Dimension and Structures of Biological Seal of Peri-Implant Tissues










Chapter 3
Dimension and Structures of Biological Seal of Peri-
Implant Tissues
Wen Lin Chai, Masfueh Razali and
Wei Cheong Ngeow
Additional information is available at the end of the chapter
http://dx.doi.org/10.5772/63950
Abstract
Over
the years, improved understanding of the nature of bone-implant interface is
among the important contributors to the success of osseointegration in modern dental
implantology. The focus has since shifted to the assessment of the soft tissue-implant
interface to better understand the mechanism of biological seal in the transmucosal
region. The importance of peri-implant mucosal region lies in the need to establish a
tight seal that isolates implant and the bone from the oral environment via epithelial
and connective tissue attachment, thus preventing ingrowth of bacterial plaque. Many
factors may influence the soft tissue attachment at this peri-implant interface. In this
chapter, the dimension of peri-implant tissues and the factors affecting the biological
seal, namely surface topography and physicochemical properties, are discussed. The
review also looks into the impact of the type of materials and surface modifications of
dental implant, all of which may influence the formation of biological seal of soft tissue
around the dental implant.
Keywords: implant-soft tissue interface, biological seal, peri-implant tissues, surface
topography, three-dimensional oral mucosal model
1. Introduction
The success of dental implant in the oral cavity depends on direct bone-implant surface contact
as
well as the soft tissue attachment surrounding the implant abutment (and dental im‐
plant), which the latter acts as biological seal against external oral environment. Much of the
attentions in the early dental implant studies were given to the bone-to-titanium interface.

These studies range from clinical [1, 2] to molecular levels [3], and from animal model [4, 5]
to human biopsies [6, 7]. In all studies, the bone appears to be in direct contact with implant
without the presence of any connective tissue or fibrous tissue encapsulating the implant.
The extensive and well-established researches on bone-implant interface have led to the wide
acceptance of the concept of osseointegration. Presently, more focuses are placed on under‐
standing and improving the implant-soft tissue interface. The biological seal of the soft tissue-
implant interface is created by epithelium and connective tissue. The presence of keratinized
mucosa surrounding an implant is thought to be one of the important factors in maintaining
peri-implant soft tissue health. Moreover, materials and surface topography of implant
abutment materials may also influence the biological seal formed at the implant-soft tissue
interface. The available data from animal studies and emerging information from human
investigations suggest that different material with different surface energy and enhanced
surface topography is associated with increased soft tissue-to-implant contact [8, 9]. The nature
of soft tissue-implant against normal periodontium is compared in the subsequent paragraph.
By understanding the tissue around transmucosal region, the factors influencing this biological
seal will be better appreciated.
2. Peri-implant tissue
The periodontium is known as a tooth-supporting structure while the peri-implant mucosa is
the structure and function of the mucosa that surrounds the abutment of a dental implant.
Clinically, both tooth and prosthesis of the dental implant will emerge from the gingival tissue
with tight gingival cuff. Figure 1 features the clinical pictures of healing abutment in situ and
the appearance of peri-implant mucosa following removal of the healing abutment. The
mucosa surrounding the dental implant formed tight gingival cuff consists of epithelium and
connective tissues established during healing after the surgery. Many studies provide
information on similarities and differences between peri-implant soft tissue and tissue at the
dento-gingival junction. The similarities and differences of both periodontium and peri-
implant mucosa are depicted in Table 1.
Figure 1. The clinical pictures of healing abutment in situ and mucosa at the implant neck. (Courtesy of Dr. Masfueh
Razali).
Dental Implantology and Biomaterial40

Features Periodontium Peri-implant tissue
Gingival sulcus
depth
Shallow Dependent upon abutment length and restoration
margin
Junctional
epithelium
Hemidesmosome attachment to enamel Hemidesmosome attachment on titanium [10, 11]
Gingival fibres Complex array of fibres, some inserted
into cementum above crestal bone,
and onto periosteum
- Lack of fibres insertion on implant surface
‐Fibres orientated longitudinally, parallel or
circumferential to the long axis of the implant [12]
Connective tissue
attachment
Well-organised collagen fibre
bundles, running perpendicular
to root cementum
- A scar-like structure that is rich in collagen but
deficient in fibroblasts and vascular systems [13, 14]
Blood supply Numerous vasculatures from periodontal
ligament space and gingival connective
tissue which formed anastomoses
Fewer capillaries compared to tissue surrounding tooth.
[14–16]
Biologic width JE = 0.97 mm, CT = 1.07 mm JE = 1.88 mm (average), CT = 1.05 mm [12]
Table 1. Comparison of periodontium and peri-implant tissue.
Figure 2. A schematic drawing of similarities and differences between dentogingival tissue and peri‐implant mucosa
(Prepared by Dr. Masfueh Razali).
Macroscopically, a tooth-supporting structure comprises the gingiva, connective tissues and
periodontal ligament, which connects tooth to bone via cementum. There are three types of
gingival epithelium covering the underlying connective tissue of a tooth. These are junctional
epithelium, which provides the contact between the gingiva and the tooth; sulcular epithelium,
which faces the tooth surfaces without any contact being made with the tooth surface; and
lastly, oral epithelium, which faces the oral cavity. The oral epithelium is a keratinized,
stratified squamous epithelium. The junctional epithelium, which is structurally different, is
formed from the reduced enamel epithelium during tooth eruption and from dividing basal
cells of the oral epithelium. The junctional epithelium forms a collar around the tooth and is
about 2 mm high and 100 μm thick. It is composed of only two cell layers, namely a basal layer
Dimension and Structures of Biological Seal of Peri-Implant Tissues
http://dx.doi.org/10.5772/63950
41

and a supra basal layer. The inner cells of the junctional epithelium form and maintain a tight
seal against the tooth surface. The connective tissue is composed of gingival fibres, which runs
in many directions, from tooth and/or bone to gingival tissues. Similarly, the supporting
structures of dental implant also consist of gingival epithelium and connective tissue attach‐
ment but without periodontal ligament. The epithelial part resembles the junctional epithelium
around natural teeth [10, 12, 18, 19]. The features of both normal periodontium surrounding
teeth and peri-implant tissue are illustrated in Figure 2.
