Bio Film in Implantology

Original article of Dr.
Komagal
BIOFILM IN IMPLANTS
By: Dr.B.Komagal
0 Original article of Dr. Komagal |

Original article of Dr.Komagal
CONTENT
Introduction 2
Biofilm: nature and properties 4
Factors affecting biofilm development 9
Attachment of bacteria 12
Biocompatability 16
Material interaction with biological systems 17
Designing Biocompatable material 20
Instrumentation for analysis of biomaterial surfaces 22
Formation of biofilm in implant surface 26
Dental plaque in implant vs tooth surface 31
Biofilm an Surface roughness 35
Surface modifications- effecting P.gingivalis adherence 40
Titanium implant biomaterial: effecting LipopolySachharide(LPS) adherence 43
Clinical implications 46
Surface free energy 48
Chemical composition of surface 50
Implant abutment fit 51
Future directions 52
Conclusion 55
Reference 56
1
INTRODUCTION
Biofilms are fascinated structures and may be found virtually anywhere. Biofilm
consist of one or more communities of microorganisms, embedded in a glycocalyx, that
are attached to a solid surface. The function of biofilms depend on the ability of bacteria
and microcolonies within the biofilm to communicate with one another( Quarun sensing)
The oral environment is unique because it is the only area of the body where biomaterials
are routinely placed for long-term use in nonsterile environment. Colonization or oral
biomaterials by various microorganism is inevitable.
Dental implants are root analogues that are surgically placed into the jaw bone and
used to support crowns, bridges and dentures. The use of dental implant is now
widespread. The excellent biocompatibility of implant material results mainly from it’s
surface characteristics. Surface roughness and modifications, surface free energy,
chemical composition of surface, implant abutment fit are vital properties of surface
characteristics of implant prosthesis that creates an impact on the biofilm formation on
implant biomaterial.
Periimplantitis is an inflammatory process affecting the tissues around an

osseointegrated implant, resulting in the loss of supporting bone leading to implant
failure. The factors associated with peri-implantitis appear to be related to the
composition of the bacterial environment around an implant and the ability of bacteria to
adhere to implant material.
Hence the importance of biofilm in implant prosthetics is a field to facilitate the

development of new biologically based rational for treatment modalities in dentistry.
2
BIOFILM
Biofilms may be found virtually anywhere. They colonize a widely diverse set of
moist surfaces, including the oral cavity, the bottom of boats and docks, the inside of
pipes and rocks in streams. Infectious disease investigators are interested in biofilms that
colonize a wide array of artificial devices that have been implanted in the human,
including catheters, hip and voice prostheses and contact lenses. Biofilms consist of one
or more communities of microorganisms, embedded in a glycocalyx, that are attached to
a solid surface. The reason for the existence of a biofilm is that it allows microorganisms
to stick to and to multiply on surfaces. Thus, attached bacteria (sessile) growing in a
biofilm display a wide range of characteristics that provide a number of advantages over
single-cell (planktonic) bacteria.1
3
THE NATURE OF BIOFILMS
Biofilms are fascinating structures. They are the preferred method of growth for
many and perhaps most species of bacteria This method of growth provides a number of
advantages to colonizing species. A major advantage is the protection the biofilm
provides to colonizing species from competing microorganisms from environmental
factors such as host defense mechanisms, and from potentially toxic substances in the
environment, such as lethal chemicals or antibiotics. Biofilms also can facilitate
processing and uptake of nutrients, cross-feeding (one species providing nutrients for
another), removal of potentially harmful metabolic products (often by utilization by other
bacteria) as well as the development of an appropriate physicochemical environment
(such as a properly reduced oxidation reduction potential).
A crude analogy to the development of a biofilm might be the development of a

city. Successful human colonization of new environments requires several important
factors including a stable nutrient supply, an environment conducive to proliferation and
an environment with limited potential hazards. Cities (like biofilms) develop by an initial
"attachment" of humans to a dwelling site followed by multiplication of the existing
inhabitants and addition of new inhabitants. Cities and biofilms typically spread laterally
and then in a vertical direction, often forming columnar habitation sites. Cities and
biofilms offer their inhabitants many benefits. These include shared resources and
4
interrelated activities. Inhabitants of cities or biofilms are capable of "metabolic

processes" and synthetic capabilities that could not be performed by individuals in an
unattached (planktonic) or nomadic state. An important benefit provided by a city or
biofilm is protection both from other potential colonizers of the same species, from
exogenous species and from sudden harmful changes in the environment. Individuals in
the "climax community" of a flourishing city or biofilm can facilitate joint activities and
live in a far more stable environment than individuals living in isolation. Cities, like
biofilms, require a means to bring in nutrients and raw materials and to remove waste
products. In cities, these are usually roads, water or sewage pipes; in biofilms they may
be water channels such as those described below. Cities have maximum practical sizes
based on physical constraints and nutrient or waste limits; so do biofilms.
Cities that are mildly perturbed, such as by a snowstorm or a local fire, usually
reform a climax community that is similar to that which was present in the first place, as
do biofilms. However, major perturbations in the environment such as prolonged drought
or a radioactive cloud can lay waste to a city. Major perturbations in the environment
such as a toxic chemical can severely affect the composition or existence of a biofilm.
Communication between individuals in a city is essential to allow inhabitants to interact
optimally. This is usually performed by vocal, written or pictorial means. Communication
between bacterial cells within a biofilm is also necessary for optimum community
development and is performed by the production of signaling molecules such as those
found in "quorum sensing" or perhaps by the exchange of genetic information. The long-
term survival of the human species as well as a species in a biofilm becomes more likely
if that species (or the human) colonizes multiple sites. Thus, detachment of cells from
biofilms and establishment in new sites is as important for the survival of biofilm-
dwellers as the migration of individuals and establishment of new cities is for human
beings. Thus, we may regard mixed species biofilms as primitive precursors to the more
complex organizations observed for eukaryotic species.1
5
PROPERTIES OF BIOFILM
STRUCTURE
Biofilms are composed of microcolonies of bacterial cells (15–20% by volume)

that are non-randomly distributed in a shaped matrix or glycocalyx (75–80% volume).
Earlier studies of thick biofilms (>5 mm) that develop in sewage treatment plants
indicated the presence of voids or water channels between the microcolonies present in
these biofilms. The water channels permit the passage of nutrients and other agents
throughout the biofilm acting as a primitive "circulatory" system. Nutrients make contact
with the sessile (attached) microcolonies by diffusion from the water channel to the
microcolony rather than from the matrix. Other models of biofilms, however, have been
suggested, including the heterogeneous mosaic and the dense biofilm models.
Microcolonies occur in different shapes in biofilms which are governed by shear forces
due to the passage of fluid over the biofilm. At low shear force, the colonies are shaped
liked towers or mushrooms, while at high shear force, the colonies are elongated and
capable of rapid oscillation. Individual microcolonies can consist of a single species but
more frequently are composed of several different species.
Exopolysaccharides – the backbone of the biofilm
As mentioned above, the bulk of the biofilm consists of the matrix. It is composed
predominantly of water and aqueous solutes. The "dry" material is a mixture of
exopolysaccharides, proteins, salts and cell material. Exopolysaccharides, which are
6
produced by the bacteria in the biofilm, are the major components of the biofilm, making
up 50–95% of the dry weight. They play a major role in maintaining the integrity of the
biofilm and confer other beneficial properties. Bacteria can produce several different
polysaccharides depending on the physiological state of the bacteria and the presence of
specific substrates. All biofilms contain exopolysaccharides, although they can vary quite
markedly in both bacterial composition and the composition of the extracellular matrix.
Some exopolysaccharides are neutral, such as the mutan from Streptococcus mutans,
whereas others are highly charged polyanionic macromolecules. Different ionic charge
and concentrations of exopolysaccharides will alter the confirmation and cause rapid
changes in the three-dimensional gel network of polysaccharides. Similar effects may
also be produced by provision of sucrose or other sugars. The exopolysaccharides can be
degraded and utilized by bacteria within the biofilm. One distinguishing feature of oral
biofilms is that many of the microorganisms can both synthesize and degrade the
exopolysaccharides.
Exopolysaccharides can exist in both ordered or disordered forms. At high

temperatures and often at very low ionic concentrations, the disordered form
predominates, although few biofilms exhibit total absence of an ordered structure.
Biofilm matrices are complex structures that contain masses of fibers of varying size,
structure, composition and rigidity that interact with each other, with cells and with
surface matrices. A wide range of possible conformations, flexibility and configurations
can be expected among different classes of polysaccharides. The density of the fibrillar
masses will affect accessibility of both cells and surfaces to nutrients and other solutes. 1
The chemical composition and tertiary structure of the exopolysaccharides will

