrier when placing four different implant systems immediately in fresh extraction sockets (3i Implant System, Astra Tech

Fig. 8. a. Implant into fresh extraction sockets. Two-piece implant (3i Osseotite Certain, Biomet 3i, USA). Biological width formation at 1 week of healing. A junctional epithelium can be clearly detected. Toluidine blue staining. Original augmentation 2.5X. b. Detail of (a). The connective tissue immediately apical of the junctional epithelium is rich in inflammatory cells. Toluidine blue staining. Original augmentation 5X. Fig. 8. a. Implant dans une alvole dextraction frache. Implant en deux parties (3i Osseotite Certain, Biomet 3i, tats-Unis). Formation dun espace biologique au bout de 1 semaine de cicatrisation. Un pithlium de jonction peut tre clairement dtect. Coloration au bleu de toluidine. Grossissement 2,5. b. Dtail. Le tissu conjonctif situ immdiatement apicalement lpithlium de jonction est riche en cellules inflammatoires. Coloration au bleu de toluidine. Grossissement 5.

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M. DE SANCTIS, N. BALDINI, F. VIGNOLETTI

Fig. 9. Implant into fresh extraction sockets. Twopiece implant (3i Osseotite Certain, Biomet 3i, USA). Biological width formation at 2 weeks of healing. A gap is present between the implant shoulder and the abutment. Proliferation of the junctional epithelium can be detected at the implant-abutment interface. Toluidine blue staining. Original augmentation 2.5X. Fig. 9. Implant dans une alvole dextraction frache. Implant en deux parties (3i Osseotite Certain, Biomet 3i, tats-Unis). Formation dun espace biologique au bout de 2 semaines de cicatrisation. Il existe un hiatus entre lpaulement de limplant et le pilier. La prolifration de lpithlium de jonction peut tre dtecte linterface implant/pilier. Coloration au bleu de toluidine. Grossissement 2,5.

Implants, Thommen Implant System, ITI Implant System) (Vignoletti et al., 2009). The biological width 6 weeks after implant placement averaged between 3.5-4.1 mm and

2.8-3.2 mm at the buccal and lingual aspects, respectively. On the buccal aspect the soft tissues barrier was comprised of a junctional epithelium that measured between 2-2.7 mm and a connective tissue that ranged between 1-1.8 mm. The corresponding values at the lingual side were 1.6-2 mm of epithelium and 0.9-1.4 mm of connective tissue. The study failed to demonstrate differences in the healing pattern when placing four different implant systems in fresh extraction sockets. Nevertheless, the obtained length of the epithelium with the four implant systems is longer than what has been reported when placing implants in healed-ridge experimental models.

Fig. 10. Implant into fresh extraction sockets. a. Two-piece implant (3i Osseotite Certain, Biomet 3i, USA). Biological width formation at 4 weeks of healing. Note the mature connective tissue. b. Detail of (a). No inflammatory cells can be detected at the implant-abutment interface. Fig. 10. Implant dans une alvole dextraction frache. a. Implant en deux parties (3i Osseotite Certain, Biomet 3i, tats-Unis). Formation dun espace biologique au bout de 4 semaines de cicatrisation. Noter le tissu conjonctif mature. b. Dtail. Aucune cellule inflammatoire ne peut tre dtecte linterface implant/pilier.

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Biological width around implants. Histological evidence: a review on animal studies

In conclusion, contradictory data are available on the influence of placing implants immediately upon tooth extraction on the biological width. The limited experimental evidence available seem to indicate a tendency towards higher dimensions of the mucosal seal around implants placed according to this surgical protocol.

Conclusion
The structure of peri-implant mucosa has many similarities with periodontal tissues. The soft tissue barrier is composed by a sulcus with a non keratinized sulcular epithelium, a junctional epithelium, and a supracrestal connective tissue with an area of dense circular fibers near to the implant surface. The presence of a junctional epithelium facing the titanium surface, similar to the one around teeth, has been evidenced by a large number of studies. Nevertheless recent ultrastructural studies on rats models have failed to demonstrate the presence of a true junctional epithelium, because of the absence of the basal lamina and of the hemidesmosomes in the samples 8 weeks after implant insertion. Further researches are needed to clarify these issues. Connective fibers orientation represents the most important difference between periodontal and periimplant tissues that is while in the periodontal structure, fibers run perpendicular the long axis of the tooth, in perimplant tissue, the fibers from the bone crest run parallel to the implant surface. The dimension the soft tissue barrier around implant seems to be constant, similarly to what has been

described around teeth. This dimension has been described as periimplant biologic width: This is composed by the dimension of the sulcus, and by the supra-crestal epithelial and connective tissue component. Most studies report bigger values for peri-implant biologic width than the ones reported for periodontal biologic width. The difference is generally related to a bigger epithelial component at implant sites when compared to the tooth. A minimum dimension of the biological width is needed in order to accommodate for the soft tissue healing process: when this dimension is not present, bone resorption may occur, to allow for an appropriate biological dimension of the peri-implant soft tissue barrier. The influence of five different factors on peri-implant biologic width dimensions has been evaluated reviewing the available literature, these are: surgical technique, loading time, titanium surfaces and abutment materials, implant structure and position, immediate post-extractive insertion. Surgical technique, one-stage or two-stage surgery and loading time, both immediate or delayed, do not influence the dimensions of the soft tissue barrier around the implants. More controversial is the issue of the influence of titanium surfaces and abutment materials. Titanium is the only material that demonstrated soft tissue biocompatibility, zirconium and aluminum oxide have showed favorable histological outcomes whereas dental porcelain and gold were less biocompatible. There is no agreement in literature on the influence of titanium surface,

in fact while there are studies indicating a smaller dimension of the biologic width in smooth surfaces nevertheless while other studies suggest that these differences are not present. Further research is needed to allow for a conclusion. The type of implant (i.e. one- or twopiece implants) and the surgical procedure (i.e. one- or two-stage surgical protocol) do not influence the dimensions and composition of the biological width. Nevertheless limited data are available on the influence of the position of the implant shoulder in relation to the bone crest. It may be suggested that the deeper the implant shoulder position is, the longer the biological width. Microgap between implant and abutment when present can modify the dimension of biologic width, the longer epithelial component described may be determined by bacterial colonization or abutments micro movements. The clinical consequences of such histological findings are still unknown. Contradictory data are present on dimension of the biological width when an immediate postextractive approach is utilized: differences between buccal and lingual sites have been documented, demonstrating a bigger biologic width in buccal sites of immediately placed implants. This difference is related to a bigger connective component in buccal sites. The limited experimental evidence available seems to indicate a tendency towards larger dimensions of the mucosal seal around implants placed according to this surgical protocol.

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