Generally, the macroscopic and microscopic features of peri-implant mucosa are almost
similar to the tooth-supporting tissue (at the dento-gingival junction) with few exceptions.
1. The junctional epithelium: the junctional epithelium faces the implant smooth surfaces or
abutment of an implant is less thick, and consists of only a few cell layers especially at the
apical region.
2. Biologic width: both consist of junctional epithelium and connective tissue attachment,
but the junctional epithelium of an implant is longer [10, 14, 17] than that around teeth.
Variation in height of these two attachments is noted between human and animal studies.
3. The gingival fibres connecting the periosteum to bone run parallel to the long axis of the
implant, as compared with those around a tooth, where the gingival fibres consists of
complexes arrays running from many direction including from tooth to gingival tissues,
some of which perpendicular to the tooth. There were also fibres running circumferen‐
tially as shown by [15]. The arrangement of the fibres is schematically illustrated in Figure
2. The histological sections of transmucosal region of peri-implant soft tissue are shown
in Figure 3. Note that there was a cell-free area adjacent to implant, and fibres appear
running parallel to long axis of implant.
4. No periodontal ligament—bone is present.
Figure 3. Transmucosal region of peri-implant mucosa demonstrating fibres of gingival connective tissue, no attach‐
ment of fibres on implant surface. (Reproduced with permission from [15])
Dental Implantology and Biomaterial
42
So far, the structure, dimensions and the composition of gingival and implant transmucosal
regions have been investigated by many researchers. These include early animal models
studies in dogs [10, 11, 14] and in human [20, 21]. From those studies, a few conclusions have
been made which included:
1. On average, the attachment between mucosa and a titanium implant comprises junctional
epithelium about 1.4–2.9 mm high, and a connective tissue zone approximately 0.7–2.6
mm high [10, 12]
2. The periodontium and peri-implant mucosa have common characteristics, but they differ
in terms of composition of the connective tissue, the alignment of the collagen fibre
bundles and the distribution of vascular structures apical to the junctional epithelium.
The connective tissue-implant interface commonly consists of a non-infiltrated, densely
structured, collagen-rich connective tissue. It can be divided into two zones: outer zone
(located beneath the junctional epithelium) and inner zone (positioned above the bone
crestal and in direct contact with implant surface) [14]
A qualitative analysis of the subepithelial connective tissue showed a cell-rich, well-
vascularized outer zone with fibres running in many different directions and a poorly
vascularized inner zone consisting of numerous dense collagen fibres running close to the
implant surface, predominantly in a parallel direction [14, 15, 22]. The inner zone is in
direct contact with the implant/abutment surface and is 50–100 mm thick. It is rich in fibres,
with few scattered fibroblasts that appear to be in close contact with the transmucosal
component. The peri-implant mucosa generally resembles and is recognized as a scar
tissue, exhibiting an impaired resistance towards bacterial colonization [13, 16]. As a
consequence, the connective tissue adhesion at implant has a poor mechanical resistance
as compared to that of natural teeth.
As suggested by some studies mentioned earlier, the biological seal of the peri-implant tissue
formed by both epithelial and connective tissue attachments is weak and poor in mechanical
resistance [13, 16]. Hence, this area is subjected to increased risk of peri-implant diseases, as
the bacterial assault begins in this area. It is important to understand the nature of both
attachments as it may lead to an enhancement this biological seal. Various models been used
to evaluate the implant-soft tissue interface. These models are reviewed in the next section.
3. Evaluation of implant-soft tissue response
The soft tissue interface especially the structure of collagen fibre bundles received more
attention over the past 10 years with studies that include animal models such as dogs [10–12,
14] and monkeys [23, 24] and human [25, 26] used to explore the structure and dimension of
soft tissue-implant interface. Recently, Chai and co-workers [18] have ventured upon the use
of three-dimensional oral mucosal models by using the tissue engineering technology to
investigate the nature of the peri-implant biological seal.
Dimension and Structures of Biological Seal of Peri-Implant Tissues
http://dx.doi.org/10.5772/63950
43
www.ebook3000.com

3.1. Implant-soft tissue interface models
The advantages and disadvantages of each implant-soft tissue interface study models are
described in the next section. These models were developed in order to enhance our under‐
standing of the soft tissue response on various materials with different surface topography
and to establish best methods to evaluate the biological seal of peri-implant tissue. Generally,
an in vitro study using monolayer cell culture model is conducted to assess the cytotoxicity of
the cells and quickly observe cell activities and behaviour towards new dental implant
materials. Histomorphometric analysis of en bloc tissue consisting of both soft tissue and
implant body is the best method to demonstrate the presence of epithelial and connective tissue
attachment at the soft tissue-implant interface. Yet, due to limited opportunity to obtain
histological section from human, animal models were developed.