determine whether it forms an effective adhesive. It will also affect the hydrophilic or
hydrophobic nature of the surface. Exopolysaccharides aid in protecting microbial cells
within the biofilm by preventing desiccation and attack by harmful agents. They may
also bind essential nutrients such as cations to create a local nutritionally rich
environment favoring specific microorganisms. The exopolysaccharide matrix could also
act as a buffer and assist in retaining extracellular enzymes (and their substrates),
7
enhancing substrate utilization by bacterial cells. Exopolysaccharides are effective in

maintaining biofilm structure through the formation of networked, cross-linked linear
macromolecules. In most mixed biofilms, numerous types of polysaccharide are found,
complicating the network structure. The quantity of exopolysaccharides in a biofilm does
not necessarily reflect the proportion of the bacterial species that produce it. Loss or
removal of one type of exopolysaccharide may have a more drastic effect on the biofilm
matrix than another even if the removed polymer is not dominant.
Physiological heterogeneity within biofilms
Bacterial cells within biofilms can produce enzymes such as β-lactamase against
antibiotics or catalases, superoxide dismutases against oxidizing ions released by
phagocytes. These enzymes are released into the matrix, producing an almost
impregnable line of defense. Bacterial cells in biofilms can also produce elastases and
cellulases, which become concentrated in the local matrix and produce tissue damage.
Measurement of oxygen and other gases has demonstrated that certain microcolonies are
completely anaerobic even though composed of a single facultative species and grown in
ambient air. Carbon dioxide and methane can reach very high concentrations in specific
microcolonies in industrial biofilms. Thus, studies to date indicate that sessile cells
growing in mixed biofilms can exist in an almost infinite range of chemical and physical
microhabitats within microbial communities.
8
FACTORS AFFECTING BIOFILM DEVELOPMENT AND

BEHAVIOR
Hydrodynamics affect both the physical shear stress at a wetted surface and the
rate at which nutrients are transported to the surface of the biofilm. Both shear and mass
transfer can influence biofilm development. Biofilms grown under high shear are
generally thinner and denser than those grown under lower shear. Hydrodynamics can
also affect biofilm structure.
Biofilms can also be affected by changes in nutrient concentration. Stoodley et al.

demonstrated that adding nutrients to a biofilm increased both mass and structure. A ten-
fold increase in carbon and nitrogen concentration to a mixed species biofilm grown
under turbulent flow changed the structure from one of ripples and streamers to large cell
clusters. In addition, the thickness of the biofilm increased five-fold within 24 hours.
Return to the original nutrient levels resulted in an immediate loss of biomass and the
reappearance of ripples and streamers.
Detachment of cells from biofilms
The detachment of cells from biofilms is essential to allow colonization of new

habitats by bacteria. Detachment, however, is probably the least well understood biofilm
phenomenon. It appears from in vitro studies that cells detach in different fashions. Some
of these include the detachment of single cells in a continuous predictable fashion
(erosion), the sporadic detachment of large groups of cells (sloughing) or an intermediate
process whereby large pieces of biofilm are shed from the biofilm in a predictable
manner. The more predictable intermediate process results in detached clusters consisting
of about 104 cells. In vitro, the detachment rate was shown to be about six clusters per
mm2 of surface per hour. Unlike single cells, the detached cell cluster may be protected
from the host defense systems similar to the protection afforded the biofilm from which it
was shed. 1
9
The rate of detachment of bacteria from biofilms in the oral cavity is not clear. It
has been argued that the rate of growth in many biofilms grown in vitro may be very slow
and that detachment may be an uncommon event. The cells in such biofilms may be
metabolically active and capable of growth once released from the biofilm. Other
considerations suggest that the detachment of cells from intraoral biofilms may be an
active ongoing process. The mean total viable counts of bacteria in saliva average about
108 per ml. The total volume of saliva secreted per day is approximately 1500 ml,
suggesting that as many as 1.5×1011 bacteria are swallowed per day. These bacteria must
come from somewhere, either the biofilms growing on the teeth and soft tissues or
perhaps from some limited growth in saliva itself. Thus, the number of organisms in
saliva and the critical importance of transmission of bacterial species reinforces the
notion that detachment of bacteria from intraoral biofilms is an important event about
which too little is known. 1
Related to the phenomenon of detachment of biofilm cells in groups is the

possibility of bodily movement of biofilm structures en masse on solid surfaces. This
possibility has been demonstrated in in vitro studies of mixed biofilms that showed
movement of intact biofilm structures across solid surfaces while remaining attached to
them. This has implications for the colonization of surfaces by biofilms. The formation of
a biofilm is generally thought to be initiated by colonization of planktonic cells that
become attached and then multiply. The data of Stoodley et al. suggest that the
colonization of "preformed" biofilm structures can occur as these structures move to
adjacent areas. This may provide certain advantages in that formation of the biofilm is
not reliant on planktonic cells, which are known to be more susceptible to antimicrobial
agents.
10
Quorum sensing
Some of the functions of biofilms depend on the ability of the bacteria and
microcolonies within the biofilm to communicate with one another. Quorum sensing in
bacteria "involves the regulation of expression of specific genes through the accumulation
of signaling compounds that mediate intercellular communication" . Quorum sensing is
dependent on cell density. With few cells, signaling compounds may be produced at low
levels; however, autoinduction leads to increased concentration as cell density increases.
Once the signaling compounds reach a threshold level (quorum cell density), gene
expression is activated. Cell signaling has been studied extensively in luminescent
bacteria and appears to be mediated by an N-acyl homoserine lactone encoded by a lux1
gene. A similar system has been shown to exist in certain other gram-negative species.
The high cell concentrations in biofilms present an ideal situation for quorum sensing, as
even small microcolonies (<10 cells) may induce gene expression since the signaling
compounds may be concentrated within the microcolony and are not degraded. Quorum
sensing may give biofilms their distinct properties. For example, expression of genes for
antibiotic resistance at high cell densities may provide protection. Quorum sensing also
has the potential to influence community structure by encouraging the growth of
beneficial species (to the biofilm) and discouraging the growth of competitors. It is also
possible that the physiological properties of bacteria in the community may be altered
through quorum sensing. The possible role of quorum sensing in influencing the
properties of biofilms was first suggested by Cooper et al. 1
11
Signaling is not the only way of transferring information in biofilms. The high
density of bacterial cells growing in biofilms facilitates the exchange of genetic
information between cells of the same species and across species or even genera.
Conjugation, transformation, plasmid transfer and transposon transfer have all been
shown to occur in naturally occurring or mixed-species biofilms prepared in vitro. Of
particular interest was the demonstration of transfer of a conjugative transposon
conferring tetracycline resistance from cells of one genus, Bacillus subtilis, to a
Streptococcus species present in dental plaque grown as a biofilm in a constant-depth
film fermenter.
Attachment of bacteria
The key characteristic of a biofilm is that the microcolonies within the biofilm
attach to a solid surface. Thus, adhesion to a surface is the essential first step in the
development of a biofilm. In the mouth, bacteria can attach to a wide variety of surfaces,
including the oral soft tissues, the pellicle-coated teeth and other bacteria. Many bacterial
species possess surface structures such as fimbriae and fibrils that aid in their attachment
to different surfaces. Fimbriae (pili) are proteinaceous hair-like appendages 2–8 nm in
diameter composed of protein subunits called fimbrillins (fimbrins). Some fimbriae also
carry adhesins. Fimbriae have been detected on a number of oral species including
Actinomyces naeslundii, Porphyromonas gingivali, and some strains of streptococci such
as Streptococcus salivarius, Streptococcus parasanguis and members of the
Streptococcus mitis group. Examination of the fimbriae of oral strains indicate that they
12
are very thin, flexible and 2–3 nm in diameter, thus differing from the larger more rigid
fimbriae found on other bacteria such as Escherichia coli. Fibrils can also be found on a
number of oral bacterial species. They are morphologically different and shorter than
fimbriae and may be densely or sparsely distributed on the cell surface. Oral species that
posses fibrils include S. salivarius, S. mitis group, Prevotella intermedia, Prevotella
nigrescens and S. mutans.
As will be discussed later, A. naeslundii is one of the most important colonizing

species on tooth surfaces. This species, together with other Actinomyces, comprises a
major segment of the microbiota attached to the tooth and may be thought of as part of
the "scaffolding" structure of dental plaque. For this reason, the intense examination of
its means of attachment has been warranted. The fimbriae of A. naeslundii are the best
characterized of the gram-positive oral bacteria. They are about 3–4 nm wide and <1.5
μm long. Two major types of fimbriae have been identified although they cannot be
distinguished ultrastructurally from one another. Some strains of A. naeslundii carry
both, whereas others carry only one. Type 1 fimbriae are associated with adhesion of A.
naeslundii to salivary acidic proline-rich proteins and to statherin deposited within the
salivary pellicle. Type 2 fimbriae are associated with attachment of A. naeslundii to
glycosidic receptors on epithelial cells, polymorphonuclear leukocytes and oral
streptococci. This lectin-like adhesion to these substrates is inhibited by galactose and N-
acetyl galactosamine. 1
The best characterized fimbriae of the oral gram-negative bacteria are those of P.
gingivalis. Three types of fimbriae have been identified that are up to 3 μm long and 5
nm wide, the major class of which is composed of fimbrillin. Fimbriae appear to be the
major adhesion-mediating determinants of P. gingivalis. The fimbrillin polypeptide
binds proline-rich proteins, statherin, lactoferrin, oral epithelial cells, oral streptococci,
A. naeslundii, fibrinogen and fibronectin. The fimbriae of P. gingivalis have chemotactic
properties and demonstrate cytokine induction, both of which are necessary for P.
gingivalis to invade epithelial cells and endothelial cells. Recent studies have suggested
13
the presence of fimbrial-like structures on Peptostreptococcus micros, although their role

