1

SOFT TISSUE ATTACHMENT TO TITANIUM

IMPLANTS COATED WITH GROWTH
FACTORS

A report submitted to the University of Adelaide in partial

fulfilment of the requirements of the Degree of Doctor of
Clinical Dentistry (Periodontology)


Christopher William BATES BDS (Adel), MClinDent (Pros) (Lond)

9
Chapter 1.
A REVIEW OF THE STRUCTURE OF THE PERI-IMPLANT
SOFT TISSUES AND THE POSSIBLE IMPLICATIONS OF
COATING TITANIUM IMPLANTS WITH GROWTH
FACTORS ON SOFT TISSUE ATTACHMENT
CW BATES
1
, V MARINO
2
, PM BARTOLD
3

1
Post Graduate Student (Periodontology), School of Dentistry, University of Adelaide.
2
Research Assistant, Colgate Australian Clinical Dental Research Centre, School of
Dentistry, University of Adelaide.
3
Professor of Periodontology, Colgate Australian Clinical Dental Research Centre, School
of Dentistry, University of Adelaide.





10
1.1 INTRODUCTION
The process of osseointegration described by Brnemark (Brnemark et al 1969, 1977) and
Schroeder (Schroeder et al 1981) plays an integral role in dental rehabilitation. Since the first
observation 40 years ago, osseointegrated titanium implants have been used predictably in the
dental rehabilitation of fully edentulous patients. The application of dental implants has
evolved, and from the 1980s dental implants have been used increasingly in the treatment of
partially edentulous patients, with equal or better long-term success (Buser et al 1990, 1997,
Lekholm et al 1994, Behneke et al 2000, Bornstein et al 2005).
The surgical procedures for the placement of endosseous dental implants are based on the
original work by Brnemark and colleagues approximately 40 years ago. The two-stage
surgical procedure was originally advocated to obtain an optimal process of new bone
formation and remodelling after implant placement (Brnemark et al 1977). Osseointegration
and good long-term success was also found to be achievable with non-submerged implants
(Buser et al 1990, 1992, 1997, Ericsson et al 1997) with the added advantage of avoiding a
second surgical procedure (Buser et al 1999). Implant dentistry has evolved over the last 15
years and has benefited from significant progress made in associated treatment protocols and
the development of bone augmentation procedures (guided bone regeneration (GBR) and
sinus floor elevation) allowing for correction of alveolar bone deficiencies. Additionally,
improved osteophilic microtextured implant surfaces have been developed to accelerate
healing, significantly reducing treatment time.
Research and clinical focus in dental implantology in the last two decades has primarily
concentrated on the bone-to-implant interface of osseointegrated implants. The soft tissue
profile and seal around implants have been investigated to a much lesser degree. This interest
has been largely due to the fact that a successfully osseointegrated implant depends on
anchorage in bone and requires a direct bone-to-implant interface to provide long-term
support for a prosthesis.
11
Both bone and soft tissue integration onto dental implants are wound healing processes
whereby several stages of tissue formation and degradation are involved (Berglundh et al
2003, Abrahamsson et al 2004). Osseointegration is the result of the modelling and
remodelling of bone tissue that occurs after implant placement whilst the wound healing that
occurs following the closure of mucoperiosteal flaps during implant surgery results in the
establishment of a mucosal attachment (transmucosal attachment) to the implant. The
establishment of the mucosal barrier around the implant is characterised by the gradual shift
from a coagulum to granulation tissue followed by the formation of a barrier epithelium and
the maturation of the connective tissue (Berglundh et al 2007). Like natural teeth,
osseointegrated implants are transmucosal masticatory devices that penetrate the oral
mucosa with the periodontal and peri-implant tissues expected to perform a protective
function (Weber & Cochran 1998).

1.2 DENTOGINGIVAL JUNCTION
The importance of the dentogingival tissues and the various functional components of the
barrier properties are fairly well understood. The dentogingval junction, the interface between
the tooth and gingiva that plays a role in tissue homeostasis and protection of the underlying
periodontal soft and mineralised connective tissue from the oral environment, is an adaptation
of the oral mucosa that comprises epithelial and connective tissue components. The
epithelium is divided into three functional compartments, namely the oral, sulcular and
junctional epithelium whereas the connective tissue is divided into the superficial and deep
components (Nanci & Bosshardt 2006).
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Figure 1. Schematic illustration of the dentogingival junction (Image adapted from Padbury et al
(2003)).

1.2.1 Oral Epithelium
The oral epithelium is a keratinized, stratified squamous epithelium. Based on the extent to
which the keratin-producing cells are differentiated, the oral epithelium can be divided into
the following cell layers; the basal layer (stratum basale/germinativum), the prickle cell layer
(stratum spinosum), the granular cell layer (stratum granulosum) and the keratinised cell layer
(stratum corneum).
When cell nuclei are lacking in the outer cell layers, the epithelium is denoted as
orthokeratinized. Often however, the cells of the stratum corneum in the epithelium of the
human gingiva contain remnants of nuclei and thus are denoted as parakeratinized. In addition
to the keratin-producing cells which account for about 90% of the cell population, the oral
epithelium contains cells such as melanocytes, Langerhans cells (which function as antigen
presenting cells), Merkels cells (suggested to have a sensory function) and inflammatory
cells (only in an inflammation response).
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The cells of the basal layer are in contact with the basement membrane that separates the
epithelium and the underlying connective tissue. The basal cells possess the ability to undergo
mitotic cell division and therefore it is from the basal layer that the epithelium is renewed and
can be considered the progenitor cell compartment of the epithelium. When two daughter
cells have been formed by cell division, the adjacent older basal cell is pushed into the
spinous cell layer and starts to traverse the epithelium as a keratinocyte. It takes
approximately one month for a keratinocyte to reach the outer epithelial surface, where it is
shed from the stratum corneum.
There are no fibrous protein components of the epithelial extracellular matrix, and the non-
fibrous epithelial components include water and a variety of glycoproteins, lipids,
proteoglycans, and extensions of intercalated cell surface molecules (Bartold 1987). Gingival
epithelial cells are able to synthesize and secrete sulphated molecules that contribute to the
make-up of the intercellular cementing substance of gingival epithelium (Weibkin & Thonard
1981). Hyaluronan, and the proteoglycans decorin, syndecan and CD44 have all been
identified in human gingival epithelial intercellular spaces (Tammi et al 1990) and it has been
shown that gingival keratinocytes synthesize and secrete several proteoglycans containing the
glycosaminoglycan (GAG) heparan sulphate and other molecular species (Potter-Perigo et al
1993).

1.2.2 Oral Sulcular Epithelium
Clinically healthy oral sulcular epithelium is characterized by squamous epithelial cells
joined by tight junctions and an inconspicuous surface lacking distinct papillary formation.
Electron microscopy studies have shown that even though there are large quantities of
bacteria adhering to the tissues, no significant defence response is seen in healthy oral sulcular
epithelium (Vitkov et al 2005). Although they are both supported by the same connective
tissue the oral sulcular epithelium is different to the oral epithelium in that it is
nonkeratinized. This difference may be attributed to inflammation, because even in normal
14
circumstances the connective tissue associated with the dentogingival junction is slightly
inflamed (Nanci and Bosshardt 2006). Studies on monkey tissues have shown that a
meticulous regime of oral hygiene combined with antibiotic cover can halt the inflammatory
process leading to oral sulcular epithelium keratinization (Bye et al 1980, Caffesse 1980).

1.2.3 Junctional Epithelium
Basement membrane structures separate the underlying CT from the gingival epithelium,
endothelial cells in blood vessels and the surrounding nerves. These basement membrane
structures are similar in composition to other basement membranes with type IV collagen and
laminin being the two major components. An internal basal lamina lies in between the
epithelium and tooth surface and acts as an interface through which the junctional epithelium
is attached to the root surface. The junctional epithelium plays a crucial role in sealing off
periodontal tissues from the oral environment and thus its integrity is essential for maintaining
a healthy periodontium. Periodontal disease sets in when the structure of the junctional
epithelium begins to fail (Nanci and Bosshardt 2006).
The junctional epithelium arises from the reduced enamel epithelium as the tooth arises
from the oral cavity, forming a collar that follows the cementoenamel junction. This
epithelium is a nondifferentiated, stratified squamous epithelium with a high rate of cell
turnover. The squamous cells are orientated parallel to the tooth surface and are derived from
a layer of cuboidal basal cells situated away from the tooth surface resting on a basement
membrane. The suprabasal cells have a similar ultrastructure and maintain the ability to
undergo mitosis. The cell layer facing the tooth provides attachment of the gingiva to the
tooth surface by a structural complex called the epithelial attachment (Nanci and Bosshardt
2006). The epithelial attachment consists of a basal lamina that adheres to the tooth surface
and the attachment of the superficial cell layer to the tooth surface and basement membranes
is mediated by hemidesmosomes. The basal lamina is a specialized extracellular matrix that
15
contains laminin 5, a matrix protein that mediates cell adhesion and regulates the polarization
and migration of keratinocytes (Frank and Carter 2004).
Cells of the junctional epithelium differ from those of the oral and sulcular epithelium in
that they contain more cytoplasm and organelles but have wider intercellular spaces with
fewer desmosomes and tonofilaments. The wider fluid filled intercellular spaces normally
contain polymorphonuclear leukocytes and monocytes that pass from the subepithelial
connective tissue through the junctional epithelium and into the gingival sulcus (Nanci &
Bosshardt 2006). These mononuclear cells and the junctional epithelial cells secrete
molecules such as - and -defensins, cathelicidin LL-37, interleukin (IL)-8, IL-1 and IL-1,
tumour necrosis factor alpha (TNF-), intercellular adhesion molecule-1 (ICAM-1), and
lymphocyte function antigen-3 (LFA-3) (Nanci & Bosshardt 2006). These secreted molecules,
as well as mononuclear cells, junctional epithelial cells, blood and tissue fluid make up the
first line of defence in the control of the constant microbial challenge.
The junctional epithelium can be considered an incompletely developed stratified
squamous epithelium and a structure that evolves along a different pathway, producing
components contributing to epithelial attachment rather than progressing further into a
keratinized epithelium (Nanci & Bosshardt 2006). The special nature of the junctional
epithelium is believed to reflect the fact the connective tissue supporting it is functionally
different than that of sulcular epithelium, a difference with important implications for
understanding the progression of periodontal disease and the regeneration of the dentogingival
junction after periodontal surgery (Nanci & Bosshardt 2006).