3.1.1. In vitro studies
Presently, in vitro testing is performed as a prerequisite to in vivo evaluation. However, the
in vitro techniques do not reflect the clinical situation and the progress in our understanding
of extra- and intracellular processes that occur in connective tissue attachment. Thus, the data
cannot be extrapolated into clinical applications. Nevertheless, the study involving monolayer
cells is by far the most popular and easy-to-conduct study although more sensitive in vitro
evaluations are now available. The cell shape, activities and response can be evaluated
morphometrically via immunocytochemical staining [27], or by analysis using scanning
electron [28, 29] or fluorescent [30, 31] miscroscopies. Additionally, the gene and protein
expressions for cell adhesion and attachment can also be carried out [27, 32, 33]. Most studies
used primary human gingival [29, 32, 34] and periodontal [35] fibroblasts as a cell model, which
are cultured directly onto the dental implant materials surface. Keratinocytes are also
frequently used [27, 36, 37]. Compared to fibroblasts, keratinocytes by far is most difficult to
culture. Cochran et al. [35] compared the behaviour of periodontal and gingival fibroblast as
well as keratinocytes towards the titanium with different surface textures. They found that
human fibroblast and epithelial cell attachment and proliferation are significantly affected by
surface characteristics of titanium. Of three cell types, gingival fibroblasts appeared to attach
best, followed by periodontal ligament fibroblasts and epithelial cells. Both types of fibroblasts
grow and proliferate well on both rough and smooth titanium surfaces compared to epithelial
cells once they are attached to the surface [35]. Other study found a significant decrease in the
number of gingival fibroblasts on rough titanium (Ti) surfaces compared with smooth polished
Ti surfaces [30, 34]. On the other hand, Oates et al. [32] found that the fibroblasts adhesion and
attachment are enhanced in rougher surface than smooth surface, in contrast to other findings
[33] where focal adhesion kinases were immunogold labelled. In a different study using
ceramic, fibroblasts attached more on the milled ceramic and appeared to follow the direction
of the fine irregularities on the surface [38]. Nevertheless, most common finding of those
studies is that cells were oriented in a parallel order along the grooves of the machined surface
but arranged randomly when in contact with a rough surface. Hence, the in vitro models
appear to be able to provide an insight and could be used to guide specific cell attachment or
Dental Implantology and Biomaterial
44

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Chapter 3Dimension and Structures of Biological Seal of Peri-Implant TissuesWen Lin Chai, Masfueh Razali andWei Cheong NgeowAdditional information is available at the end of the chapterhttp://dx.doi.org/10.5772/63950AbstractOver the years, improved understanding of the nature of bone-implant interface isamong the important contributors to the success of osseointegration in modern dentalimplantology. The focus has since shifted to the assessment of the soft tissue-implantinterface to better understand the mechanism of biological seal in the transmucosalregion. The importance of peri-implant mucosal region lies in the need to establish atight seal that isolates implant and the bone from the oral environment via epithelialand connective tissue attachment, thus preventing ingrowth of bacterial plaque. Manyfactors may influence the soft tissue attachment at this peri-implant interface. In thischapter, the dimension of peri-implant tissues and the factors affecting the biologicalseal, namely surface topography and physicochemical properties, are discussed. Thereview also looks into the impact of the type of materials and surface modifications ofdental implant, all of which may influence the formation of biological seal of soft tissuearound the dental implant.Keywords: implant-soft tissue interface, biological seal, peri-implant tissues, surfacetopography, three-dimensional oral mucosal model1. IntroductionThe success of dental implant in the oral cavity depends on direct bone-implant surface contactas well as the soft tissue attachment surrounding the implant abutment (and dental im‐plant), which the latter acts as biological seal against external oral environment. Much of theattentions in the early dental implant studies were given to the bone-to-titanium interface. These studies range from clinical [1, 2] to molecular levels [3], and from animal model [4, 5]to human biopsies [6, 7]. In all studies, the bone appears to be in direct contact with implantwithout the presence of any connective tissue or fibrous tissue encapsulating the implant.The extensive and well-established researches on bone-implant interface have led to the wideacceptance of the concept of osseointegration. Presently, more focuses are placed on under‐standing and improving the implant-soft tissue interface. The biological seal of the soft tissue-implant interface is created by epithelium and connective tissue. The presence of keratinizedmucosa surrounding an implant is thought to be one of the important factors in maintainingperi-implant soft tissue health. Moreover, materials and surface topography of implantabutment materials may also influence the biological seal formed at the implant-soft tissueinterface. The available data from animal studies and emerging information from humaninvestigations suggest that different material with different surface energy and enhancedsurface topography is associated with increased soft tissue-to-implant contact [8, 9]. The natureof soft tissue-implant against normal periodontium is compared in the subsequent paragraph.By understanding the tissue around transmucosal region, the factors influencing this biologicalseal will be better appreciated.2. Peri-implant tissueThe periodontium is known as a tooth-supporting structure while the peri-implant mucosa isthe structure and function of the mucosa that surrounds the abutment of a dental implant.Clinically, both tooth and prosthesis of the dental implant will emerge from the gingival tissuewith tight gingival cuff. Figure 1 features the clinical pictures of healing abutment in situ andthe appearance of peri-implant mucosa following removal of the healing abutment. Themucosa surrounding the dental implant formed tight gingival cuff consists of epithelium andconnective tissues established during healing after the surgery. Many studies provideinformation on similarities and differences between peri-implant soft tissue and tissue at thedento-gingival junction. The similarities and differences of both periodontium and peri-implant mucosa are depicted in Table 1.Figure 1. The clinical pictures of healing abutment in situ and mucosa at the implant neck. (Courtesy of Dr. MasfuehRazali).Dental Implantology and Biomaterial40 Features Periodontium Peri-implant tissueGingival sulcusdepthShallow Dependent upon abutment length and restorationmarginJunctionalepitheliumHemidesmosome attachment to enamel Hemidesmosome attachment on titanium [10, 11]Gingival fibres Complex array of fibres, some insertedinto cementum above crestal bone,and onto periosteum- Lack of fibres insertion on implant surface‐Fibres orientated longitudinally, parallel orcircumferential to the long axis of the implant [12]Connective tissueattachmentWell-organised collagen fibrebundles, running perpendicularto root cementum- A scar-like structure that is rich in collagen butdeficient in fibroblasts and vascular systems [13, 14]Blood supply Numerous vasculatures from periodontalligament space and gingival connectivetissue which formed anastomosesFewer capillaries compared to tissue surrounding tooth.