in adhesion, if any, has yet to be elucidated.
Factors other than fimbriae or pili are important in the initial attachment of other
bacterial species. Force-generating movement is thought to be an important first step in
biofilm formation by gramnegative bacteria such as E. coli, P. aeruginosa,
Pseudomonas fluorescens and V. cholerae. Active motility due to the production of
flagella or twitching motility due to type IV pili are thought to increase the number of
initial interactions between bacterial cells and solid surfaces and to help to overcome
initial repulsive forces between bacteria and the surface. Mutants that do not possess
flagella or type IV pili show delayed biofilm formation or do not produce biofilms. This
mechanism is not an absolute requirement by all taxa, since gram-positive species such
as those that predominate on tooth surfaces are not motile.
Although fimbriae or pili are important for the attachment of certain species to
solid surfaces, they are not the only means of initial attachment. Cell surface proteins of
Staphylococcus epidermidis and Caulobacter crescentus are important in the initial
attachment of these species to solid surfaces, and a capsular, polysaccharide adhesin of
S. epidermidis can also mediate the attachment of this species to solid surfaces.
Recent studies have suggested that the adhesins involved in initial attachment to
solid surfaces are in many instances different from the molecules involved in forming a
multi-layered structure. While one set of proteins mediates attachment of S. epidermidis
to solid surfaces, a second set of proteins or polysaccharide mediate cell-to-cell
attachment in building a 3 dimensional structure. Similarly, E. coli adheres to surfaces
by means of Type 1 fimbriae, but the development of a complex three-dimensional
structure appears to be due the production of an exopolysaccharide, colanic acid.
Biofilm as a community
Bacteria within dental plaque do not exist as independent entities but rather
function as a coordinated, metabolically integrated microbial community. This
14
community life-style within dental plaque provides enormous potential benefits to the
participating organisms including:
 A broader habitat range for growth (the metabolism of early colonizers alters the
local environment, making conditions suitable for attachment and growth of later
more fastidious species)
 An increased metabolic diversity and efficiency (molecules that are normally

recalcitrant to catabolism by individual organisms can be broken down by the
microbial consortia)
 An enhanced resistance to environmental stress, antimicrobial agents, and the host

defences.
Recent studies suggest that the environmental heterogeneity generated within

biofilms promotes accelerated genotypic and phenotypic diversity that provides a form of
'biological insurance' that can safeguard the 'microbial community'. This diversity can
affect several key properties of cells, including motility, nutritional requirements,
secretion of products, detachment, and biofilm formation.
15
BIOCOMPATIBILITY
The consensus definition of biocompatibility is “the ability of a material to

perform with an appropriate host response in a specific application. 9
The biomaterial is “a nonviable material used in a medical device, intended to

interact with biological systems”.
Materials designed with an emphasis on biologic response centered on coating

medical polymer surfaces is being introduced called bioactive materials: introducing
implants materials that have osteogenic potential.
Technological surface : is defined as a surface whose properties and behavior predict its
function in service. Primarily produced via surface coating of industrial materials.
3 interactive and dynamic components are involved in designing material or biologic

systems:
 Chemical nature of biomaterial surface

 Mediating pellicle layer
 Microbial and host cellular response
16
MATERIAL INTETRACTION WITH BIOLOGICAL

SURFACES
CHEMICAL NATURE OF THE BIOMATERIAL SURFACE
Synthetic material placed in mouth is exposed to salivary protein rich solution

from which selected molecules will be absorbed onto its surface adsorption is via
physical forces that reduce the interfacial free energy of biomaterial.
Each material has its own intrinsic adsorptive properties which is based on
chemical composition. The unique surface energy profile is determined by nature and
packing of atoms or group of atoms at the surface. 9
Molecules are absorbed to a surface by physical forces which includes hydrogen

bonding, electrostatic interactions. hydrophobic interactions ore believed to be the major
adsorptive force, thermodynamically driven as a result of a gain in entroply when
structured water at a surface is displaced by an absorbing protein.
Selective adsorption of protein at interfaces is uniquely determined both by

surface composition of material and composition of proteins in solution. This was
elegantly demonstrated in a study by Horbett and Schwag in which large changes in
protein adsorption (and subsequent cell attachment and spreading) were observed when
either serum concentration or substrate surface chemistry was altered.
MEDIATING PELLICLE LAYER
17
The fluids coming in contact with natural and artificial oral surfaces are
predominantly compromised of saliva, serum transudates such as gingival fluids,
microbial and host cell constituents.
A dental implant placement can be surrounded by mixture of plasma and cell

matrix proteins. These fluids consist of heterogenous mixture of macromolecules, such as
proteins, glycoproteins, lipids, and electrolytes (ions) which are selectively absorbed onto
different surfaces to form an acquired pellicle. Significantly differences were found in
amino acids compositions between pellicles collected from natural teeth and plastic film
applied to tooth surfaces in the same individual.
Individual variation in salivary content of specific proteins may also dictate the
composition of in vivo-pellicle. High molecular weight salivary mucin is found as a
component of in vivo enamel and in vivo denture pellicles, and both mucins are found in
vivo cementum pellicles. Salivary cystatin has been detected in vivo enamel pellicle but
not in vivo denture pellicle.
Selective macromolecules also show a pattern of selective adsorption to metal

surfaces used as implant materials.
Albumin, prealbumin, immunoglobuling have been identified as proteins

adsorbed to titanium oxide surfaces.
Albumin and fibrinogen have been reported to adsorb to titanium alloys.
18
Thus the acquired pellicle can serve as a dynamic mediator or effector system that
regulates interactions or interfacing of biomaterials with surrounding tissues.
These pellicles can be considered dynamic systems, and they appear to undergo
continual remodeling either by chemical or enzymatic modification of adsorbed
molecules or through complexing with additional molecules. Remodeling processes may
be mediated by host, or microbial enzymes, environmental pH, or ionic concentration.
Tissue components involved in these interactions can be derived from adhesions or
receptors of host cell and microbial origin. Since pellicle formation is the outcome of
chemical nature of biomaterial surface, biocompatible materials are designed to alter
composition of biological pellicle, which is achieved by using entirely new materials,
modifying the surface of existing materials, or coupling specific biologic molecules to the
surface of a material (a designed pellicle). 9
MICROBIAL AND HOST CELL RESPONSE
The oral environment is unique in that it is the only area of the body where
biomaterial are routinely placed for long-term use in a nonsterile environment.
Colonization of oral biomaterial and oral surfaces by various microorganisms is routine.
Most oral bacteria display distinct tropisms for colonizing specific oral surfaces. Its
determined by compositional differences in pellicles. Streptococcus sanguis,
streptococcus mutans, and actinomyces viscos preferentially colonize tooth surfaces,
where as streptococcs salivorius and candida albicons mainly colonize the tongue
dorsum. Staphylococcus epidermidis preferential colonize polymer biomaterials, and
staphylococcus aureus is frequently the major pathogen cultured from metal implants
associated with bone, joint and soft tissue infections. Significantly lower numbers of
A.viscosus were found to bind to saliva coated titanium than to saliva coated enamel, this
suggest that microbial colonization may differ-on implant surfaces.
These correlation provide evidence for the fact that bacteria possess a recognition
system that is capable of interacting with specific molecules on a biomaterial surface.
Receptor adhesion interactions based on stereochemical specifity are believed to be most
19
critical in bacterial adhesion to oral pellicles. Bacteria possess protein-like surface

structures known as adhesions that bind to specific glycoprotein or protein receptors
found as a part of the biomaterials pellicle. Conversely, pellicles themselves contain
ligands that serve recognition system for microbial receptors. A multitude of specific
adhesion receptor pairs have been reported. Among these are salivary praline-rich
proteins with A.viscosus, fibronectin with porphyromonos gingivalis, and salivary
mucins or amylase with viridans streptococci. 9
DESIGNING BIOCOMPATIBLE MATERIAL
SURFACES CAN BE MODIFIED TO ALTER PELLICLES
Surfaces exhibit unique properties from the bulk material. Each biomaterial has
its own unique chemical, physical, and mechanical properties. However, it must be
recognized that surface properties of a material may exhibit marked differences from
that of the bulk material. These differences may be due to fabrication or sterilization
processes. Many examples of this phenomenon may be found. Machining metals
into desired shapes may enrich the surface in more reactive elements of the metal.
Sterilization of metal implants may result in adsorption of iron or other metals from
the sterilization unit onto the surface of the material. The fabrication process may
also alter the physical condition of the surface by introducing microcracks, porosity,
or altered texture at the surface layer. Little is known about the effects of surface
morphology or roughness on the interaction of a material with its biologic
environment. 9
Schemes for surface modification.
After recognition of surface changes that may have occurred as a result of