1.2.4 Connective Tissue
The subepithelial connective tissue layer, the lamina propria, is the principal tissue
component of the dentogingival complex. The gingiva is attached to the tooth surfaces and the
alveolar bone through fibrous attachments of the connective tissue and the subepithelial
16
connective tissue is thought to provide signals for the normal development of stratified
squamous epithelium (Karring et al 1971, 1975).
Collagen fibres are the predominant component in gingival connective tissue and constitute
the most essential component of the periodontium. These tend to be arranged in groups of
bundles with a distinct orientation. Circular fibres run their course in the free gingiva and
encircle the tooth in a cuff- or ring-like fashion, whereas the dento-gingival, dento-periosteal
and trans-septal fibres at some point embed themselves into the cementum of teeth.
One-tenth of gingival connective tissue volume is occupied predominantly by fibroblasts,
which are the cells responsible for producing the connective tissue elements in both normal
and diseased gingiva (Nanci & Bosshardt 2006). Other cells present in gingival connective
tissue are mainly derived from the blood and blood vessels. These include endothelial cells,
polymorphonuclear leukocytes (PMNs), macrophages, lymphocytes, plasma cells and mast
cells. These inflammatory cells are usually present in relatively small numbers in normal
healthy gingival CT, but their numbers increase in inflamed tissues, the relative proportions
dependent on the type and severity of the inflammation (Bartold & Narayanan 1998).
Gingival connective tissue has a high turnover rate with turnover rates of gingival and
periodontal ligament collagen higher than most other connective tissue types. This high
turnover rate of matrix components does not decrease significantly with age (Sodek & Ferrier
1988).
The boundary between the oral and sulcular epithelium and the subepithelial connective
tissue has a wavy course. The portion of connective tissue that project into the epithelium are
called connective tissue papillae and are separated from each other by epithelial ridges known
as rete pegs. In normal non-inflamed gingiva, rete pegs and connective tissue papillae are
lacking at the boundary between the junctional epithelium and its underlying connective
tissue. Therefore, a characteristic morphologic feature of the oral epithelium and the oral and
sulcular epithelium is the presence of rete pegs, whilst these structures are lacking in the
17
junctional epithelium. The basal lamina of the epithelium is invested into the underlying
connective tissue through anchoring fibrils containing type VII collagen.
The connective tissue supporting the junctional epithelium is structurally different in
another way from that supporting the oral epithelium in that even in clinically normal
conditions it shows an inflammatory cell infiltrate (Nanci & Bosshardt 2006). The connective
tissue adjacent to the junctional epithelium contains an extensive vascular plexus.
Inflammatory cells such as PMNs and T-lymphocytes continually extravasate and migrate
across the junctional epithelium into the gingival sulcus.

1.3 DENTAL IMPLANT / PERI-IMPLANT MUCOSA INTERFACE
1.3.1 Histology
The epithelial-tooth and epithelial-implant interface have many common features. The
results of a cell culture study by Gould et al (1981) wherein oral epithelial cells were grown
on epoxy resin discs with a thin film of titanium showed that epithelial cells attached to the
titanium surface by means of basal lamina and hemidesmosomes, similar to how epithelial
attachment occurs on the surface of a tooth. This cell culture experiment was followed by an
in vivo study with titanium coated epoxy resin discs implanted for 4 weeks into intrasulcular
incisions in the palatal and interproximal papilla on the palatal aspects of the left maxillary
second molar and premolars of a 40-year old male periodontal patient (Gould et al 1984).
Under scanning electron microscopy multilayers of epithelial cells were observed to have
formed against the surface of the titanium, exhibiting the characteristic interdigitation of cell
membranes and intercellular desmosomal attachments. There was clear evidence of
desmosomal attachment at the epithelial titanium interface and the appearance of a basal
lamina. The results of this study showed that the epithelial cells attached to titanium similar to
that observed in vitro and the way that the epithelium attaches to the tooth in vivo. An analysis
of machined surface commercially pure titanium implants in function for up to 7 years and
subsequently trephined from patients indicated that epithelial cells were regularly observed to
18
form a tight collar around the titanium implant and that hemidesmosomes attached the
bordering epithelial cells to the implant (Hansson et al 1983).

1.3.2 Animal and Human Studies
Further studies in man as well as in different animal models have investigated the mucosa
that surrounds titanium dental implants (Berglundh et al 1991, 1992, 1994, 2003, 2007; Buser
et al 1992, Ericsson et al 1996, 1997; Abrahamsson et al 1996, 1997, 1998, 1999, 2002, 2004;
Berglundh & Lindhe 1996, Cochran et al 1997, Moon et al 1999, Glauser et al 2005,
Schpbach & Glauser 2007, Welander et al 2007, 2008; Allegrini J r et al 2008, Nevins et al
2008). In an early study using a beagle dog model, Berglundh et al (1991) compared the
gingiva around teeth and the mucosa around two-stage implants (Branemark System, Nobel
Biocare, Gothenburg, Sweden). Abutment connection was carried out 3 months after implant
placement followed by 2 months of healing and then another 8 week plaque control period
before biopsies of the implant site and contralateral premolar tooth region were harvested. It
was found that the peri-implant mucosa consisted of a keratinized oral epithelium forming a
continuous 2 mm long barrier/junctional epithelium and a zone 1-1.5 mm high where
connective tissue was in direct contact with the titanium oxide (TiO
2
) layer of the implant,
described as a zone of connective tissue integration. They stated that the main difference
between the mesenchymal tissues present at a tooth surface and at an implant site is the
occurrence of cementum (acellular or cellular) on the root surface.
It has been shown histologically that the epithelial structures and the surrounding lamina
propria of dental implants cannot be differentiated from those structures around teeth.
Undecalcifed sections from rough surface titanium implants with a polished collar indicated
that the epithelial structures show a peri-implant sulcus with a non-keratinized sulcular
epithelium and a junctional epithelium. In the supracrestal area, direct connective tissue
contact to the implant was observed. The connective tissue in the zone of integration exhibited
a low density of blood vessels but a large number of fibroblasts and collagen fibres appearing
19
to originate from the periosteum of the bone crest and extend towards the margin of the soft
tissue in a direction parallel to the surface of the abutment (Buser et al 1992). However,
unlike the junctional epithelium around teeth which originally arises from the reduced enamel
epithelium, the junctional epithelium around implants originates from the epithelial cells of
the oral mucosa (Bosshardt & Lang 2005). Structurally, from the histological findings
previously mentioned, the peri-implant epithelium closely resembles the junctional epithelium
around teeth. Abrahamsson & Soldini (2006) found that in healthy conditions, both the
periodontal probing characteristics of the peri-implant and periodontal tissues were similar
and that the probe extension corresponded to the extension of the barrier epithelium with the
distance between the probe tip and bone being approximately 1 mm for both the tissue types.
Functionally, Berglundh et al (1992) found that the masticatory mucosa around teeth and
implants reacted in a similar fashion to early plaque formation and that the inflammatory
response in terms of polymorphonuclear leukocyte transmigration in the junctional epithelium
of the peri-implant mucosa and gingiva were almost identical.