[14–16]Biologic width JE = 0.97 mm, CT = 1.07 mm JE = 1.88 mm (average), CT = 1.05 mm [12]Table 1. Comparison of periodontium and peri-implant tissue.Figure 2. A schematic drawing of similarities and differences between dentogingival tissue and peri‐implant mucosa(Prepared by Dr. Masfueh Razali).Macroscopically, a tooth-supporting structure comprises the gingiva, connective tissues andperiodontal ligament, which connects tooth to bone via cementum. There are three types ofgingival epithelium covering the underlying connective tissue of a tooth. These are junctionalepithelium, which provides the contact between the gingiva and the tooth; sulcular epithelium,which faces the tooth surfaces without any contact being made with the tooth surface; andlastly, oral epithelium, which faces the oral cavity. The oral epithelium is a keratinized,stratified squamous epithelium. The junctional epithelium, which is structurally different, isformed from the reduced enamel epithelium during tooth eruption and from dividing basalcells of the oral epithelium. The junctional epithelium forms a collar around the tooth and isabout 2 mm high and 100 μm thick. It is composed of only two cell layers, namely a basal layerDimension and Structures of Biological Seal of Peri-Implant Tissueshttp://dx.doi.org/10.5772/6395041 and a supra basal layer. The inner cells of the junctional epithelium form and maintain a tightseal against the tooth surface. The connective tissue is composed of gingival fibres, which runsin many directions, from tooth and/or bone to gingival tissues. Similarly, the supportingstructures of dental implant also consist of gingival epithelium and connective tissue attach‐ment but without periodontal ligament. The epithelial part resembles the junctional epitheliumaround natural teeth [10, 12, 18, 19]. The features of both normal periodontium surroundingteeth and peri-implant tissue are illustrated in Figure 2.Generally, the macroscopic and microscopic features of peri-implant mucosa are almostsimilar to the tooth-supporting tissue (at the dento-gingival junction) with few exceptions.1. The junctional epithelium: the junctional epithelium faces the implant smooth surfaces orabutment of an implant is less thick, and consists of only a few cell layers especially at theapical region.2. Biologic width: both consist of junctional epithelium and connective tissue attachment,but the junctional epithelium of an implant is longer [10, 14, 17] than that around teeth.Variation in height of these two attachments is noted between human and animal studies.3. The gingival fibres connecting the periosteum to bone run parallel to the long axis of theimplant, as compared with those around a tooth, where the gingival fibres consists ofcomplexes arrays running from many direction including from tooth to gingival tissues,some of which perpendicular to the tooth. There were also fibres running circumferen‐tially as shown by [15]. The arrangement of the fibres is schematically illustrated in Figure2. The histological sections of transmucosal region of peri-implant soft tissue are shownin Figure 3. Note that there was a cell-free area adjacent to implant, and fibres appearrunning parallel to long axis of implant.4. No periodontal ligament—bone is present.Figure 3. Transmucosal region of peri-implant mucosa demonstrating fibres of gingival connective tissue, no attach‐ment of fibres on implant surface. (Reproduced with permission from [15])Dental Implantology and Biomaterial42 So far, the structure, dimensions and the composition of gingival and implant transmucosalregions have been investigated by many researchers. These include early animal modelsstudies in dogs [10, 11, 14] and in human [20, 21]. From those studies, a few conclusions havebeen made which included:1. On average, the attachment between mucosa and a titanium implant comprises junctionalepithelium about 1.4–2.9 mm high, and a connective tissue zone approximately 0.7–2.6mm high [10, 12]2. The periodontium and peri-implant mucosa have common characteristics, but they differin terms of composition of the connective tissue, the alignment of the collagen fibrebundles and the distribution of vascular structures apical to the junctional epithelium.The connective tissue-implant interface commonly consists of a non-infiltrated, denselystructured, collagen-rich connective tissue. It can be divided into two zones: outer zone(located beneath the junctional epithelium) and inner zone (positioned above the bonecrestal and in direct contact with implant surface) [14]A qualitative analysis of the subepithelial connective tissue showed a cell-rich, well-vascularized outer zone with fibres running in many different directions and a poorlyvascularized inner zone consisting of numerous dense collagen fibres running close to theimplant surface, predominantly in a parallel direction [14, 15, 22]. The inner zone is indirect contact with the implant/abutment surface and is 50–100 mm thick. It is rich in fibres,with few scattered fibroblasts that appear to be in close contact with the transmucosalcomponent. The peri-implant mucosa generally resembles and is recognized as a scartissue, exhibiting an impaired resistance towards bacterial colonization [13, 16]. As aconsequence, the connective tissue adhesion at implant has a poor mechanical resistanceas compared to that of natural teeth.As suggested by some studies mentioned earlier, the biological seal of the peri-implant tissueformed by both epithelial and connective tissue attachments is weak and poor in mechanicalresistance [13, 16]. Hence, this area is subjected to increased risk of peri-implant diseases, asthe bacterial assault begins in this area. It is important to understand the nature of bothattachments as it may lead to an enhancement this biological seal. Various models been usedto evaluate the implant-soft tissue interface. These models are reviewed in the next section.3. Evaluation of implant-soft tissue responseThe soft tissue interface especially the structure of collagen fibre bundles received moreattention over the past 10 years with studies that include animal models such as dogs [10–12,14] and monkeys [23, 24] and human [25, 26] used to explore the structure and dimension