processing, two basic approaches may be used to further selectively modify material
surfaces.
Chemical alteration of surface layers to optimize adsorption of desired

pellicle components or decrease adsorption of unfavorable pellicle components.
20
This may be achieved by chemical etching, vapor deposition of new layers, or ion
implantation. The drawback of chemical alterations is that predicting or predetermining
that will be formed as a result of these changes is difficult.
Physical immobilization in which molecules remain at surfaces by electrostatic or

ionic interactions and chemical immobilization in which molecules are joined to the
surface by covalent bonds are other means of designing pellicles.
Modification of metal surfaces
The interaction between metal surfaces and tissues or biologic solutions is more
complex, because degradation and metal ion release (or corrosion) occurs continuously in
an aqueous environment. It is likely that the biologic effects of metal components with
tissues in many environments are the result of interaction of metal ions with tissue
components. In examining a variety of metal surfaces, it is apparent that protein
adsorption behavior is different for different metal surfaces and that the adsorption
process is not controlled by surface energy alone.
Many methods are available to produce surface coating on metal surfaces. The
most common is chemical vapor deposition and physical vapor deposition (evaporation,
sputter deposition, and ion plating). Although it is technically possible to coat the surface
of metals with a wide range of desired materials, little is known about the potential
biologic response of these coated materials. Titanium that was modified with surface
organic layers showed differences in bacterial and human cell adhesion. In addition, in
vitrostudies reported increased migration and attachment of human gingival fibroblasts to
titanium alloy disks coated with collagen and platelet-derived growth factor (PDGF).
Sietz et al. found that precoating bioactive glass with fibronectin reduced the time
required for cell spreading and altered the morphology of adherent cells. These studies
suggest that application of biologic molecules to the surface of implant materials may
have applications in clinical implantology.
21
New materials and tissue analogs
Synthetic materials that are designed to mimic natural tissues have been termed
“tissue analogs”.
Other tissue analogs include liposomes that are constructed from a mixture of
lipids and are used as models of cell membranes. Liposomes have been widely used in
pharmaceutics as a vehicle for in vivo drug delivery. Liposome-encapsulated drugs
or bioactive peptides could be added to a wound site or coated onto an implanted
material to enhance biocompatibility of the implant with the surrounding tissues. 9
INSTRUMENTATION FOR ANALYSIS OF

BIOMATERIAL SURFACE
Surface analytic techniques to measure chemical changes
Because surface chemistry and morphology ultimately influence

biocompatibility, it is imperative to have analytic tools that accurately assess surface
properties. Analysis of a biomaterial's surface structure is necessary after its
processing to monitor the nature and extent of surface modification. Surfaces present
unique analytical problems because they are covered with pellicle proteins that may
be associated with water. Because many spectroscopic measurements require a dry
or vacuum surface, this presents additional difficulties in the assessment of in vivo
situations. Most surface spectroscopic techniques can provide three basic types of
information: 9
 Identification of surface chemical constituents, such as atoms;

 Determination of structural and compositional changes that result from
chemical or mechanical treatment
 Detection of environmental effects or biologic reactions that result in chemical

changes or coatings at the surface of the material.
22
Bioanalytic and microscopic techniques are used primarily in detection and

analysis of pellicle proteins and can be applied to studies of microbial and cellular
adhesion. 8
23
Vibrational spectroscopy
Fourier transform infrared spectroscopy with attenuated total reflectance

(FTIR-ATR) uses vibrational spectra generated by infrared light to identify
constituent chemical groups at a surface of ,material or examine adsorbed materials
or thin films at a surface. This is a nondestructive technique that can provide both
quantitative and qualitative information about chemical groups, such as hydroxyl,
amide, nitrate, as well as functional interactions between chemical groups that
suggests molecular conformation. FTIR-ATR has been used to monitor the nature
and degree of chemical modification of synthetic polymers in preparation for
collagen coating, after plasma modification or to follow fibronectin adsorption onto
plasma- . derivatized polystyrene.
Much research has focused on surface modification of characterized

biomaterials to permit immobilization of enzymes, antibiotics, or cellular growth
factors. FTIR-ATR is capable of providing useful data about the extent and chemical
nature of modifications produced on a variety of biomedical polymers. These
surface modifications may be tailored for specific applications depending on the
biologic response required. FTIR-ATR may be used to monitor Surface molecules
that are adsorbed in vitro or in vivo on retrieved or in situ implant materials. 9
24
Electron spectroscopy
Electron spectroscopy for chemical analysis (ESCA), also known as x-ray

photoelectron spectroscopy (XPS), is an ultra high vacuum surface spectroscopic
technique. These measurements are based on photoelectric effects created by subjecting
the sample to a beam of soft x-rays. The energy of the incident x-ray photons produces
excitation of atomic orbitals, which them emit photoelectrons with measurable specific
kinetic energies. The advantage of this instrument is that it permits nondestructive
measurement of the chemical composition of a surface to a specified depth with excellent
spatial resolution. Quantitative analysis of any atom (except hydrogen and helium) and
assessment of the chemical state of the atom (involvement in chemical bonds) can be
performed with ESCA. Other techniques that provide similar or complimentary chemical
information are auger electron spectroscopy and secondary ion mass spectroscopy
(SIMS). 9
Some current clinical applications of ESCA are to determine the surface

composition and purity of implant materials. Surface contamination, thickness and
chemical composition of the titanium oxide layer, and protein adsorption to the surface
are information that can be obtained. These related techniques have also proved useful in
examining surface chemical heterogeneity of commercial biomedical polyurethanes,
defining the microdomin structure of segmented copolymers and their relationship to
thromboresistance, and examining the ability of implantable synthetic polymers to resist
degradation.
25
Future studies could include examination of potential corrosion products that

result from clinical apposition of nonprecious framework alloys with titanium
transmucosal elements or examination of the chemical homogeneity of hydroxyapatite,
ceramic, or titanium coatings on implants or other prosthetic surfaces.
Microscopy
Transmission and scanning electro microscopy have been widely applied in dental
biomaterials. These methods have been used to examine the surface morphology of
materials, the adaptation of restorative materials to substrates, and the histologic analysis
of bone-implant relationships. Both light and electron microscopy may be used in
immunocytochemcial studies that localize proteins or molecules specific to discrete types
of tissues or examine the temporal production of biologic molecules in cells. Examples
of this are identification of differences in junctional epithelial cells from human gingival
and peri-implant mucosa on the basis of differential expression of proteins. Scanning
tunneling microscopy makes possible the three-dimensional microscopic imaging of the
surface of materials. This technique has been used to study the surface morphology of
titanium implants and the surface topography of living cells on an oriented surface. It is
likely that further refinements and new techniques will further enhance the ability to
analyze biomaterials surfaces. 9
Bioanalytic techniques
The isolation and identification of biologically important proteins from the

surface of materials and determination of structural information are the primary goals of
bioanalytic techniques. Pellicles are complex mixtures consisting primarily of proteins
and glycoproteins. Separation of individual components may be achieved by sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), high-performance
liquid chromatography (HPLC), or capillary electrophoresis (CE) in which molecules are
separated on the basis of their size and/or net charge. Electroblotting of proteins from the
SAS-PAGE to inert matrices such as nitrocellulose or polyvinylidenedifluoride
26
membranes (Western blot) permits identification of single components by either probing

with a specific antiserum to salivary or serum components or by determining the N-
terminal peptide sequencer. Incubation of blotted pellicle molecules with various
microorganisms can determine whether specific proteins function as receptors for
bacterial adhesion. Purified proteins may be further characterized with regard to their
conformation by the use of various spectroscopic methods such as circular dichroism or
nuclear magnetic resonance.
FORMATION OF BIOFILM IN IMPLANTS
The pellicle coating
When microorganisms and substratum surfaces are in an aqueous environment, in

which organic material is present (e.g. sea water, milk, tear fluid, urine, blood or saliva),
they immediately (within seconds) become covered with a layer of adsorbed, organic
molecules. This is commonly called 'conditioning film', simply because transport and
adsorption of molecules to a substratum proceed relatively fast compared with that of
microorganisms. This conditioning film in the oral cavity, called pellicle, consists of
numerous components including glycoproteins (mucins), proline-rich proteins,
phosphoproteins (e.g. statherin), histidine-rich proteins, enzymes (e.g. α-amylase), and
other molecules that can function as adhesion sites for bacteria (receptors). The bacterial
27
adhesion thus occurs between a with pellicle-coated bacterium and a with pellicle-coated
surface. 1
The mechanisms involved in pellicle formation include electrostatic, van der