1.3.3 Mucosal Barrier at Single- and Two-Stage Implant Abutments
The mucosal barrier established following both two-stage and single-stage approaches has
been found to be similar in terms of compositions and dimensions of their epithelial and
connective tissue components (Abrahamsson et al 1996, 1999, Ericsson et al 1996, 1997).
However, it has been recognised that the second surgical procedure in a two-stage implant
approach could reduce or eliminate the amount of keratinised mucosa in the peri-implant
tissues (Han et al 1995, Tinti & Parma-Benfenati 1995). Although the need for keratinised
mucosa around dental implants remains a debated issue, it is generally accepted that having an
adequate band of keratinised mucosa is desirable as the thickness of keratinized tissue around
an implant may determine the soft tissue response around dental implants to either mucosal
recession where the mucosa is of a thin biotype or the formation of a peri-implant pocket in
areas where the mucosa is of a thick biotype (Zigdon & Machtei 2008).
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Warrer et al (1995) demonstrated in a non-human primate model that the absence of
keratinised mucosa around dental implants increases the susceptibility of the peri-implant
region to plaque induced periodontal destruction. Chung et al (2006) showed that mucosal
inflammation and plaque accumulation were significantly higher around implants where the
thickness of the keratinised mucosa was less than 2 mm and/or the attached mucosa was less
than 1 mm.
Garcia et al (2008), found that although there were no significant differences in terms of
probing pocket depths, gingival health and plaque retention between single-stage and two-
stage implants, there was a greater tendency of single-stage implants to retain a band of
keratinised mucosa, indicating possible benefits of one implant surgical protocol over another.
Abrahamsson et al (1996) also observed when comparing the mucosal barriers of one- and
two-stage implants that when the mucosa of the alveolar ridge is thin, angular defects
occurred at the marginal border of the implants but that the dimensions of the mucosal
attachment at sites with a thin mucosa were similar to that at implant sites where the mucosa
was thick. Abrahamsson et al (1996) thus suggested that a certain width of the peri-implant
mucosa is required to enable a proper epithelial connective-tissue attachment and that if this
soft tissue dimension is not satisfied, bone resorption will occur to ensure the establishment of
attachment with an appropriate biological width. This hypothesis was corroborated by
Berglundh & Lindhe (1996) who found that if the mucosa was thin (2 mm) prior to abutment
connection, wound healing consistently included bone resorption and the establishment of an
angular defect. It was found that the minimal soft tissue dimensions to prevent bone
resorption was a junctional epithelium of 2 mm to 2.1 mm in length and supracrestal
connective tissue 1.3 mm to 1.8 mm in height, further reinforcing the earlier results of
Berglundh et al (1991).



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1.3.4 Vascular Supply of the Peri-Implant Mucosa
More detailed analyses of the soft tissue/implant interface using transmission electron
microscopy found that the zone of connective tissue directly adjacent to the implant surface
has a large number of round and flat-shaped fibroblasts with their long axes parallel with the
implant surface but virtually no blood vessels. Further away from this zone the number of
fibroblasts decreases but there are more collagen fibres and there is an increase in vascularity
(Moon et al 1999, Abrahamsson 2002). The vasculature of the gingiva and supracrestal
connective tissue at teeth is derived from the supraperiosteal vessels lateral of the alveolar
process and the vessels of the periodontal ligament. The vascular supply of the peri-implant
mucosa however, is derived solely from the supraperiosteal blood vessels due to a lack of a
periodontal ligament at the implant site. In both the gingiva and peri-implant mucosa, the
blood vessels lateral to the junctional epithelium form a characteristic crevicular plexus
(Berglundh et al 1994).

1.3.5 Morphogenesis of the Peri-Implant Mucosa
Berghlundh et al (2007) recently studied the histological morphogenesis of the mucosal
attachment to titanium implants in a canine model employing a single-stage implant
placement protocol. 2 hours after surgery, a coagulum was observed between the mucosa and
implant and also between the mucosa and alveolar process. After 4 days, the blood clot had
been infiltrated by numerous neutrophil granulocytes and an initial mucosal seal had been
established by the clustering of leukocytes in a dense fibrin network. The initial mucosal seal
was still present after one week although the area occupied by the leukocyte-infiltrated fibrin
tissue had decreased and was confined to a marginal portion of the soft tissue interface. The
apical part of the mucosal interface was dominated by fibroblasts and collagen. A week later,
the peri-implant mucosa was seen to adhere to the implant surface by connective tissue rich in
cells and vascular structures and the first signs of a junctional epithelium were also observed.
At 4 weeks after surgery, the junctional epithelium was formed and occupied 40% of the
22
mucosal surface and the connective tissue was well organised with a large proportion of
collagen and fibroblasts. Bone remodelling resulted in a distinct crestal bone portion at a
position about 3 mm apical to the soft tissue margin. The formation of the junctional
epithelium was completed at 6 to 12 weeks after surgery. At this stage a dense layer of
fibroblasts was seen to form a connective tissue interface with the titanium and these
fibroblasts were interposed between thin collagen fibres parallel to the implant surface.
From this study it can be seen that the establishment of the mucosal barrier around the
implant is characterized by the gradual shift from a coagulum to granulation tissue followed
by the formation of a barrier epithelium and the maturation of the connective tissue.
Berglundh et al (2007) thus concluded that the peri-implant mucosa exhibited minor signs of
inflammation during the first 2 weeks of healing but from 4 weeks, the mucosa was stable and
well attached to the bone and that the soft tissue barrier adjacent to titanium implants placed
using a non-submerged protocol takes about 6 to 8 weeks to establish the proper dimensions
and tissue organisation.

1.4 INFLUENCE OF IMPLANT SURFACE CHARACTERISTICS ON SOFT TISSUE
INTEGRATION
There is no doubt that the peri-implant tissues form a crucial seal between the oral
environment, the bone and the implant surface (Cochran et al 1994, 1997). This seal is fragile
and when subject to bacterial or mechanical challenge, due to the absence of periodontal
ligament fibres, the destruction of peri-implant tissue can be a faster and more devastating
process than in periodontal tissue (Salcetti et al 1997, Maksoud 2003). A number of studies
have examined the changes in soft tissue levels after implant placement (Bengazi et al 1996,
Ekfeldt et al 2003, Grunder 2000). Despite significant differences in experimental designs, the
majority of studies conclude that gingival recession varying between 0.6 mm to 1.5 mm is
unavoidable. While multiple factors can influence gingival recession around transmucosal
implants, there is little doubt that the low level of connective tissue attachment to implant
23
surfaces is important (Rompen et al 2006). Thus, enhancing the seal formed by the peri-
implant soft tissues, especially that of the titanium/connective tissue interface, may be an
important factor in implant survival and success. Various methods have been proposed to
improve the quality of the soft tissue interface including micro and macro design features of
the transmucosal portion of the implant (Glauser et al 2005).

1.4.1 Influence of Material Type
Most studies looking at the effect of implant material type on soft tissue integration to date
have been in vitro studies investigating epithelial and fibroblast attachment to different
materials such as titanium alloys, gold, aluminium oxide and dental ceramics and animal
studies investigating soft tissue attachment to abutments made from the abovementioned
materials and non-titanium implants (Rompen et al 2006). Whether the type of material had
an influence on soft tissue integration was investigated in a canine model where gold alloy
abutments were connected to Branemark System (Nobel Biocare AB, Gothenburg, Sweden)
implants at second-stage surgery (Abrahamsson et al 1998). Smaller soft tissue dimensions,
soft tissue recession and increased bone level height reductions were observed when
compared to titanium alloy abutments after a 6-month healing period. More recently,
Welander et al (2008) also using a 2-stage protocol on 4 implants (Osseospeed, Astra Tech
Dental, Mondal, Sweden), but placing the abutments after only one month, found that
titanium and zirconium oxide abutments induced a favourable soft tissue healing response but
gold alloy abutments failed to establish appropriate soft tissue integration. The use of a gold
abutment resulted in an apical shift of the junctional epithelium and the crestal bone during
the soft tissue healing process. It was also observed that decreased amounts of collagen and
fibroblasts and increased proportions of leukocytes were present in the connective tissue
adjacent to gold abutments (Welander et al 2008). However, Abrahamson & Cardapoli (2007)
in a canine model using custom machined surface implants that had separate titanium and
gold portions (Straumann AG, Waldenburg, Switzerland), effectively creating a one-piece
24
implant and single-stage protocol, found that both soft tissue dimensions and marginal bone
level were similar at implants designed with the transmucosal part made of gold or titanium.
Recently, Tete et al (2009) investigated peri-implant mucosa and evaluated the collagen fibre
orientation around the necks of 10 dental implants with machined titanium necks and 20
implants with zirconia necks 3 months after insertion in a porcine model. For the
osseointegrated implants under scanning electron microscopy and polarised light microscopy,
the collagen fibres of the peri-implant mucosa showed a parallel or parallel-oblique
orientation to the implant surface for all samples regardless of the material.