ofsoft tissue-implant interface. Recently, Chai and co-workers [18] have ventured upon the useof three-dimensional oral mucosal models by using the tissue engineering technology toinvestigate the nature of the peri-implant biological seal.Dimension and Structures of Biological Seal of Peri-Implant Tissueshttp://dx.doi.org/10.5772/6395043www.ebook3000.com 3.1. Implant-soft tissue interface modelsThe advantages and disadvantages of each implant-soft tissue interface study models aredescribed in the next section. These models were developed in order to enhance our under‐standing of the soft tissue response on various materials with different surface topographyand to establish best methods to evaluate the biological seal of peri-implant tissue. Generally,an in vitro study using monolayer cell culture model is conducted to assess the cytotoxicity ofthe cells and quickly observe cell activities and behaviour towards new dental implantmaterials. Histomorphometric analysis of en bloc tissue consisting of both soft tissue andimplant body is the best method to demonstrate the presence of epithelial and connective tissueattachment at the soft tissue-implant interface. Yet, due to limited opportunity to obtainhistological section from human, animal models were developed.3.1.1. In vitro studiesPresently, in vitro testing is performed as a prerequisite to in vivo evaluation. However, thein vitro techniques do not reflect the clinical situation and the progress in our understandingof extra- and intracellular processes that occur in connective tissue attachment. Thus, the datacannot be extrapolated into clinical applications. Nevertheless, the study involving monolayercells is by far the most popular and easy-to-conduct study although more sensitive in vitroevaluations are now available. The cell shape, activities and response can be evaluatedmorphometrically via immunocytochemical staining [27], or by analysis using scanningelectron [28, 29] or fluorescent [30, 31] miscroscopies. Additionally, the gene and proteinexpressions for cell adhesion and attachment can also be carried out [27, 32, 33]. Most studiesused primary human gingival [29, 32, 34] and periodontal [35] fibroblasts as a cell model, whichare cultured directly onto the dental implant materials surface. Keratinocytes are alsofrequently used [27, 36, 37]. Compared to fibroblasts, keratinocytes by far is most difficult toculture. Cochran et al. [35] compared the behaviour of periodontal and gingival fibroblast aswell as keratinocytes towards the titanium with different surface textures. They found thathuman fibroblast and epithelial cell attachment and proliferation are significantly affected bysurface characteristics of titanium. Of three cell types, gingival fibroblasts appeared to attachbest, followed by periodontal ligament fibroblasts and epithelial cells. Both types of fibroblastsgrow and proliferate well on both rough and smooth titanium surfaces compared to epithelialcells once they are attached to the surface [35]. Other study found a significant decrease in thenumber of gingival fibroblasts on rough titanium (Ti) surfaces compared with smooth polishedTi surfaces [30, 34]. On the other hand, Oates et al. [32] found that the fibroblasts adhesion andattachment are enhanced in rougher surface than smooth surface, in contrast to other findings[33] where focal adhesion kinases were immunogold labelled. In a different study usingceramic, fibroblasts attached more on the milled ceramic and appeared to follow the directionof the fine irregularities on the surface [38]. Nevertheless, most common finding of thosestudies is that cells were oriented in a parallel order along the grooves of the machined surfacebut arranged randomly when in contact with a rough surface. Hence, the in vitro modelsappear to be able to provide an insight and could be used to guide specific cell attachment orDental Implantology and Biomaterial44 specific material with surface characteristics for in vivo models. Animal models are the mostcommon in vivo models carried out compared to human studies.3.1.2. Animal modelsStudies using animals as in vivo models for evaluation of soft tissue response around dentalimplant have been extensively conducted and are well documented. In animal models, thehistological section of peri-implant tissue was made possible, which becomes the goldstandard for the implant-soft tissue interface analysis. While dogs models such as the beagle[8, 14] being the most common animal of choice, monkeys [23, 39] and minipigs [40] were alsoused to demonstrate the presence of epithelial and connective tissue attachment aroundtransmucosal region of dental implants histologically.The experiments in animals demonstrated that the dimension of the mucosal attachment toimplants was similar to the gingival attachment at teeth and was composed of an epithelialportion about 1.5–2 mm long and a cell-rich connective tissue portion close to the implant thatwas about 1–1.5 mm high [10]. Animal models were also used to evaluate the soft tissueresponse towards different abutment materials. Abrahamsson et al. [11] investigated theinfluence of abutment material on the location and the quality of the attachment that occurredbetween the peri-implant mucosa and the implant. They found no proper attachment formedat the abutment level made of gold alloy and porcelain when compared to those made oftitanium and ceramic. In addition, similar finding was noted by Welander et al. [41] whentitanium, zirconia and Au/Pt alloy were used. The tissue around abutment made from titaniumand zirconia was stable; meanwhile, an apical migration of epithelium was noted on Au/Ptalloy. In another study, Abrahamsson et al. [42] demonstrated that the soft tissue attachmentthat formed at implants made of commercially pure titanium (c.p. titanium) was not influencedby the roughness of the titanium surface.Among many, dogs have been the most common animal of choice. This is possibly due to easyaccess with regard to clinical examinations and oral hygiene procedures of the dogs. It mustbe noted that non-human primates bear more resemblence to human anatomy and histologythan any other animal, thus may offer a higher degree of relevance to human. Nevertheless,the results from animal experiments should always be carefully interpreted since the healingresponse and immuno-reaction in animals might not be similar to human, so the data mightnot be comparable. A given sequence of soft tissue integration to implants in a dog may notcorrespond exactly to an expected outcome in humans. The differences in tissue responseduring healing between human-human subjects may sometimes become more pronouncedbetween different human to human subjects than between animals and humans. Moreover,the healing response in animals is also less predictable compared to human. In the light ofevidence-based dentistry, the result from animal studies should be interpreted cautiously.Additionally, animal studies are also bound to ethical considerations, where