Waals, and hydrophobic forces. Studies of early (2 h) pellicle on tooth enamel revealed
that for example its amino acid composition differs from that of saliva. This clearly
indicates that the pellicle is formed by a selective adsorption of environmental
macromolecules. The physicochemical surface properties of a pellicle, including its
composition, packing, density, and/or configuration, are largely dependent on the
physical and chemical nature of the underlying hard surface. Thus, the characteristics of
the underlying hard surface are transferred through the pellicle layers, and as such will
still have its influence on the initial bacterial adhesion.
Specific biochemical mechanisms of bacterial adhesion
Streptococcal and actinomyces strain, the early colonizers, bind specific salivary
molecules. Streptococci (especially Streptococcus sanguis), the principal early
colonizers, bind to acidic proline-rich proteins and to other receptors in the pellicle like α-
amylase and sialic acid. Actinomyces viscosus possesses fimbriae that contains adhesins
that specifically bind to proline-rich proteins of the dental pellicle. 12
After the formation of a monolayer on the surface, biofilm formation can start,
either by multiplication of adhering species and/or the adhesion of new species. From this
stage on, new mechanisms are involved, because each newly accreted cell itself becomes
a nascent surface and therefore may act as a coaggregation bridge to the next potentially
accreting cell type that passes by. At least 18 genera from the oral cavity have shown
some form of coaggregation. Essentially all oral bacteria possess surface molecules that
foster some sort of cell-to-cell interaction. This process occurs primarily through the
highly specific stereo-chemical interaction of protein and carbohydrate molecules located
on the bacterial cell surfaces in addition to the less specific interactions resulting from
hydrophobic, electrostatic, and Van der Waals forces. Most coaggregations among strains
of different genera are mediated by lectin-like adhesins and can be inhibited by lactose
28
and other galactosides. Secondary colonizers (e.g. Prevotella intermedia, Prevotella

loescheii, Capnocytophaga spp., Fusobacterium nucleatum, and P. gingivalis) do not
initially colonize clean tooth surfaces but adhere to bacteria already in the plaque mass. 12
Non-specific physicochemical mechanisms of bacterial adhesion
In the formation of a biofilm to a non-shedding surface in an aqueous

environment, such as the oral cavity, four well-defined stages have been described.
Phase 1: Transport to the surface
The initial transport of a bacterium to the surface may occur through Brownian
motion (average displacement of 40 μm/h), through sedimentation of the bacterium in the
solution, through liquid flow (several orders of magnitude faster than diffusion), or through
active bacterial movement (chemotactic activity). Alternatively, microorganisms in
suspension may also be transported towards each other from microbial (co)aggregates.
Phase 2:Initial adhesion
29
This stage results in a weak and reversible adhesion of the bacterium via its
interaction with the surface at a certain distance (50 nm) through long- and short-range
forces. The organisms will be attracted or repelled by the surface, depending on the
resultant of the different non-specific interaction forces. Two physicochemical
approaches, initially considered distinctly different, are available to describe microbial
adhesive interactions: the thermodynamic and the DLVO approach.
1. Thermodynamic approach is based on the SFE of the interacting surfaces and does not
include an explicit role for electrostatic interactions. Before a bacterium can come in
direct contact with a surface, the water film between the interacting surfaces has to be
removed. The interaction energy for this process can be calculated from the assumption
that the interfaces between bacterium/liquid (bl) and surface/liquid (sl) are replaced by a
surface/bacterium (sb) interface. The change in the interfacial excess Gibbs energy upon
adhesion is described by the formula: ΔGadh=γsb−γsl−γbl in which the interfacial free
energy of adhesion for bacteria (ΔGadh) is correlated with the surface–bacterium
interfacial free energy (γsb), the surface–liquid interfacial free energy (γsl), and the
bacterium–liquid interfacial free energy (γbl). 12
If ΔGadh is negative (nature tends to minimize free energy), adhesion is

thermodynamically favoured and will proceed spontaneously.
2. Classical DLVO approach describes the interaction energies between surface and
bacterium. When a bacterium approaches a surface, it will interact with that surface by
means of two forces: the Lifshitz–van der Waals attractive forces (GA: the first force
becoming active at distances even above 50 nm), and the electrostatic repulsive forces
(GF: available at a closer distance). The latter force occurs due to the formation, in water,
of a counter-charged layer, diffusely distributed around the particle, to neutralize the
negative charge of the bacterium and of the surface (the electrical double layer or Stern
layer, Figs 1a and b). When this double layer overlaps the double layer of the surface (the
pellicle coating confers a negative charge to all surfaces), an electrostatic interaction will
take place. As both surfaces have the same charge, this electrostatic interaction is
repulsive in nature. The distance at which this interaction appears depends on the
30
thickness of the double layers, which themselves depend on the ionic charge of the
surface and the ionic concentration of the suspension medium.
DLVO have postulated that, above a separation distance of 1 nm, the summation of the
above-mentioned two forces describes the total long-range interaction between bacterium
and surface Figure 1b shows the total interaction energy (also called the total Gibbs
energy (GTOT), as the result of this summation of the above-mentioned forces
(GTOT=GA+GE), and in function of the distance between bacterium and surface.
Both approaches have proven merits for microbial adhesion, when certain collections of
strains and species are considered. They have, however, failed so far to yield a
generalized description of all aspects of microbial adhesion valid for each and every
strain therefore introduced a so-called extended DLVO theory. This theory considers four
fundamental, non-covalent interactions: Lifshitz–van der Waals, electrostatic, Lewis
acid–base and Brownian motion forces. The acid–base interactions are based on electron-
donating and electron-accepting interactions between polar moieties in aqueous solutions.
The polar or acid–base interfacial free energy balance ΔGadhAB is incorporated into the
extended DLVO approach by attributing a decay function to this balance. The influence
of the acid–base interactions is enormous when compared with electrostatic and Lifshitz–
van der Waals interactions. However, the acid–base interactions are also relatively short
ranged, and a close approach between the interacting surfaces (less than 5 nm) is required
before these forces can become operative. This new concept has been very useful for the
prediction of bacterial adhesion in several in vitro experiments.
Phase 3: Attachment
After contact is established between bacterium and surface, either directly or via
bridging of the gap (fimbriae), a firm anchorage between bacteria and surface can be
established by specific interactions (covalent, ionic, or hydrogen bonding). After
adhesion, most organisms also start to secrete slime and embed themselves in a slime
layer, the glycocalix, which forms an important virulence factor as it provides protection
against humoral and cellular immune components. 12
31
Phase 4: colonization/plaque maturation
When the firmly attached microorganisms start growing and newly formed cells
remain attached, biofilms can develop. The growth rate of sessile microorganisms has
been found to be partially depending on the biomaterial involved. At this stage, other
processes, including quorum-sensing, start to play an additional role. On a rough surface,
bacteria are better protected against shear forces so that a change from reversible-to-
irreversible bonding occurs more easily and more frequently.
DENTAL PLAQUE IN IMPLANT Vs TOOTH SURFACE
The microflora at both tooth and implant sites can be identified as biofilms. The
microbiota of healthy periodontal sites and that of diseased sites have been shown to
differ from each other. Small numbers of microorganisms and fewer morphological types
can be found in healthy gingival sulci. Diseased sites harbor a complex microflora with a
large proportion of Gram-negative anaerobic microorganisms.Poryphyromonas
gingivalis, Tannerella forsythia, Actinobacillus actinomycetemcomitans confers an
increased risk for periodontitis.7
The adhesion of bacteria depends on surface characteristics (Carlen et al.

2001Therefore, one might anticipate that the microflora in the biofilm should be different
at implant vs. tooth surfaces as well in gingival crevicular fluid (GCF), and on the
subgingival epithelial surface. The adhesion of bacteria depends on surface
characteristics (Carlen et al. 2001).
MICROFLORA ASSOCIATED WITH TITANIUM
Microflora associated with titanium implants studied from GCF collected from
implants is function for more than 24 months included P. gingivalis, Fusobacterium
nucleatum, Micromonas micros, and Actinomyces israelii (Buchmann et al. 2002) .
32
The pattern of bacterial presence 6 months after implant placement is stabilized,

containing predominantly M. micros, F. nucleatum, and Prevotella intermedia (van
Winkelhoff et al. 2000).
Failing implants as demonstrated by Leonhardt et al. (1999) showed the presence

of P. gingivalis, P. intermedia, Prevotella nigrescens, and A. actinomycetemcomitans.
Subjects with peri-implantitis, GCF samples yield very high counts of F.