1.4.2 Effect of Surface Topography
The surface topography of implants can be altered by a number of surface treatment
processes; machining/micromachining, particle blasting, hydroxyapatite plasma spraying,
chemical/electrochemical etching, and anodization. The resulting topographical features
obtained on the implant surface can range from nanometres (below cell-size) to millimetres
(tissue-size) (Rompen et al 2006). The titanium/connective tissue interface, certainly for
smooth, machined surface dental implants, lacks the mechanical attachment of inserting
collagen fibres unlike that of periodontal tissues of teeth (Schpbach & Glauser 2007,
Welander et al 2007). Up until recently most dental implants were designed such that the
transmucosal portion of the implant was of a smooth or polished nature. These design
concepts have recently changed, with several implant designs allowing crestal placement and
incorporating roughened surfaces into the coronal portion of the body of the implant up to the
level of the implant-abutment platform (eg. Nobel Replace, Straumann Bone-level, Astra
Osseospeed). Whether the lack of mechanical attachment at the titanium/connective tissue
interface differs for roughened surface implants has not been extensively investigated.
Abrahamsson et al (2002) compared the composition of the soft tissue barriers to implant
abutments with a machined surface with abutments with a dual, thermal acid-etched surface
using a canine model over a 6-month period. They found that the roughness of the titanium
25
surface did not influence the soft tissue attachment that formed on commercially pure titanium
in terms of the dimensions of the epithelial-connective tissue barrier and the composition of
the connective tissue attachment, with the inner zone of the connective tissue attachment at
both types of abutments was composed of about 30-33% fibroblasts and 63-66% collagen.
Furthermore, in a similar experimental model, Zitzman et al (2002) found that the roughness
of the titanium abutments did not influence plaque formation or the inflammatory response of
the peri-implant mucosa. In a recent animal trial, Allegrini J r et al (2008) compared the soft
tissue integration in the neck area of two implant types from the same manufacturer (Nobel
Biocare AB, Nobel ReplaceTapered Groovy and ReplaceSelect Tapered), inserted with
the shoulders at ridge crest level after placement . These two implant types were similar in
design except for the thread pitch and that one implant type had a smooth machined collar and
the other a microgrooved, roughened TiUnite surface. Under polarised light microscopy, the
histological observations of both implant types indicated that there was no difference in terms
of sulcus depth and junctional epithelium attachment and that collagen fibre orientation was
similar for both implant types, running in a direction from the periosteum and alveolar crest
towards the oral epithelium (Allegrini J r et al 2008).
Some clinicians however, have advocated that roughened implant surfaces may in fact be
conducive to very good soft tissue adherence in dehiscence type defects and as such
placement of implants into dehiscence type defects may not always require osseous grafting
procedures to correct these defects (Dragoo, personal communication). The composition of
the protein film and the orientation of the molecules that are absorbed on the implant surface
may be influenced by the surface roughness of the titanium (Rompen et al 2006). In an in
vitro study, Di Iorio et al (2005) evaluated the fibrin clot extensions on machined surface and
aluminium-oxide blasted/acid-etched titanium disks, and found that an improvement in
microtexture complexity determines the formation of a more extensive and three-
dimensionally complex fibrin scaffold. Recent in vivo studies provide evidence that
microtexturing of the implant surface can be used to control the soft tissue response (Glauser
26
et al 2005, Schpbach & Glauser 2007, Nevins et al 2008). The influence of surface
modifications on interactions between the implant surface on both the junctional epithelium
and connective tissue was evaluated in a human study using one-piece experimental mini-
implants (Nobel Biocare AB, Gothenburg, Sweden) with either a machined surface, acid-
etched surface or a surface with an oxidised and microporous TiO
2
layer (Glauser et al 2005,
Schpbach & Glauser 2007). There was shorter epithelial attachment and a longer connective
tissue seal with the acid-etched and oxidised implants compared to the machined surface
implants (Glauser et al 2005). Furthermore, it was found that with machined and acid-etched
mini-implants, the adherence of the junctional epithelium to the implant surface was
characterised by a basal lamina and numerous hemidesmosomes but the interface between the
connective tissue and the implant surface was smooth, with collagen fibres running a course
more or less parallel to the implant surface, indicating poor mechanical resistance. However,
with the microtopographically complex oxidised implant surface, the junctional epithelium
exhibited attachment by hemidesmosomes together with mechanical interdigitation of the
innermost cell layer with the open pores of the implant surface, with the connective tissue
showing functionally oriented collagen fibrils towards the implant surface under polarised
light microscopy, indicating a less vulnerable seal (Schpbach & Glauser 2007). More
recently, Nevins et al (2008), employing a single-stage protocol using implants with Laser-
Lok microchannels at the collar (Biohorizons Implant Systems, Birmingham AL, USA)
observed under light microscopy that the junctional epithelial cells were in close contact with
the implant surface and that the microgrooved area of the implants were covered with
connective tissue. Polarized light and scanning electron microscopy of the microgrooved area
showed functionally oriented collagen fibres running toward and attaching to the grooves of
the implant surface (Nevins et al 2008). The Laser-Lok microchannels consist of precise,
three-dimensional microstructures that are formed by a computer-controlled laser ablation
technique. The rationale and dimensions of the microchannels were based on an earlier series
of in vitro studies whereby the effect of microgrooved surfaces were investigated with respect
27
to the attachment, spreading, orientation, and growth of fibroblast and osteoblast cell types
(Soboyejo et al 2002, Ricci et al 2008, Grew et al 2008). Soboyejo et al (2002) observed
using mouse calvarial cells that cells on titanium alloys with a microgroove 8 to 12 m deep
undergo contact guidance and limited cell spreading and more recently it was found that rat
fibroblast cells grown on microgrooved surfaces were well aligned and elongated in the
direction parallel to the grooves (Ricci et al 2008) and that these cells had profoundly altered
cell morphologies with respect to cytoskeletal and attachment proteins (Grew et al 2008).

1.4.3 Effect of Surface Modification
Epithelial cells and fibroblasts have different affinities for adhesive proteins of the
extracellular matrix and hence surface modification of titanium implants may improve the
ability of the epithelial and connective tissue components in the peri-implant mucosa to attach
to the implants. Currently, many dental implants incorporate a roughened surface as part of
their macro design. Many of these surfaces are able to absorb proteins and thus act as either a
reservoir or carrier for attachment proteins, growth factors or other biological agents which
may be of assistance for soft or hard tissue integration.
Coating machined, plasma-sprayed and hydroxyapatite titanium surfaces with fibronectin
and laminin-1, a component of epithelial cell basement membranes, have been observed in
vitro to enhance gingival fibroblast and epithelial cell attachment respectively by about
threefold (Dean et al 1995). Coating titanium alloy with laminin-5 has also been observed to
enhance gingival epithelial cell attachment and hemidesmosomes assembly in vitro (Tamura
et al 1997). Other in vitro studies have shown that cell adhesion to titanium discs coated with
collagen was enhanced in comparison with uncoated titanium (Roessler et al 2001, Nagai et al
2002), with type IV collagen shown to provide an excellent substrate for epithelial cell
attachment to titanium surfaces (Park et al 1998). However, in a recent study investigating
soft tissue healing around implants in a canine model, it was found that vertical dimensions of
the epithelial and connective tissue components as well as the composition of the connective
28
tissue zone directly adjacent to the implant were similar at collagen-coated and non-coated
implants after 4 and 8 weeks of healing (Welander et al 2007).
To date there have been few studies investigating the effect of surface modification with
growth factors such as enamel matrix derivatives (EMD) but none with platelet-derived
growth factor (PDGF) on the connective tissue attachment to titanium implants. A number of
previous studies have investigated the effects of PDGF and EMD on bone healing around
dental implants. Whilst PDGF has been shown to be influential in improving the regeneration
of peri-implant bone (Lynch et al 1991b, Becker et al 1992, Meraw et al 2000), the use of
EMD does not appear to contribute to the amount of bone-to-implant contact around titanium
implants (Franke Stenport & J ohansson 2003, Cangini & Cornelini 2005). More recently, a
pilot study conducted on a minipig reported on the effects of autogenous periodontal cell
grafts (periodontal ligament and gingival connective tissue cultures), with and without the
application of EMD, on the implant-connective tissue interface (Craig et al 2006). It was
proposed that a periodontal connective tissue attachment could be formed on dental implants
provided a source of periodontal regeneration competent cells was present in the wound
healing environment and that the application of EMD might aid in the formation of this
attachment. However, in this pilot study, with and without the application of EMD, an
implant-connective tissue interface morphologically consistent with a periodontal connective
tissue attachment was not observed in sections from any of the implant or autogenous cell
grafts (Craig et al 2006).

1.5 GROWTH AND DIFFERENTIATION FACTORS
Growth factors are proteins that may act locally or systemically to affect the growth and
function of cells in a number of ways; by promotion of fibroblast proliferation, pre-
osteoblast/osteoblast proliferation, extracellular matrix synthesis, mesenchymal cell
differentiation and vascularisation (Cochran & Wozney 1999). Growth factors may have an
autocrine effect but more commonly they have a paracrine effect, such that the production of
29
a growth factor by one cell type affects the function of a different cell type. These factors may
control the growth of cells and hence the number of available cells, and they can also control
the metabolism of a particular cell type. This therefore can influence the rate of production of
extracellular matrix components such as collagen. Differentiation factors control the
phenotypic state of cells, causing precursor cells to become fully functional mature cells of a
particular type.
A number of wound healing events and cellular activities (particularly during the
demolition phase and the formation of granulation tissue, organization, and contraction phase)
are controlled by growth factor polypeptides. Therefore, it is logical to utilize the potential of
growth factors to promote periodontal (Caffesse & Quinones 1993, Giannobile 1996) and
peri-implant regeneration. Several growth factors, as single agents or in combinations have
been investigated for their periodontal regenerative potential in animal and clinical studies.