study design andcalculation of sample size of animals in experiments are to be carried out with caution.Essentially, to have more clinical validity, human randomized control trials should be carriedout to obtain more information on the peri-implant tissues.Dimension and Structures of Biological Seal of Peri-Implant Tissueshttp://dx.doi.org/10.5772/6395045 3.1.3. Human studiesThe composition of the connective tissue interface towards implants was studied in bothanimal experiments and human biopsy materials. While human studies are very limited dueto ethical issues, the evidence of epithelial and connective tissue attachment around peri-implant regions are obtained mostly from failed implant [43], autopsy[44, 45] or clinical studies[1, 46], where the presence of connective tissue attachment on these studies is still difficult todemonstrate. Most of the human studies that have been carried out were clinical studies inwhich the traditional periodontal parameters were used for monitoring the soft tissueresponses around dental implants intra-orally. According to clinical studies that involve themarginal bone levels, we can conclude that bone level is stable as it implies that the soft tissueintegration has not migrated apically [1, 46, 47]. Liljenberg et al. [48] in their study of soft tissuebiopsies of edentulous ridge mucosa and peri-implant mucosa revealed that the compositionof both tissues were nearly identical in terms of collagen, cells and vascular structures. Theperi-implant mucosa harboured a junctional epithelium that contained significantly enhancednumbers of different inflammatory cells infiltration. On the other hand, Piatelli et al. [44] foundthat there was no inflammatory infiltrate in epithelium or connective tissue in human autopsybiopsies of titanium dental implants. It is also interesting to note that the collagen fibres in thecoronal part were parallel to implant surface while in the apical region the fibres were in aperpendicular fashion was found. Additionally, Glauser et al. [49] used both hard and softtissue biopsies of mini titanium implants with different surface characteristics to demonstratethe establishment of junctional epithelium attachment to the implant surfaces. They noted thatcollagen fibres and the fibroblasts were oriented parallel to the implant surface. The oxidizedand acid-etched implants revealed less epithelial downgrowth and longer connective tissuethan machined implants [49]. As for different types of materials, Vigolo et al. [46] assessed theperi-implant mucosa around abutments made of gold alloy and titanium and found nodifference between the two types of abutments with regard to peri-implant marginal bone leveland soft tissue parameters. Meanwhile, Nevins et al. [26] using en bloc biopsy demonstratedintimate contact of junctional epithelium cells to implant surface and connective tissue withfunctionally oriented collagen fibres running towards the implant surface designed with Laser-Lok microchannels. Nonetheless, it is unethical to remove implant in order to attain en bloctissue for histological analyses in human, and data from autopsy did not necessarily representthe ultrastructural nature of the peri-implant interface. In addition, not all animal experimentscan be replicated in human samples due to cost and ethical considerations. For this reason, theinvestigation of peri-implant interface for improvement of connective tissue attachment israther difficult to conduct in human. Thus, the need of development of different models forhistological analyses may be essential.3.1.4. Three-dimensional oral tissue engineeringAs the opportunity to undertake human studies is limited, many studies that evaluated theperi-implant interface were carried out using animal models. With advances in knowledge ontissue regeneration, tissue-engineered oral mucosal equivalents (three-dimensional oralmucosal model, 3D OMM) have been developed for clinical applications and also for con‐Dental Implantology and Biomaterial46 ducting in vitro studies on biocompatibility, mucosal irritation, disease and other basic oralbiological phenomena such as for grafting of oral mucosal defects [50, 51]. The 3D OMMconsists of both epithelium and connective tissue layers, grown in the laboratory using collagenmembrane as the scaffold. Therefore, evaluation of cell-cell interaction between epithelium,connective tissue and implant surface using 3D OMM is possible and could become analternative method to study the nature of peri-implant interface. The use of 3D OMM willpermit histological preparation and histomorphometric analysis of the interface. With themodification of culture technique, Chai et al. [18] have constructed 3D OMM and havedemonstrated the presence of peri-implant tissue with features that mimicked those seen invivo when tested with titanium. Chai and co-workers [19] further developed the 3D OMM andsucceeded in obtaining formed peri-implant-like-epithelium (PILE) on the polished, ma‐chined, sand-blasted and TiUnite titanium surfaces. Using the 3D OMM, ultrastructuralinvestigation of the soft tissue-implant interface with transmission electron microscopy (TEM)is also possible. It is also interesting to note that the presence of hemidesmosome-like structureas an epithelial attachment to the material surface is shown using this model (Figure 4).Moreover, the biological seal of peri-implant tissue can also be demonstrated quantitativelywith 3D OMM [52, 53]. This can be carried out via assessment of penetrative behaviour ofradioisotope material through the 3D OMM model [52]. Alternatively, the biological seal ofperi-implant can also be assessed through the measurement of degree formed by pocket ornon-pocket epithelial attachment at the oral mucosal model-material interface [18, 53].Although only limited study is available on the use of 3D OMM for evaluating the peri-implantinterface, this model appears to have a more promising prospect than the monolayer cellculture model. This model is a useful method to evaluate the soft tissue response prior toinvestigation with an animal model.Figure 4. Hemidesmosome-like structures (black arrows) formed from 3D OMM and specimens (Ti). P = polished andM = machined surfaces. (Reproduced with permission from [19]).Dimension and Structures of Biological Seal of Peri-Implant Tissueshttp://dx.doi.org/10.5772/6395047 3.2. Analyses of the soft tissue-implant interfaceThe soft-tissue implant interface can be investigated through histomorphometric and histo‐logic analyses. Of both, the preparation for latter analysis is very difficult to carry out especiallyif the implant is attached to the tissue. The histological studies also allow identification ofspecific protein markers expressed by any of the tissue or cells in response to dental implants.The histological sections can then be analysed under different types of microscopies. Amongthe known microscopic analyses for assessing the peri-implant interface are light microscopy(LM), scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), focusion