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)

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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 BATES1, V MARINO2, PM BARTOLD3

1Post Graduate Student (Periodontology), School of Dentistry, University of Adelaide.

2Research Assistant, Colgate Australian Clinical Dental Research Centre, School of

Dentistry, University of Adelaide.

3Professor of Periodontology, Colgate Australian Clinical Dental Research Centre, School

of Dentistry, University of Adelaide.

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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.

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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

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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

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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

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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

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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

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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 Jr 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 (TiO2) 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

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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

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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

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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

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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

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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 Jr et al (2008) compared the soft

tissue integration in the neck area of two implant types from the same manufacturer (Nobel

Biocare AB, Nobel Replace Tapered Groovy and Replace Select 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 Jr 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

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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 TiO2 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 & Johansson 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, Jovanovic 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 Emdogain versus 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 Emdogain and GTR does not seem to additionally improve

44

the outcomes compared to treatment with Emdogain alone or GTR alone (Esposito et al

2005, Sculean et al 2007, 2008). Moreover, the treatment of periodontal intrabony defects

with a combination of Emdogain and bone-graft biomaterial does not appear to additionally

improve the clinical outcomes or bone-formation when compared to using Emdogain alone

(Dori et al 2005, Sculean et al 2007, 2008). Hence, Emdogains advantage 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 Emdogain may also enhance periodontal regeneration in mandibular

class II furcations, with the clinical results obtained comparable with those obtained with

GTR (Jepsen et al 2004, Meyle et al 2004). Jepsen 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 Emdogain reduced

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,

Emdogain has 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 Emdogain in 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 Emdogain would enhance soft tissue adhesion to

exposed implant surfaces in dehiscence type defects remains to be established.

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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-implan