1.6 PLATELET-DERIVED GROWTH FACTOR (PDGF) AND INSULIN-LIKE
GROWTH FACTOR (IGF)
Platelet-derived growth factor (PDGF) is secreted locally during clotting by blood platelets
at the site of soft- or hard-tissue injury and it stimulates a cascade of events that lead to the
wound healing response. PDGF has also been shown to influence periodontal ligament
fibroblast migration, proliferation and synthetic activity (Matsuda et al 1992, Dennison et al
1994, Boyan et al 1994). PDGF is one of the bodys main initiators of healing in injury to soft
tissues and hard tissues, stimulating a cascade of events that leads to the wound healing
response. It is a protein naturally found sequestered in blood platelets as well as bone matrix
in 3 different forms: PDGF-AA, PDGF-AB, and PDGF-BB (Ross et al 1986). While it was
originally identified in platelets, many cell types have been identified to release PDGF. The
primary effect of PDGF is that of a mitogen, initiating cell division. The PDGF-BB form in
particular is a potent stimulator of many types of connective tissue cells including periodontal
ligament (PDL) fibroblasts, cementoblasts and osteoblasts (Lynch et al 1989, Piche & Graves
30
1989). It promotes rapid cell migration (chemotaxis) in to the site of injury with subsequent
proliferation (mitogenesis) of the PDL fibroblasts and osteoblasts by binding to well
characterized cell surface receptors (Lynch et al 1989, Pinche & Graves 1989).
PDGF has been characterized as a competence factor in studies using fibroblastic cell
types. A competence factor is classically a growth factor that makes a cell competent for cell
division and a progression factor such as insulin-like growth factor-1 (IGF-1) or
dexamethasone is then necessary to induce mitosis (Cochran & Wozney 1999). Hence, with
these cell types the growth factors function in a synergistic manner. However, other cell types
such as osteoblasts are able to proliferate in response to PDGF alone (Graves et al 1989) and
isolated PDL cells have been found to behave in a similar fashion (Oates et al 1993).
IGF-1 and IGF-2 are mitogenic polypeptide growth factors that in fibroblastic systems
appear to be progression factors. In bone cell systems, IGF stimulate both proliferation of pre-
osteoblasts as well as the differentiation of osteoblasts, including type I collagen synthesis
(Baylink et al 1993). IGF therefore increases the number of bone synthesizing cells and the
amount of extracellular matrix deposited by each cell.

1.6.1 Animal and Human Studies using PDGF and IGF
The first studies published to show an effect of growth factors in relation to periodontal
regeneration used a combination of PDGF-BB and IGF-1 to treat naturally occurring
periodontal defects in beagle dogs (Lynch et al 1989, 1991a). Following surgical access and
debridement of the root surface, the roots were treated with a combination of PDGF-BB and
IGF-1 in an aqueous methyl cellulose gel. Control was application of the aqueous gel alone.
The authors observed increased cellular activity after applying PDGF-BB, leading to
significant cementum and bone deposition and the formation of a physiologic PDL space.
When used around implants, direct application of PDGF-BB in combination with IGF
produced 2 to 3 times more new bone at 7 days than controls in a canine model (Lynch et al
1991b). However, at 21 days, although significant amounts of new bone were found around
31
the dental implants treated with growth factors, there was no significant difference found
between the growth factor and control sites. This suggests that the control sites had enough
time to form bone in sufficient quantities and that the use of PDGF/IGF only accelerates the
process. Promising results were also seen in immediate extraction socket implants treated
with PDGF-BB/IGF in combination with a non-resorbable membrane whereby bone density
and bone-to-implant contacts were increased two times for sites treated with the growth
factors compared with sites treated with the membrane alone or membrane combined with
demineralized freeze-dried bone when analysed histologically after a healing period of 4.5
months (Becker et al 1992).
From these early studies, it was important to determine whether similar results with growth
factors could be obtained in a non-human primate model. Rutherford et al (1992) in a study
using 3 cynomolgus monkeys (Macca fascicularis) investigated the healing following the
surgical implantation of PDGF-AA/IGF-1, PDGF-BB/IGF-1 or a 3% methylcellulose control
gel in ligature-induced periodontitis. Using histological analysis they found that both forms of
PDGF appeared to stimulate one half of the new reattachment, including bone formation in
septal areas with horizontal bone loss. In a subsequent study using 4 monkeys and 8
interproximal sites, a combination of PDGF/dexamethsone in a collagen matrix carrier was
placed in debrided lesions of experimental periodontitis (Rutherford et al 1993). These were
then assessed histologically after 4 weeks and it was observed that the lesions treated with
PDGF/dexamethasone/collagen matrix induced a 5-fold increase in new cementum and PDL
and a 7-fold increase in supracrestal bone formation compared to the controls of lesions
treated with collagen matrix or root debridement alone.
Giannobile et al (1994) compared the regeneration following surgical implantation of
PDGF/IGF-1 into natural periodontitis defects in canines and ligature-induced periodontitis
defects in non-human primates. Positive results occurred in the two models after one month,
with the osseous response being greater in the canine model than in the primate model and
new attachment was more substantial in the primate model than the canine model.
32
PDGF/IGF-1 implantation resulted in a 64.1% and 51.4% increase in new attachment and a
21.6% and 65% osseous fill in the primate and canine models respectively. Interestingly the
primate controls showed a much higher new attachment formation (34.1%) compared to the
canine model with the naturally occurring periodontitis (8.6%). It was also subsequently
found that in a study in 10 cynomolgus monkeys that on its own, IGF-1 did not significantly
alter wound healing (Giannobile et al 1996). PDGF-BB on its own significantly stimulated
new attachment (70% new attachment) and it also resulted in an increase in bone formation
but not to a significant degree. The combination of PDGF/IGF-1 resulted in significant
increases in new attachment (75% new attachment) and bone formation (43% osseous fill).
These studies indicated that these agents would be suitable for use in human clinical trials.
Howell et al (1997) published the results of a human clinical trial involving 38 subjects
whereby a combination of PDGF-BB/IGF-1 in a methylcellulose carrier gel was applied to
osseous periodontal defects. A significant increase in alveolar bone formation was observed in
the sites treated with PDGF-BB/IGF-1 when compared to the control sites at 9 months post
operatively. The sites treated with PDGF-BB/IGF-1 showed an increase of 2.08 mm in new
vertical bone height as compared to a 0.75 mm new bone height increase in sites treated with
open flap debridement.