beam (FIB) and transmission electron microscopy (TEM). Similar to SEM, CLSM allowsassessment of peri-implant interface and cell-cell interaction without the need of histologicalprocessing as for light microscopy. With these two, direct visualization of implant-soft tissueinterface is possible with appropriate preparation for each microscopy. The tissue or specimenscan also be fluorescently labelled for the identification of adhesion molecules or cells andexamined under CLSM [43]. While the use of 3D oral mucosal model with implant materialintact may allow direct examination of the connective tissue attachment, the method to preparethe specimen still remains challenging and technically demanding. More studies in term ofoptimization of certain promising technique such as FIB for TEM analysis must be exploredin order to obtain the ultrastructural nature of the implant-soft tissue interface.4. Factors influencing biological seal of implant-soft tissue interfaceThe existence and function of biologic width around dental implant are well documented inanimal and human histological studies. Any factors affecting soft tissue reaction around dentalimplant might also affect the biologic width, thus the biologic seal of the peri-implant region.As mentioned earlier in the text, the nature and health of soft tissue surrounding an implantmay be influenced by many factors. The presence of keratinized mucosa surrounding animplant is thought to influence the dimension of biological seal [54]. Moreover, the attachmentof epithelial and connective tissues may also be influenced by material properties and surfacemodifications of implant abutment materials. Within the context of this chapter, how soft tissueresponds to material and surface modification of implant/implant abutment is only discussedbriefly.4.1. Bulk of materialsMaterial properties appear to affect the attachment formed by epithelial tissue. Most often,titanium is the material used for dental implants and abutments, and is therefore the mostextensive and widely studied material. Commercially pure titanium (Grade 2 and Grade 4) iscommonly used in the fabrication of dental implants and implants abutments. Recently,zirconia is gaining more popular and seems to be a suitable implant material because of itsexcellence aesthetics, mechanical properties and biocompatibility. The presence of zirconia indentistry is now being embraced, with the manufacturers promoting the esthetic, biomechan‐ical and biological qualities of the material. Despite the extensive literature in the field ofDental Implantology and Biomaterial48 osseointegration of zirconia [39, 55], the response of soft tissue towards zirconia is starting togain attention from many researchers [9, 11, 40, 41]. In animal experiments, Abrahamsson etal. [11] showed that an epithelial downgrowth occurred and migrated towards the implantneck and associated bone loss which was noted around the abutments of gold and gold alloysfused with dental ceramics, as compared to abutments made of pure titanium and aluminiumoxide (Al2O11) ceramics where peri-implant cuff of about 3.5 mm width was noted to be present.Kohal et al. [39] also reported a satisfactory soft-tissue formation on both titanium andzirconium oxide (ZrO2) surfaces, without evidence of perpendicular fibres on the monkeymodel. Likewise, another study showed that the soft-tissue dimension at Ti and ZrO2 abut‐ments remained stable after 5 months of healing, meanwhile at gold/platinum alloys abutmentsites, an apical shift of the barrier epithelium and marginal bone loss occurred [41]. In contrast,a human clinical study conducted by Vigolo et al. [46] revealed no significant differencesregarding peri-implant bone loss and soft-tissue level when abutments of titanium and goldalloy were used with cemented single implant crown. Similarly, Linkevicius and Apse [56] intheir systematic review concluded that available data failed to give evidence that titaniumabutments are better at maintaining stable peri-implant tissues as compared to gold, alumi‐nium oxide and zirconium oxide abutments. The performance of zirconia vs titanium abut‐ments over long term is yet to be available. Recently, Zembic et al. [57] has published a 5-yearcomparison of the clinical performance of both titanium and zirconia abutments, and theyfound no statistically and clinically relevant difference between the survival rates, andtechnical and biological complication of these two abutment types.4.2. Surface modificationsSurface modifications of titanium dental implants or implant abutment are performed toimprove the biological, chemical and mechanical properties of implants. Over the years,specific surface properties such as topography, structure, chemistry, surface charge andwettability have been investigated to help enhance the soft tissue attachment. Commonly, thesurface modification can be broadly classified into modification of physical properties of thesurface or chemical properties of the surface. In the subsequent paragraphs, the surfacemodifications of titanium dental implant/abutment are divided into surface topography andsurface/chemical composition of the material. The surface topography of the implant can bealtered in many ways. However, the methods of surface modifications of dental implant arenot discussed since they are not within the scope of this chapter.4.2.1. Surface topographyDifferent materials exhibit different surface energy. The differences in surface free energy mayreflect their wettability characteristics. The higher the hydrophilicity of the material, the betteradhesion of the cells thus enhancing the attachment formed by these cells [58]. Improving thesurface texture with various techniques, thus altering the surface chemistry also enhances thewettability of certain material. Modification of surface texture will create different surfacetopography of dental implant material including abutment materials. Analysis of surfacetopography can be obtained from scanning profilometer (Figure 5) or SEM (Figure 6) in whichDimension and Structures of Biological Seal of Peri-Implant Tissueshttp://dx.doi.org/10.5772/6395049 the surface details can be visualized three dimensionally. The definition of surface roughnessof dental implant has been proposed by Albrektsson and Wennerberg [59, 60]. This definitioncan be used for study of osseointegration or implant-soft tissue interface. Accordingly, thecharacterization of surface topography is shown in Table 2. The values of Sa were determinedby optical interferometry using Gaussian filters. There is a need to emphasize that Table 2shows a summary of several studies cited in this chapter.Figure 5. A light interferometry micrograph showing the surface topography of the four types of Ti surfaces. (a) Pol‐ished, (b) machined, (c) sandblasted, and (d) TiUnite. Scale bar: (a) 21.06–0.95 mm, (b) 21.65–2.15 mm, (c) 211.39–6.70mm, (d) 23.28–4.82 mm. (Reproduced with permission from [52]).Figure 6. Scanning electron micrographs of the four types of Ti surface topographies.