nucleatum, A. actinomycetemcomitans, and P. gingivalis (Hultin et al. 2002).
Salcetti et al. (1997) highlighted that the presence of P. nigrescens and M. micros
could be associated with failing titanium implants.
MICROFLORA ASSOCIATED WITH TEETH
Increased risk for periodontitis seen with the presence of Poryphyromonas

gingivalis, Tannerella forsythia, Actinobacillus actinomycetemcomitans. Diseased sites
harbor a complex microflora with a large proportion of Gram-negative anaerobic
microorganisms.
Study: Funded byClinical research Foundation(CRF) Uni of Berne.
-The study made by using checkerboard DNA–DNA hybridization method to assess the
composition of the microflora of patients who had at least one oral osseo-integrated
implant and who were otherwise dentate in order to study the following:
 Microbial composition at teeth and oral osseo-integrated implants assessed from

samples taken by means of curettes toward tooth/implant surfaces as well as from
the sulcus/pocket junctional epithelium.
 Microbial composition in gingival fluid from samples taken at implant sites but
collected with paper strips.
SUBJECT SELECTION
33
The inclusion criteria were as follows:
 Subjects must be dentate

 Must have at least one osseo-integrated oral titanium implant.
 The implant must have definitive constructions for at least 6 months.
The primary exclusion criteria were:
 Patients with a clinical diagnosis of peri-implantitis

 Patients who had taken any antibiotics within 6 weeks prior to clinical examination
and microbial sampling.
 Pregnant or nursing women.
Comparison of the microflora at tooth and implant sites collected as curette samples
No differences in total DNA bacterial counts were found between curette samples
from tooth vs. implant. However, the proportion of Streptococcus oralis and
Fusobacterium periodonticum was significantly higher at tooth sites
Studies of the microflora comparing GCF samples with curette samples aimed at
implant surfaces
In the red complex, no difference was found for P. gingivalis, whereas the
proportions of T. forsythia and Treponema denticola were both higher in GCF.
In the orange complex, with the exceptions of P. intermedia and Capnocytophaga

gracilis significantly higher proportions of the individual bacteria were found in the GCF
samples
In the green complex, 50% or more of the pathogens in this group were not found
in GCF samples, and were not found in 70% or more from the curette samples.
A. actinomycetemcomitans could not be detected in 30% of the GCF samples and in 60%
of the curette samples.
34
In the yellow complex, the proportions of all the streptococci spp. were
significantly higher in the GCF samples.
In the blue and violet complexes, A. israelii and Actinomyces odontolyticus were
found in higher proportions in GCF over curette samples. 7
Studies of the microflora comparing curette samples aimed at the implant surfaces vs.
curette samples aimed at the sulcular junctional epithelium
No differences in total DNA bacterial counts for any of the individual bacteria
were found when curette samples toward implant surfaces were compared with curette
samples toward soft gingival tissues.
Studies of the microflora from implants comparing GCF with curette samples aimed at
the sulcular junctional epithelium
The difference in the total DNA bacterial counts was significantly higher in GCF
samples (P<0.001). Specifically, P. gingivalis and T. denticola were found in higher
proportions from GCF samples.
Impact of probing depth and Bleeding On Probing at implant test sites on bacterial
yield
The data demonstrated that the prevalence of A. actinomyctemcomitans, P.

nigrescens, and T. denticola was significantly higher in sites with BOP that in sites
without evidence of BOP.
Draw back in the study method:
 Difficulty in collecting samples from implants with curretes.
 Currete sample may be contaminated with GCF and juctional-epithelium

containing bacteria.
35
 Study was a cross-sectional by design and won’t provide information to predict

outcome of implant success. 7
36
BIOFILM AND SURFACE ROUGHNES
It is generally believed that roughened surfaces influence microbial colonisation by

enhancing microbial retention within surface irregularities. Verran & Boyd (2001) have
proposed three categories of surface roughness, termed as
macro- roughness (Ra 10 μm)
micro- roughness (Ra 1 μm)
nano-roughness (Ra 0.2 μm).
Micro-roughness has been suggested to be appropriate for dental implants (Bollen

et al. 1997) and it has been postulated that a surface with an Ra below 0.2 μm is unlikely
to promote microbial adherence due to the larger size of most bacteria (NB P. gingivalis
is approximately 1.5 × 1 μm in size).5
Interestingly, increasing the surface roughness above 155 nm did not enhance the
adherence of P. gingivalis and it could be postulated the increased size of surface
irregularities was then too large to offer increased bacterial retention. Importantly,
commercially available Brånemark-type dental implants display a range of surface
roughness (350–2500 nm) exceeding the value determined in this study to reduce
bacterial adhesion.
Implants display a range of surface roughness (350–2500 nm) exceeding the value

determined in this study to reduce bacterial adhesion.
Rougher surfaces (crowns, implant abutments, and denture bases) accumulate and
retain more plaque (applying parameters such as thickness, area, and colony-forming
units).
37
Several days of undisturbed plaque formation, rough surfaces harbour a more

mature plaque characterized by an increased proportion of rods, motile organisms, and
spirochetes.
Tooth surfaces with rough surfaces are more frequently surrounded by an

inflamed periodontium, characterized by a higher bleeding index, an increased crevicular
fluid production, and/or an increased inflammatory infiltrate. 12
Threshold surface roughness as postulatd by: Bollen etal 1997
Two later studies examined the effect of smoothening the abutment surface. A
smoothening below an Ra=0.2 μm showed no further significant changes, either in the
total amount of or in the pathogenicity of adhering bacteria. An Ra value of 0.2 μm was
therefore suggested as a threshold surface roughness, below which bacterial adhesion
cannot be reduced further.12
The clinical impact of surface roughness is, for example, illustrated. It shows the
clinical picture of two strips that were divided into two halves: a rough (Ra=2 μm) and a
smooth region (Ra=0.1 μm). Whereas the smooth regions were only for 1/4 covered with
plaque, the rough parts were completely colonized within 3 days of undisturbed plaque
formation. The impact of surface roughness becomes especially important when larger
shear forces are active.
38
Impact of surface roughness on Biofilm is explained by:
 The initial adhesion of bacteria preferably starts at locations where they are
sheltered against shear forces so that they find the time to change from reversible to
irreversible attachment.
 Roughening of the surface increases the area available for adhesion by a factor 2-3.
 Rough surfaces are difficult to clean, resulting in a rapid re-growth of the biofilm
by multiplication of remaining species, rather than by recolonization.
Study: The aim of the study was to determine three different surface coatings
(titanium, TiN and ZrN on glass sheets) on the colonization of plaque-building bacteria in
the oral cavity.
To eliminate the influence of surface roughness in this study, coated glass sheets were
used as substrates (with an Ra value close to zero). 12
Bacterial cell counts
Four glass sheets with different surface coatings (two each of titanium, TiN and
ZrN), in two different volunteers (A and B), were used to assess variation in bacterial cell
numbers. Because they had the same surface and the same position in the oral cavity,
identical surfaces were dealt with in parallel. The total bacterial cell counts of the eight
different samples incubated in the oral cavity differed, ranging from 1.2 × 107 (ZrN-A26)
to 9.8 × 1010 (Ti-B17) cells per glass sheet (Fig. 1). Bacterial cell counts were higher on
all titanium-coated glass sheets than on TiN- or ZrN-coated glass. The fewest bacterial
cells were present on the ZrN-coated glass sheet in both volunteers.2
Conclusion: The number of adherent bacterial colonies (Streptococcus sanguis,

S. mutans) was significantly reduced on titanium surfaces coated with inherently stable
titanium hard materials, such as ZrN, compared to a polished titanium surface. Similar
results were achieved by TiN coating and by a thermic oxidation. These results were
confirmed by an in vivo study of Scaranoet al. who showed that bacterial adhesion on
39
implant surfaces coated with TiN was significantly lower compared to an uncoated
implant surface.
Bacterial cell counts determined by SYBR Green staining and epifluorescence

microscopy, of the biofilms grown on discs with different coatinsgs.
SSCP fingerprints based on 16S rDNA and 16S rRNA
Total DNA and RNA extraction was performed in order to compare bacterial
communities of biofilms on glass sheets coated with different surfaces. 16S rDNA genes
were amplified by the PCR, and 16S rRNA was amplified by RT–PCR. The amplicons
generated were subjected to SSCP analysis to obtain fingerprints of the bacterial
community of the biofilms (Fig. 2A)PCR products of the 16S rRNA genes; Fig. 2B, RT
products of the 16S rRNA.
rRNA fingerprints are considered to reflect the composition of the metabolically

active members of the community, whereas the rDNA fingerprints reflect the population
composition of the community. 2
40
Original( not normalized) single strand conformation polymorphism(SSCP) fingerprints

from bacterial biofilm communities were generated using
a) Polymerase chain reaction (PCR)products of 16s RNA genes.