1.6.2 Animal and Human Studies using PDGF-BB
More recently, PDGF-BB has been combined with tissue-specific scaffolds such as bone
grafts or bone substitutes. A clinical trial investigated the effect of surgically implanted
PDGF-BB mixed with demineralized freeze-dried bone allograft for the treatment of
interproximal intrabony defects and class II furcation defects associated with severe
periodontitis (Camelo et al 2003, Nevins et al 2003). Nine-month post surgical results show
significant improvements in clinical parameters. A mean probing depth reduction of 6.42 mm,
a mean clinical attachment level (CAL) gain of 6.17 mm, and a mean radiographic bone
height gain of 2.14 mm were observed for intrabony defects treated with PDGF-BB and bone
33
allograft. A mean probing depth reduction of 3.4 mm and a mean CAL gain of 4.0 mm were
observed with class II furcation defects treated with PDGF-BB and bone allograft. Evaluation
of histological biopsies showed the formation of new bone, cementum and PDL coronal to a
reference notch placed at the time of treatment in the base of the calculus in both the
interproximal and class II furcation defects.
Recently a growth-factor enhanced matrix (GEM) has become available for clinical use.
This graft material has consists of a concentrated solution of pure recombinant human
platelet-derived growth factor (rhPDGF-BB), and an osteoconductive (bone scaffold) matrix
composed of -tricalcium phosphate. Marketed as GEM 21S (BioMimetic Inc, USA), it is the
first available purified, recombinant (synthetic) growth factor product (Lynch et al 2006). A
large multi-centre, randomized blinded human clinical trial of 180 participants investigated
the effectiveness of PDGF-BB with a porous -tricalcium phosphate (TCP) matrix (Nevins et
al 2005). The subjects had at least one interproximal periodontal defect 4 mm after
debridement and were divided into three treatment groups: Group 1 -TCP plus 0.3 mg/ml
rhPDGF-BB (GEM 21S); Group 2 -TCP plus 0.1 mg/ml rhPDGF-BB; and Group 3 -
TCP and buffer alone. At 3 months post surgically, GEM 21S showed a significantly greater
CAL gain than the -TCP alone but at 6 months, although the CAL gain for GEM 21S
continued to be greater than the -TCP alone, this was found not to be statistically significant.
GEM 21S however did show significantly less gingival recession at 3 months when compared
to the -TCP alone. The differences between Group 1 and Group 2 were not deemed to be
significant. Radiographic assessment at 6 months indicated that the linear bone growth and
percentage bone fill was significantly higher for the GEM 21S group when compared to
Group 2 and the group with -TCP alone. A subgroup analysis indicated that treatment with
rhPDGF-BB (Groups 1 and 2) improved bone fill in smokers and for all defect types (1, 2, 3-
wall and circumferential). Recent follow-up results for patients from centres participating in a
24 month evaluation of sites treated in the initial clinical trial demonstrated that the
significantly enhanced results in sites treated with 0.3 mg/ml rhPDGF-BB (GEM21S), which
34
were observed at the initial 6 month period, continued to improve and remained significantly
improved over results seen in the -TCP control group. The follow-up results indicated that
there was a continued increase in bone fill in treated defects and that there were increasing
levels of radiopacity and patterns of trabeculation (McGuire et al 2006, Nevins et al 2007).
Simion et al (2006) has also recently used rhPDGF-BB with a bovine derived xenogenic
scaffold to study periodontal bone regeneration in surgically created severe alveolar ridge
defects using a canine model. A rhPDGF-BB infused deproteinized bovine cancellous bone
block was placed in the defect site and stabilised with two titanium implants. The effect of
rhPDGF-BB, with and without a bilayer collagen membrane placed between the periosteum
and the bone graft block, was investigated and compared with untreated bone graft blocks
implanted with the collagen membrane. The rhPDGF-BB infused matrix significantly
enhanced bone formation and gingival healing in large, critical sized alveolar bone defects.
Histological and radiographic analyses indicated that the greatest bone regeneration occurred
with the rhPDGF-BB infused bone graft block without the interstitial membrane as the
membrane appeared to inhibit penetration of the graft by osteogenic cells. The rhPDGF-BB
appeared to exert a potent chemotactic effect when there was direct access to the periosteum
and its rich supply of osteogenic cells.
Hence, improvement in periodontal wound healing has been observed after applying
PDGF-BB, leading to significant bone, cementum and periodontal ligament regeneration
(Lynch et al 2006). The results from a case series recently published further illustrate the
beneficial effect of PDGF on both soft and hard tissue healing and potential in promotion of
periodontal and peri-implant regeneration (McGuire & Scheyer 2006). McGuire and Schyer
(2006) evaluated rhPDGF-BB plus -TCP and a collagen membrane with a coronally
repositioned flap and compared the results with those obtained with a subepithelial connective
tissue graft with a coronally repositioned flap in patients with recession type defects.
Statistical comparisons were not made due to the limited number of cases treated and the
small differences in clinical results in this feasibility study. Nevertheless, both procedures
35
predictably achieved root coverage with patients having no more than 1 mm of residual
recession at the end of 6 months. This case series provided proof-of-principle for successful
treatment of periodontal recession-type defects with rhPDGF-BB plus -TCP and a collagen
membrane without the need for autogenous tissue harvested from a second surgical site. This
finding, as well as the other animal and clinical studies concerning rhPDGF-BB, indicate that
it is capable of simultaneously promoting wound healing, regeneration of bone, and
acceleration of gingival attachment in periodontal and peri-implant defects and therefore may
be of particular relevance to the above stated clinical observations that implants can be placed
into dehiscence defects without the need for osseous correction.

1.7 TRANSFORMING GROWTH FACTOR (TGF) AND FIBROBLAST GROWTH
FACTOR (FGF)
Other growth factors may have potential for regeneration of periodontal and peri-implant
tissues. TGF- is a multifunctional growth factor synthesized by many cell types and
generally it increases extracellular matrix formation, such as type I collagen, and leads to an
increase in fibrosis (Cochran & Wozney 1999). It has been shown to be chemotactic for bone
cells and may increase or decrease their proliferation, depending on the state of differentiation
of the cells, culture conditions and the concentration of TGF- (Bonewald & Mundy 1990). In
vivo, TGF- has been shown to produce new cartilage and/or bone when injected in proximity
to bone, but it does not induce bone formation when implanted away from a bony site (Beck
et al 1991). There have not been any data reported on in vivo healing with TGF- in a
periodontal situation.
FGF has general growth-promoting effects on most fibroblastic cell types and it stimulates
angiogenesis, wound healing and cell migration. Studies on individual cell types indicate FGF
can stimulate endothelial and PDL cell migration and proliferation (Terranova et al 1989). It
has been shown to increase bone formation and accelerate the rate of fracture repair. Some of
36
these effects may be mediated through increases in TGF-, but there are no in vivo data
available for the use of FGF in periodontal repair (Cochran & Wozney 1999).

1.8 BONE MORPHOGENETIC PROTEINS (BMP)
The only other growth factors studied in detail in humans are bone morphogenetic proteins
(BMP). BMP are a group of 20 to 30 related differentiation factors of the TGF- superfamily
that appear to be intimately associated with epithelial-mesenchymal signalling (Bartold &
Narayanan 1998). Each of the BMP molecules is unique biochemically and biologically and
the BMP family of proteins sequestered in bone includes several subfamilies. BMP-2 and
BMP-4 are closely related molecules with more than 90% amino acid identity. BMP-5, BMP-
6 and BMP-7 (also called osteogenic protein 1) make up a subgroup that share approximately
70% amino acid identity with BMP-2 and BMP-4 (Lynch et al 2008).
In vivo studies have documented the ability of BMP to induce bone formation (Wozney
1995) and BMP are the only known factors that are capable of inducing bone formation at
extraskeletal sites, causing the differentiation of cells derived from the soft tissue into bone-
producing cells (Reddi 1981). Additionally, BMP are also required for the embryonic
development of many organs and tissue types, including the skeleton and teeth. These factors
make BMP a candidate for the regeneration of alveolar bone and regeneration of other aspects
of periodontal and peri-implant tissue formation.
BMP-3, the most abundant protein in the purified bone-inductive extract, shares about a
50% amino acid identity with other BMP (Lynch et al 2008). However, an early study
indicated that osteogenin (BMP-3) did not have a stimulatory effect in the healing of
periodontal defects (Bowers et al 1991).
BMP-2 and BMP-4 are expressed within the thickened layers of the budding odontogenic
epithelium and later these two molecules are expressed by the underlying mesenchyme
(Bartold & Narayanan 1998). BMP-2 has been characterised as a bone inductive molecule,
because it is a single bioactive factor demonstrating the same inductive activity present in
37
bone and bone extracts. A large number of studies, mainly using canine and non-human
primate models, have been carried out to evaluate BMP-2 combined with various scaffolds
and carriers for periodontal regeneration, alveolar augmentation and dental implant
osseointegration.

1.8.1 Animal Studies using BMP
Ripamonti and co-workers (1994) found that the use of BMP-2 significantly increased
alveolar bone healing in surgically created class II furcation defects in 4 baboons (Papio
ursinus). BMP-2 has also been found to significantly increase bone and cementum formation
in surgically created periodontal defects in a canine model, when used in a carrier gel or
underneath barrier membranes (Sigurdsson et al 1995, 1996). Kinoshita and co-workers
(1997) reported on BMP-2 induced periodontal regeneration of ligature induced horizontal
circumferential defects in a canine model. Considerable new bone formation was observed in
rhBMP-2 treated sites and new cementum with Sharpeys fibres was observed on the
instrumented root surfaces. Root resorption was a rare finding and ankylosis was not observed
in BMP or control sites. The results from this study indicate that suitable application of
rhBMP-2 can produce considerable periodontal tissue regeneration, even in cases of
horizontal circumferential defects.
The combination of BMP-2 with other growth factors has also been used synergistically to
improve tissue regeneration. Using a canine model, Meraw et al (2000) investigated the
effects of a combination growth factor cement (GFC) containing BMP-2, TGF-, PDGF and
FGF in a bioabsorbable, non-hydroxyapatite, calcium phosphate cement on guided bone
regeneration in circumferential defects around the coronal portion of machined surface
titanium dental implants. It was found that the use of GFC significantly increased the bone-to-
implant contact and the amount of bone per surface area compared to plain cement and no
cement. The authors concluded that in order to address the complexity of the required cellular
38
events and interactions that normally occur in the healing process, combinations of growth
factors may be of extended benefit over use of single growth factors in early bone healing.
Since these initial studies, the research focus using BMP-2 has shifted towards alveolar
ridge augmentation when used as an inlay or onlay graft biomaterial (Sigurdsson et al 1997,
2001; Wikesjo et al 2004, J ovanovic et al 2007) and in sinus augmentation techniques
(Nevins et al 1996, Hanisch et al 1997).

1.8.2 Reosseointegration with BMP
It has also been shown that BMP-2 could be useful in promoting reosseointegration of peri-
implantitis defects around titanium implants (Hanisch et al 1997). Hanisch et al (1997), using
a non-human primate model, surgically debrided ligature-induced peri-implantitis defects
created over 11 months around hydroxyapatite-coated titanium implants and placed BMP-2 in
an absorbable collagen sponge carrier. This was compared to control defects that received a
buffer with the collagen sponge carrier. Histometric analysis following a 16-week healing
interval showed that vertical bone gain was 3-fold greater in BMP-2 treated sites than in
control sites, suggesting that the use of BMP-2 may have the potential in the regeneration of
peri-implantitis defects and thus the regeneration of peri-implant soft tissues.