(Reproduced with permissionfrom [52]).Dental Implantology and Biomaterial50 Characteristics  Roughnessvalue(Sa)Clinical Use Findings (cell behavior or soft tissue response)Monolayer studiesAnimal studiesHuman studies 3D OMMSmooth 0.0–0.4 μm Machinedsurface,abutmentsGenerally, twotypes of cell used(epithelial andgingivalfibroblastcells)No value ofsurface roughnessstatedMost studiescomparedpolished,sandblasted andplasma-sprayedtitanium surfacesNoted higheradhesion andproliferation ofboth cells onpolished titaniumsurface [34, 35]Surfacetopographyhasno influenceonsoft tissueattachment(epithelial andconnectivetissue).Connectivetissuefibres werefoundparallelboth atsmooth(turned)and at rough(acid-etched)abutments[42].Conflictingevidencefoundthatperpendicularfibres are infavour of moreporous implantsurface [13]Surfacecompared:turned,oxidized andacid etchedSoft tissue sealwas almost thesame for allsurfaces comparedThe length of thejunctionalepitheliumappearedhigher/longer onsmooth titanium(2.9 mm) thanfor rough surfaces(1.4–1.6 mm),and in reversefor the lengthof the connectivetissue [49]Surfacecompared:polished,machined,sandblastedand anodizedNo differentfoundin term ofcontourof soft tissueattachment [52,53].Minimallyrough 0.5–1.0 μm Turned implant,Osseotite™(dual acidetched)Moderatelyrough1.0–2.0 μm Tioblast™ andOsseospeed™,sandblasted andacid etched(SLA), TiUnite™(anodized)(most commonimplanttopography)Rough >2.0 μm Plasma-sprayed,hydroxyapatite-coatedTable 2. Implant surface roughness.Surface texture is known to influence epithelial cells and fibroblast attachment, although thereis no complete agreement in the literature on the exact effect. One report found no significantdifferences concerning soft tissue reactions between roughed or smoothed surface implant[13], whereas Cochran et al. [35] found that smooth surfaces were more favourable forepithelial cell proliferation, as the fibroblasts appear to attach and proliferate better on roughsurfaces. Simion et al. [61] reported that epithelial cells adhered and spread better on metallicDimension and Structures of Biological Seal of Peri-Implant Tissueshttp://dx.doi.org/10.5772/6395051 surfaces than on ceramic surfaces with well-organized focal contacts and pre-hemidesmo‐somes found on metallic surfaces, but not on porcelain and aluminium oxide.Brunete and Chehroudi [62] in their review have suggested that the micro-fabricated groovedsurfaces are able to inhibit epithelial downgrowth on implants depending on the dimensionof the grooves in vitro. Similarly, fibroblasts also exhibit contact guidance on grooved surfaces,although its shape in vitro differs from that found in vivo. Delgado-Ruiz and co-workers [63]noted that micro-grooved surfaces were able to induce transverse collagen fibre formation,thus supporting two studies [26, 64]. It is also important to include a study by Nevins et al. [26]who demonstrated that soft tissue in humans is attached mechanically by perpendicularcollagen fibre bundles on a micro-grooved pulsed laser surface.4.2.2. Surface compositionOver the years, many strategies have been explored to improve the biological seal of peri-implant tissue by changing the surface chemistry of dental implants and implant abutments.The surface chemistry of the materials may be altered by biological modification, or bychanging the chemical composition of the materials. As for biological modifications, methodsof surface modification available include adding or coating with biomimetic/bioactivesubstances such as fibronection or intergrin onto the surface with the aim of promoting cellularadhesion and controlling cell behaviour. Fibronectin is a glycoprotein present on cell surfaces,found in connective tissues, basement membranes, and extracellular fluids, and is known toplay a role in cell-to-cell and cell-to-substrate adhesion and enhances gingival fibroblastattachment. It is interesting to note that epithelial cells and fibroblasts have different affinitiesfor adhesive proteins of the extracellular matrix. Dean et al [65] noted that higher number offibroblasts bound to fibronection coated implant surface than epithelial cells, while gingivalepithelial cell binding on implant surface coated with laminin was higher in number thanfibroblasts [66, 67]. Collagen Type 1 was also used to modify surface chemistry as it was foundto improve initial fibroblasts attachment [68].The chemistry of material surfaces can also be altered by using element such as calcium ormagnesium coating. Hydrothermal treatment of titanium with CaCl2 or MgCl2 was found toenhance initial attachment of epithelial and fibroblasts cells, and may increase the quality ofthe soft tissue seal around dental implant [69]. In addition, surface chemistry of materials mayalso inadvertently altered by the presence of impurities, surface contamination and saliva. Aclean surface has a high surface free energy, while a contaminated one has a lower surfaceenergy.5. ConclusionThere is some controversy on the possible attachment of connective tissue fibres to implant,but current studies indicate the presence of a parallel orientation with no insertion of peri-implant connective tissue fibres. This difference in connective tissue attachment may affect theperi-implant tissue’s susceptibility to disease. The gold standard for evaluating the connectiveDental Implantology and Biomaterial52 tissue and epithelial attachment is assessing the histological section using various modes ofprocessing, staining and analyses. The 3D OMM mimicking the oral tissue is a promisingtechnique to be considered for evaluating the connective tissue attachment, yet the processingof the tissue/implant block is still similar to the tissue block obtained from animal/human. Thereaction of cells on biomaterials is affected by the surface topography and surface physico-chemistry of the materials. Various studies have shown that materials and surface modificationof dental implants influence cell behaviour and interaction. Some of documented data werelimited to cell response on the monolayer cell culture model and animal histological studies.Therefore, it is recommended that randomised controlled clinical trials are to be performed todetermine the effects of dental implant materials and surface modifications on the peri-implanttissues.Author detailsWen Lin Chai1, Masfueh Razali2* and Wei Cheong Ngeow3*Address all correspondence to: [email protected] Department of Restorative Dentistry, Faculty of Dentistry, University of Malaya, KualaLumpur, Malaysia2 Department of Periodontology, Faculty of Dentistry, Universiti Kebangsaan Malaysia(UKM), Jalan Raja Muda Abdul Aziz, Kuala Lumpur, Malaysia3 Department of Oro-Maxillofacial Surgical & Medical Sciences, Faculty of Dentistry, Uni‐versity of Malaya, Kuala Lumpur, MalaysiaReferences[1] Lindquist L, Carlsson G, Jemt T. A prospective 15‐year follow‐up study of mandibularfixed prostheses supported by osseointegrated implants. 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