b) Reverse transcription (RT) products of the 16s RNA genes.
Specific number of bands obtained from Polymerase chain reaction(PCR) and reverse
transcription single-strand conformation polymorphism(SSCP) fingerprints from the
bacterial biofilm communities. 2
41
Bands from the same positions in different fingerprints gave identical sequences, showing
that similar organisms – streptococci seem to be the predominant colonizing micro-
organisms – represent the most abundant members in biofilms on glass sheets coated with
different surfaces.
Streptococci were the predominant organisms and represent the main early colonizers of
all surface coatings examined. No Actinomyces species were found on any of the three
different coatings. It appears that bacterial adherence to ceramic material or to coatings
with a ceramic-like character (as hard coatings) is lower than adherence to titanium alloys
However, on intraoral hard tissues, Streptococci and Actinomyces species are considered
to be early colonizers, preparing the environment for late colonizers that require more
demanding growth conditions. On the other hand, a study conducted by Wolinskyet al.
showed that plaque-forming bacteria, such as A. viscosus, adhered to enamel in numbers
five times higher than they adhered to titanium surfaces.
Conclusion of the study is that both of the titanium hard coatings investigated in the
present study seem to reduce initial bacterial adherence, with the ZrN coating showing
the lowest number of adhering bacteria compared to pure titanium. These results are in
accordance with other studies. The physico-chemical reasons for the different bacterial
cell numbers on the titanium hard coatings are still not fully known, but strongly confirm
their suitability as implant coatings to decrease peri-implant soft tissue inflammation. 2
SURFACE MODIFICATIONS- EFFECTING

P.GINGIVALIS ADHERENCE
Periodontopathic bacteria such as P.gingivalis occur more commonly around

implants that exhibit gingival inflammation. These bacteria may contribute to
periimplantitis in partially and completely edentulous patients. P.gingivalis is reported to
42
be a significant component of the predominant cultivable microflora around failing

implants. P.gingivalis and other gram-negative bacteria produce endotoxin, which is a
cell envelope lipopolysccharide (LPS). Considerable evidence exists to implicate LPS in
the pathogenesis of chronic periodontitis. This toxin, which is found in large quantities
on the surfaces of periodontally diseased teeth, has a marked effect on cells such as
macrophages, lymphocytes, fibroblasts, and osteobalsts. In the natural sulcus these
effects may contribute to inflammation, osteoclasis inhibition of cell growth, and delayed
healing. Like the natural sulcus, LPS produced by P.gingivalis and other gram-negative
bacteria in the periimplant regionis one factor that likely influences periimplant
inflammation and implant prognosis.7
The risk factors associated with peri-implantitis appear to be related to the

composition of the bacterial environment around an implant and the ability of bacteria to
adhere to the implant material (Quirynen et al. 2002).
Peri-implantits is an inflammatory process affecting the tissues around an

osseointegrated implant, resulting in the loss of supporting bone (Albrektsson & Isidor
1994). The bacteria implicated in peri-implantitis are primarily those associated with
periodontal disease and include strictly anaerobic bacteria such as Porphyromonas
gingivalis, Actinobacillus actinomycetemcomitans, Prevotella intermedia and
spirochaetes (Mombelli 1997) Dental implant biocompatibility research has focused on
the effects of the periimplant microbial flora and restorative material surface roughness
on the immune response. In the partially edeutulous patient the periimplant sulcus and
periodontal sulcus have similar microbial flora, which could be the result of bacterial
seeding from the natural gingival sulcus to the periimplant sulcus. Porphyromonas
gingivalis, a gramnegative anaerobic rods,is associated with periimplant subgingival
plaque. P.gingivalis colonizes on implant sites in partially edentulous patients within 14
days after stage II surgery. After 28 days all implants may demonstrate the presence of
P.gingivalis at the periimplant margin, and 50% of implants may demonstrate subgingival
colonization of this species.
43
Co-adherence between bacteria often facilitates the attachment of organisms

normally incapable of binding to host surfaces. The result of such co-adherence can
ultimately lead to the development of a biofilm community. Biofilm bacteria behave
differently in terms of their physiology and resistance to antimicrobials compared with
their planktonic (free living) counterparts and therefore once established can represent a
significant challenge for removal. In such instances prevention of initial adherence of the
pioneer coloniser is applicable. 8
Study: From 16 samples of study material, four groups of materials (four

specimens in each) were obtained of different surface roughness. The groups were
categorised as being 'very smooth' (hand polished with rotary brushes for a mirror
finishing process), 'smooth' (polished only with the Eco mini dry machine), 'rough'
(sandblasted with 50 μm glass beads; 5 bars pressure) and 'very rough' (sandblasted with
50 μm aluminium oxide beads; 6.5 bars pressure).
Effect of surface roughness on adhesion of P. gingivalis
Significant differences in the bacterial adhesion to the four surface roughness

groups were. A highly significant difference between the very smooth and other sample
groups was detected. There were no differences in bacterial adherence evident between
these other groups of materials. P. gingivalis ATCC 33277 was used for all adhesion
experiments.
Porphyromonas Gingivalis adherence to
44
a) Smooth surface
b) Very smooth surface
Effect of hydrophobic changes on the adhesion of P. gingivalis
There was no significant difference in adhesion to these different surfaces.
Draw back of the study: Microbial adherence to surfaces are obviously more
complex in the presence of host conditioning films where proteins can act as receptors,
potentially masking the underlying surface characteristics of the material.
Although this study only compares the effects of two titanium surface parameters
on the adhesion of one bacterial species (significant periodontal pathogen),
considerations of these findings could offer a starting point in future design of titanium
implants/abutments aimed at reducing bacterial colonisation.8
TITANIUM IMPLANT BIOMATERIAL: EFFECTING LPS

ADHERENCE
P.gingivalis and other gram-negative bacteria produce endotoxin, which is a cell

envelope lipopolysccharide (LPS). Considerable evidence exists to implicate LPS in the
pathogenesis of chronic periodontitis. This toxin, which is found in large quantities on
the surfaces of periodontally diseased teeth, has a marked effect on cells such as
macrophages, lymphocytes, fibroblasts, and osteobalsts.10
Periodontal inflammation may result from an interaction between restorative

materials and gram-negative bacterial macromolecules such as LPS in the natural sulcus.
In vitro studies have reported that P.gingivalis and Escherichia coli LPS have an affinity
for fixed prosthodontic materials. This affinity was shown to be related to factors such as
LPS type, material type, surface properties, and pH. LPS affinity for materials, as
measured by adherence and elution of LPS molecules at the surface of the restorative
45
material. Which could influence the concentration of toxin within the sulcus. If the
subgingival interaction between a material and LPS increased toxin levels in crevicular
fluid and surrounding tissues, the biologic response could be unfavorable.
In the healthy gingival sulcus and in those associated with gingivitis, crevicular
fluid LPS levels range from 0.8 to 9.6 ug/ml. because the periimplant sulcus and the
natural sulcus have similar indigenous bacteria, this concentration is likely to be
representative of total LPS levels in periimplant crevicular fluid.
However, the P.gingivalis type LPS and E.coli type LPS portions of this level are
uncertain. Studies that evaluate LPS concentration in periimplant crevicular fluid have
not been completed. 10
Study method: The purpose of the study was to evaluate Porphyromonas

Gingivalis and Escherichia coli LPS affinity for titanium biomaterial groups that differed
in surface oxide composition and surface roughness.
The purpose of this study was to evaluate the influences of titanium surface oxide
composition and surface roughness on Porphyromonas Gingivalis and Escherichia coli
LPS affinity for titanium biomaterial groups that differed in surface oxide composition
and surface roughness. {CPI and grade 5 titanium specimen}.
Grade I commercially pure titanium (CPI) and grade 5 alloyed extra low
interstitial titanium are titanium grades used in the above study. Both grades are
commonly used in the treatment of prosthodontic patients.
CPI is 99.8% commercially pure titanium, where as certified grade 5 alloy is 89%
titanium, 6.4% aluminum, and 4.1% vanadium. Both grades of titanium contain small
quantities of nitrogen, carbon, oxygen, and iron. Because grade 5 titanium is alloyed
withaluminum and vanadium, the grade 5 surface oxide likely contains aluminum and
vanadium oxides that would not be found on the CPI surface. Thus differences in LPS
affinity for CPI and grade 5 titanium could exist on the basis of differing surface oxide
compositions.
46
Conclusion: Within the parameters of this Study surface oxide composition,

surface roughness, and LPS type did not influence the adsorption or desorption of LPS
from the surfaces of the titanium test specimens. This study confirmed that P.gingivalis
and E.coli LPS indicate a high affinity for these titanium biomaterial surfaces.
Drawback of the study:
 The lack of difference between titanium because only subtle differences in surface
oxide composition existed among experimental groups. The predominate surface
oxide for both titanium grades was titanium oxide. Because titanium oxide
dominated the grade 5 titanium specimen surface, no difference in affinity on the
basis of LPS interactions with aluminum or vanadium oxides was detectable.
 Crevicular fluid also contains a complex mixture of macromolecules that might
affect LPS affinity patterns. Moreover, we would not expect LPS to be present
without bacteria nearby. Indeed, the presence of bacteria indigenous to be gingival
sulcus could influence LPS, material, and physiologic variations within crevicular
fluid (pH) could affect LPS affinity.
Surface roughness of commercially produced implant components could influence