1.9 ENAMEL MATRIX PROTEIN DERIVATIVES (EMD)
The biologic concept behind enamel matrix-induced periodontal regeneration is based on
the discovery that enamel matrix proteins are not only are involved in enamel formation, but
also play a key role in the formation of the root and attachment apparatus. EMD, mainly
amelogenins, secreted during tooth root development by Hertwigs epithelial root sheath play
a crucial role in the formation of acellular root cementum (Slavkin and Boyde 1975, Slavkin
1976, Lindskog 1982a, 1982b; Lindskog & Hammarstrom 1982, Brookes et al 1995, Fong et
al 1996) and acellular cementum is the most important tissue for the insertion of collagen
fibres. These proteins are thought to induce the formation of the periodontal attachment
39
during tooth formation and it is believed that EMD used in periodontal lesions mimic the
development of the tooth supporting apparatus (Hammarstrm 1997).
Following the formation of the tooth crown, an extension of the dental organ, Hertwigs
epithelial root sheath begins to grow in an apical direction from the cervical area to form the
mould for the root and to interact with the surrounding mesenchymal tissues to initiate root
formation. The root sheath is a double-layered structure, the inner layer of which is an
extension of the ameloblastic layer in the crown. The ameloblasts synthesize and secrete the
proteins of the enamel matrix during enamel formation. It has been shown that continuously
during root development and always at the mineralizing front of the dentine, the cells of
Hertwigs epithelial root sheath will again enter a secretory stage during which they will
deposit enamel matrix proteins onto the root surface (Slavkin et al 1988, Hammarstrm
1997). As the enamel matrix proteins precipitate onto the developing and mineralizing dentine
surface they will induce apoptosis in the cells of the root sheath, which will then separate and
disintegrate to form the epithelial cell rests of Malassez. Following separation and fenestration
of the root sheath through the apoptotic process, the enamel matrix proteins are exposed to the
mesenchymal cells of the surrounding dental follicle. The cells will be attracted to the matrix
surface and migrate through the fenestrations in the root sheath to colonize the root surface,
which is covered with enamel matrix proteins. The mesenchymal cells then express a
cementoblast phenotype and start forming collagen and root cementum. Subsequently and in
sequence, periodontal ligament and alveolar bone will form.
The data from in vitro investigations indicate that EMD affects important wound healing
processes, although the underlying molecules and mechanisms are still not completely
understood (Sculean et al 2007). A series of laboratory studies by Gestrelius et al (1997a,
1997b) investigating the effect of EMD on cell migration, attachment, proliferation,
biosynthesis activity and formation of mineralised tissue found that in in vitro conditions,
EMD promote the proliferation of periodontal ligament fibroblasts but not epithelial cells, and
that it increased total protein synthesis and formation of mineralised nodules by periodontal
40
ligament fibroblasts. EMD are considered to stimulate the proliferation of periodontal
ligament cells, enhancing alkaline phosphatase activity and activating the signalling pathway
for the secretion of growth factors in wounds (Gestrelius et al 2000, Lyngstadaas et al 2001).
Data from another in vitro study by Suzuki et al (2005) indicate that EMD may contain
additional mitogenic factors such as TGF- and BMP-like growth factors that can stimulate
fibroblast proliferation and contribute to the process of periodontal regeneration.
In the wound healing process, macrophages participate in both the destruction and repair
processes. It has been reported that macrophages have been found to produce osteoinductive
signals, including BMP-2 (Champagne et al 2002), as well as other growth factors associated
with osteoinduction such as TGF-1 and BMP-4 (Andrew et al 1994, Blom et al 2004).
Growth factors produced by macrophages are associated with wound healing and bone
formation. During the wound healing process of the periodontium, the switch from
inflammatory macrophages to wound healing macrophages may be important (Champagne et
al 2002). Moreover, there is some evidence indicating that the application of EMD can
change the inflammatory macrophages expressing IL-1, TNF- and IL-8 to wound healing
macrophages expressing BMP-2 and -4, resulting in proliferation of collagen fibres and new
bone formation (Myhre et al 2006, Fujishiro et al 2008).
The only commercially available EMD, Emdogain, is a purified acid extract of enamel
matrix proteins from developing porcine teeth. The material is a viscous gel consisting of the
enamel matrix proteins in a polypropylene liquid and is delivered by syringe to the defect site.
Ninety percent of the protein in this mixture is amelogenin, with the rest primarily proline-
rich non-amelogenins, tuftelin, tuft protein, serum proteins, ameloblastin and amelin.

1.9.1 Animal Studies using EMD
Hammarstrm et al (1997) investigated the effect of locally applied EMD and different
protein fractions of the matrix on periodontal regeneration in surgically created maxillary
buccal dehiscence in a non-human primate model. Segments of the maxilla containing teeth
41
with buccal dehiscences were removed en block together with adjacent teeth and alveolar
bone after 8 weeks of healing and prepared for light microscopic analysis. It was found that
using EMD it was possible to obtain regeneration of 60-80% of the periodontium with
acellular cementum and inserting collagen fibres and new alveolar bone. The only EMD
carrier that allowed the regeneration process to occur successfully was the propylene glycol
alginate carrier. In the control dehiscences, the healing was characterised by a long junctional
epithelium with little cementum and new alveolar bone formation. The little cementum
formed in the controls was mostly cellular and partly attached to the root surface.
Subsequent studies using murine, canine and non-human primate models comparing the
use of EMD alone, guided tissue regeneration (GTR) alone, EMD and GTR combined, and
open flap debridement surgery as control in surgically created dehiscence and intrabony
defects have led to similar histological results. Healing in control defects has been
characterised by a long junctional epithelium and little periodontal regeneration, whereas
treatment with EMD, GTR or a combination of both has resulted in the formation of
cementum with inserting collagen fibres as well as of alveolar bone (Sculean et al 2000,
Cochran et al 2003, Sakallioglu et al 2004, Sallum et al 2004, Nemcowsky et al 2006).

1.9.2 Human Studies using EMD in Intrabony Defects
Heijl et al (1997), in a randomised, multicenter study, compared the use of EMD with a
placebo in 33 patients with 34 paired test and control sites, mostly one-wall and two-wall
defects. The results after 36 months showed a mean CAL gain of 2.2 mm in the test group and
1.7 mm in the control (open flap debridement). A statistically significant radiographic bone
gain of 2.6 mm was also found.
In a histologic study of 10 treated intrabony defects in 8 patients, Yukna & Mellonig
(2000) reported evidence of regeneration (new cementum, bone and periodontal ligament) in
3 specimens, new attachment (connective tissue attachment, adhesion only) in 3 specimens
42
and a long junctional epithelium in 4 specimens, but no evidence of root resorption or
ankylosis was found.
Froum et al (2001) compared the treatment of deep intrabony defects by open flap surgery
with and without the application of EMD in a 12-month re-entry study. Fifty-three intrabony
defects were treated with EMD and 31 defects with open flap debridement alone in 46
patients. The defects were opened again to measure the defect fill after a 12-month healing
period. It was found that the additional use of EMD resulted in a threefold larger defect fill
than treatment with open flap debridement alone. A prospective, randomised multi-centre
clinical study investigated the treatment of 166 intrabony defects with a papilla preservation
technique, 83 defects with and 83 defects without the application of EMD (Tonetti et al
2002). After 12 months, the EMD group showed a significantly higher CAL gain than in the
control group.
More recently Bosshardt et al (2005) attempted to characterise the tissues developing on
the root surface at 2 to 6 weeks following treatment of intrabony defects with EMD. It was
found that following treatment with EMD, a bone-like tissue resembling cellular intrinsic
fibre cementum develops on the root surface, instead of the acellular extrinsic fibre cementum
as previously thought. Furthermore, EMD was found to induce de novo bone formation of a
mineralised connective tissue on debrided root surfaces and stimulate matrix deposition on
old native cementum. However, the tissue gap observed between the newly formed
cementum-like tissue and the treated root surface indicated that only after 6 weeks of healing,
the bond between the newly formed tissue and the root surface was weak (Bosshardt et al
2005).
A number of clinical studies have investigated the long-term outcomes of the treatment of
intrabony defects with EMD. Heden and Wennstromm (2006) carried out a prospective study
of 114 cases that were followed up at 1 year and then at 5 years. At one year, there was a
mean gain of clinical attachment of 4.3 mm followed by a further mean gain of 1.1 mm at the
5 years. This study demonstrated the long-term stability of the gain of clinical attachment
43
using Emdogain. However, prospective, controlled, split-mouth clinical studies evaluating
the treatment of intrabony defects with EMD or GTR after 8 years (Sculean et al 2006) and
EMD alone, GTR alone, EMD and GTR combined, and open flap debridement after 10 years
(Sculean et al 2008) found that after 1 year, the mean CAL gain was not significant.
Nevertheless, the results of these studies show that the clinical outcomes of EMD are able to
be maintained over 8 to 10 years.
From a large number of clinical studies reported in the literature, it appears that surgical
periodontal treatment of deep intrabony defects (particularly 2 and 3-wall defects) with EMD
promotes periodontal regeneration and that this regenerative procedure may lead to
significantly higher improvements in clinical parameters compared to open flap debridement
alone (reviewed by Sculean et al 2007).
Comparing periodontal regeneration using enamel matrix protein derivatives and the gold
standard of periodontal regeneration (GTR), a number of studies have compared the clinical
outcome of Emdogainversus a bioresorbable membrane in conjunction with GTR. In a
prospective, controlled clinical study of 40 patients treated surgically with either EMD, GTR
with a non-bioresorbable or 2 bioresorbable membranes, and compared to open flap
debridement, Pontoriero et al (1999) showed that all 4 procedures were equally effective and
significantly better than open flap debridement in terms of probing depth reduction and CAL
gain. Subsequent studies have indicated that there is no significant difference in the CAL gain
achieved between these two regenerative procedures (Sanz et al 2004, Sculean et al 2006,
2008). A prospective multicenter, randomised, controlled clinical trial of 75 patients with
advanced chronic periodontitis compared the clinical outcomes for EMD and GTR using a
bioresorbable membrane (Sanz et al 2004). The results from this study failed to show that one
modality was superior over the other but it was observed that surgical complications,
particularly membrane exposure was a frequent finding with the GTR procedure, whilst only
6% of EMD-treated sited showed any complications. Treatment of periodontal intrabony
defects with a combination of Emdogainand GTR does not seem to additionally improve
44
the outcomes compared to treatment with Emdogainalone or GTR alone (Esposito et al
2005, Sculean et al 2007, 2008). Moreover, the treatment of periodontal intrabony defects
with a combination of Emdogainand bone-graft biomaterial does not appear to additionally
improve the clinical outcomes or bone-formation when compared to using Emdogainalone
(Dori et al 2005, Sculean et al 2007, 2008). Hence, Emdogainsadvantage over GTR is that
it is more user-friendly and less technique-sensitive than GTR and potentially associated with
less morbidity to the patient than GTR which is associated with more frequent complications
with regards to infection and membrane exposure.

1.9.3 Human Studies using EMD in Furcation Defects
The application of Emdogainmay also enhance periodontal regeneration in mandibular
class II furcations, with the clinical results obtained comparable with those obtained with
GTR (J epsen et al 2004, Meyle et al 2004). J epsen et al (2004) in a multicentre split-mouth
design study over 14 months of 45 patients and 90 mandibular class II furcation defects,
found during re-entry surgery that treatment of the furcation defects with Emdogainreduced
the open horizontal furcation depth by an average of 2.8 mm and this reduction was
significantly greater than guided tissue regeneration using barrier membranes, with a lower
incidence of post-operative complications. Using a non-human primate model, Donos et al
(2003) histologically evaluated the healing of mandibular class III furcation defects following
treatment with EMD alone, GTR alone, EMD and GTR combined, or open flap debridement
alone. The results from this study showed that sites treated only with EMD showed new
attachment and bone formation to a varying extent, whereas sites treated with GTR or EMD
with GTR combined showed formation of cementum with inserting collagen fibres and new
bone where the membrane was not exposed. With the control sites treated with only open flap
debridement, only limited new attachment and bone formation was observed. It must be kept
in mind however, that treatment of furcation defects still produces unfavourable outcomes in
45
many degree II involvements, especially in the maxilla, and in degree III furcations.
Treatment approaches for these lesions are highly technique-sensitive.

1.9.4 Human Studies using EMD in Gingival Recession Defects
Apart from its original use as an agent to enhance and promote periodontal regeneration,
Emdogainhas also been reported to be effective in the management of recession-type
defects with coronally repositioned flaps by enhancing soft tissue adherence to exposed root
surfaces (Hgewald et al 2002, McGuire & Nunn 2003, McGuire & Cochran 2003,
Nemcovsky et al 2004, Spahr et al 2005, Castellanos et al 2006, Moses et al 2006, Sato et al
2006, Shin et al 2007). In fact, the results of the first human histological biopsy using EMD
involved the treatment of a surgically created recession defect on a lower incisor (Heijl 1997).
In a split-mouth designed clinical study, Hgewald et al (2002) compared the treatment of
buccal Millers class I and II recession defects with a coronally repositioned flap and EMD
versus a coronally repositioned flap alone. No difference in amount of root coverage obtained
between the therapeutic modalities were seen after 12 months but the additional application of
EMD did result in a significantly greater formation of keratinised tissue. This study was
followed up again in 12 months by Spahr et al (2005) who found that after 24 months
complete root coverage could be maintained in 53% of the coronally repositioned flaps when
EMD was used, compared to 23% with the coronally repositioned flap on its own. Root
coverage in 47% of the non-EMD group had deteriorated compared to only 22% in the EMD
group. These results were later corroborated by Castellanos et al (2006) who found a
significantly higher percentage of vertical root coverage and gain in keratinised gingiva after
12 months with the additional use of EMD with coronally repositioned flaps, when compared
to the flap procedure alone in treating Millers class I and II buccal recession defects.
In another controlled, clinical split mouth study treating Millers class I and II recession
defects with a coronally repositioned flap and EMD versus a coronally repositioned flap and a
subepithelial connective tissue graft, McGuire and Nunn (2003) found that 12-months after
46
therapy the mean value for root coverage was 95.1% with total root coverage being achieved
in 89.5% of cases for the EMD group. The mean value for root coverage was 93.8% for the
connective tissue group but total root coverage was achieved in only 79% of cases.
Histological evaluation of two biopsies showed that treatment of the recession defects with
the coronally repositioned flap and EMD resulted in the formation of root cementum, PDL
and alveolar bone whereas treatment with a coronally repositioned flap with a subepithelial
connective tissue graft was characterised by a long junctional epithelium and some signs of
root resorption (McGuire & Cochran 2003). Similar results but favouring the coronally
advanced flap with a subepithelial connective tissue graft was reported by Nemcovsky et al
(2004). In this multicentre, controlled clinical trial the mean root coverage of the coronally
repositioned flap procedure with the additional use of EMD was 71.7% after 12 months,
compared to 87% for the coronally repositioned flap with subepithelial connective tissue
graft. A longer follow-up of 24-months of the two treatment modalities by Moses et al (2006)
found that even though treatment with a coronally repositioned flap with a subepithelial
connective tissue graft yielded significantly better root surface coverage and gain in
keratinised gingiva, EMD application is an effective long-term alternative to achieve root
surface coverage together with a gain in height of keratinised gingiva.
Hence, the use of Emdogainin these situations may promote collagen synthesis, the
formation of cementum, periodontal ligament and bone and may therefore increase the width
of keratinised tissue. These findings may be of particular relevance to the above stated clinical
observations that implants can be placed into dehiscence defects without the need for osseous
correction. Whether an agent such as Emdogainwould enhance soft tissue adhesion to
exposed implant surfaces in dehiscence type defects remains to be established.




47
1.10 CONCLUSIONS
In health, the peri-implant mucosa around osseointegrated titanium dental implants exhibit
common features to non-inflamed gingival tissues around teeth. Titanium oxide does not
appear to significantly affect the formation of the epithelium or epithelial cell structures. The
epithelial components around titanium dental implants consist of a peri-implant sulcus with a
non-keratinised sulcular and junctional epithelium and therefore appear to be consistent with
epithelial components around teeth. However, the situation in the peri-implant mucosa differs
in that there is no periodontal ligament or cementum, thus influencing its blood supply and
connective tissue fibre alignment. The connective tissue component of the peri-implant
mucosa forms a minimally-vascularised, circular, scar-like structure around the implant
abutment or supracrestal portion of the implant surface, which is in turn surrounded by a less
dense, vascularised connective tissue that derives it blood supply solely from the
supraperiosteal blood vessels of the alveolar process. Due to the absence of cementum, the
main connective tissue fibres in the peri-implant mucosa, collagen fibres run parallel to the
long axis of the implant, unlike gingival fibres. The titanium/connective tissue interface
therefore lacks a mechanical attachment of inserting collagen fibres unlike that of teeth, and
as such, forms a weak barrier to bacterial challenge or mechanical trauma.
The introduction of microtextured titanium surfaces has gone some way in addressing this
problem with some recent reports claiming a functional reorientation of collagen fibres with
certain implant types.
Roughened implant surfaces are able to absorb proteins and act as a reservoir or carrier for
attachment proteins, growth factors or biological agents. Thus, surface modification of current
commercially available roughened surface implants by surface coating with a biological agent
is another strategy that could be employed to enhance attachment at the titanium/connective
tissue interface. The use of growth and differentiation factors in periodontal regeneration of
periodontal intrabony defects and enhancement of root coverage of recession defects has been
well documented. The two most widely used classes of growth and differentiation factors are
48
PDGF and EMD. When used in periodontal regenerative therapy, these factors can result in
the regeneration of cementum, periodontal ligament and alveolar bone, as well as potentially
increasing the width of keratinised, attached gingiva.
Therefore, the potential of enhancing attachment of the peri-implant connective tissues
through surface modification of roughened titanium dental implants already in widespread
clinical use by surface coating with growth and differentiation factors currently available on
the market presents a relatively unexplored field of periodontal research with practical clinical
implications.
49
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