LPS affinity and thereby affect the LPS concentration in periimplant crevicular fluid.
Greater surface area of the rougher surface provides more space for LPS to potentially
adhere. Rougher surfaces promote plaque accumulation and maturation to a greater extent
than smoother surfaces could, and LPS-producing bacteria would be associated with this
colonization. Thus crevicular LPS levels may be influenced by the greater available
titanium biomaterial surface area and the greater number of LPS-producing bacteria
associated with and protected within the rough implant surface. 10
Roughness of machined implants and abutments can be related to material

hardness. The harder the implant biomaterial, the more resistant it would be to abrasion
during oral hygiene instrumentation. Harder material may be more abrasion resistant and
remain smoother intraorally over the life of the implant prosthesis. These smoother
47
surfaces could minimize LPS accumulation and facilitate LPS removal from the implant
surface.
LPS affinity for titanium biomaterials appears to be primarily a function of

surface energy. Materials with surface energies of 20 to 30 dynes/cm exhibit minimal
biologic adhesiveness, whereas higher surface energies support bioadhesion. Moreover,
an intraoral hard surface that exhibits a lower surface energy reduces bacterial
colonization and retards plaque maturation. Therefore surface energy of implant
materials could affect LPS adherence and elution in the periimplant sulcus.
For LPS to adhere, these implant biomaterials must exhibit greater surface
energies than LPS. CPI surface energy is approximately 33 dynes/cm after several
sterilization procedures. Because CPI and grade 5 titanium adherences and elution levels
were similar in this study, grade 5 surface energy is also likely to be approximately 33
dynes/cm. Therefore LPS surface energy is 30 dynes/cm or less. Increasing surface
roughness can increase surface energy.
Smooth surfaces with a low surface energy is desirable to minimize plaque

formation and LPS accumulation. 10
CLINICAL IMPLICATIONS
Periimplant sulcus probing depths may be greater than 3mm and result in a
situation that increases the difficulty of implant maintenance. In these deeper sulci a
pathogenic microbial flora could develop after bacterial seeding from adjacent tooth
gingival sulci. Periodontopathic bacteria including P.gingivalis produce LPS that has
been linked with dental inflammation and gingival recession. Therefore LPS in the
sulcus may be an important factor in implant prognosis. 10
48
LPS can be removed from diseased tooth surfaces. However, retoxification of

these surfaces occurs within a short period of time. Similarly, LPS can be removed from
the surfaces of implant components, but retoxification of these surfaces also likely
occurs. Inflammation and osseous saucerization often clinically observed with implant
prostheses may be related to LPS in the periimplant crevicular fluid. Because smooth
surfaces may reduce bacterial accumulation, plaque formation, and subsequent LPS
accumulation, the long-term maintenance of implant biomaterial surfaces must be
considered.
LPS decontamination of implant surfaces. LPS may be removed clinically from

machined or plasma-sprayed titanium surfaces with air powder abrasive, water or, citric
acid treatments. The efficacy of the air abrasive treatment may be equal to or greater
than other surface treatment regimens. However, if air particle abrasion affected the
surface in such a way as to increase subsequent adhesion of specific LPS concentrations,
this may not be a desirable treatment modality. Furthermore, machined implant surfaces
may show the largest reduction in surface-adhering LPS.
Maintenance: To favor long-term periimplant tissue health, implant components

with smooth surfaces must be selected and cleaning procedures that maintain surface
smoothness must be used. The surface texture of machined titanium abutments may
become smoother after treatment with a plastic scaler, rubber cup, rubber cup with tin
oxide, or air-powered abrasive. This surface smoothing may result from removal of
surface debris and rounding of sharp machined grooves present on the machined
abutment surfaces. However, this surface smoothing would likely occur only with
materials of greater hardness, such as grade 5 titanium. Air particle abrasive treatment of
materials with lower hardness could result in greater surface roughness, greater surfaces
area, and increased LPS adherence.
Some decontamination techniques may increase abutment surface roughness.

Subsequently, LPS adhering within crevices of rough implant surfaces would be
protected from mechanical removal. Care must be used with the air abrasive or the
rubber cup with pumice techniques to avoid damaging the implant component surface
49
smoothness or the soft tissues. Other techniques such as ultrasonic scaling or scaling
with metal instruments may significantly increase implant component surface roughness.
The latter two techniques should not be used with implant components. 10
In situations of limited access, one must clean with plastic scalers, which may be
ineffective in removing bacterial products. Furthermore, if the implants exposed surface
is not smooth, as in the case with plasmasprayed titanium or hydroxyapatite surfaces,
removal of bacterial products including LPS would be ineffective.
To promote a long-term favorable periimplant tissue response, the selection of

implant components should be based on two interrelated factors, surface roughness and
surface hardness. From the manufacturer, implant components fabricated from grade 5-
alloyed ELI titanium may be harder, smoother, and less susceptible to surface abrasion
during implant maintenance. Machined implant components from this material could
favor long-term soft and osseous tissue health.
SURFACE FREE ENERGY [SFE] AND BIOFILM

FORMATION
Materials with surface energies of 20 to 30 dynes/cm exhibit minimal biologic

adhesiveness, whereas higher surface energies support bioadhesion. Moreover, an
intraoral hard surface that exhibits a lower surface energy reduces bacterial colonization
and retards plaque maturation.2
Significant correlation between the substratum SFE (also called wettability) and
its plaque-retaining capacity is demonstrated. It is obvious that surfaces with a higher
SFE are more prone to bacterial adherence. Glantz 1969 was the first to recognize this in
vivo. When he followed undisturbed supragingival biofilm formation on test surfaces of
different free energies – mounted on a partial fixed bridge – he detected a 'positive'
50
correlation between substratum SFE and the weight of accumulated plaque (measured at
days 1, 3, and 7). Rolla et al 1991demonstrated that the application of a silicone oil to
teeth, which lowered their SFE, resulted in a significant reduction in plaque formation.
Quirynen and co-workers 1989,1990 studied the influence of SFE on undisturbed plaque
growth in humans over a 9-day period and reported that hydrophobic surfaces (e.g.
teflon) harboured 10 × less plaque than hydrophilic ones (enamel). The latter was well
confirmed in animal studies(Van Dijk el al 19870).
The effect of substratum SFE on supra and subgingival plaque maturation around
implants was investigated by comparing 3-month-old plaque from abutments with either
a high (titanium) or a low (teflon coating) SFE (Quirynen et al1993). Low-SFE substrata
harboured a significantly less mature plaque supra – as well as subgingivally,
characterized by a higher proportion of cocci and a lower proportion of motile organisms
and spirochetes.
These qualitative and quantitative differences clearly illustrate that the impact of
the substratum SFE remains after the pellicle formation, even though the latter had a
homogenizing effect in terms of the remaining SFE . Thus, the SFE properties are
transferred through the absorbed protein layer(Sipahi et al.2001).
The surface SFE also had an impact on the SFE of the colonizing bacteria.
Surfaces with a low SFE were preferably colonized by bacteria with a low afe, whereas
the opposite was observed for surfaces like e.g. titanium or enamel with a higher SFE.
Moreover, colonies from a specific strain, collected form surfaces with a low SFE, even
had a lower SFE than colonies of the similar strain, collected from a surface with a higher
SFE (Weekeramp et al.1989)). The latter suggests a bacterial selection by, or adaptation
to, the surfaces, up to and even within the species level. 2
 In some clinical and in vitro trials, it had been observed that the reduced biofilm
formation on surfaces with a low SFE could partially be explained by a low binding
strength between bacteria and substratum, probably because of a cohesive failure
within the conditioning layer(Christersson et al.1989:Busscher et al.1995). The
51
latter will increase the detachment of adhering bacteria. Therefore, several authors
no longer speak about bacterial adhesion and detachment, but prefer the term
bacterial retention, being the resultant of the previous phenomena.
Interaction between surface roughness and SFE
The effect of surface roughening on the resulting contact angles of droplets that
reflect the SFE has been studied extensively. Changes in solid surface Ra below 0.1 μm,
have no effect on contact angle; above 0.1 μm the effect depends on the initial contact
angle as measured on a smooth surface: if the initial contact angle is below 60° (e.g.
enamel), surface roughening will further decrease this angle; if the initial contact angle is
above 86°, surface roughening will further increase this angle; and for surfaces with
initial contact angles between 60° and 86°, surface roughening has no influence(Busscher
et al.1984).
The 'relative' importance of both parameters (SFE and roughness) on

supragingival plaque formation has been examined in vivo by (Quirynen et al.1990) They
followed undisturbed plaque formation on polymer strips with low and medium SFE,
glued to a tooth surface. Each strip had a smooth (Ra 0.1 μm) and a rough part (Ra>2 μm).
After 3 days of undisturbed plaque formation, significant inter-substrata differences were
observed on the smooth regions, while the rough regions of the strips were nearly all
completely covered with plaque. Surface roughening resulted for both materials in a
fourfold increase in plaque formation (plaque extension as well as thickness) for both
polymers. Surface roughness seems prominent towards SFE where bacterial adhesion is
concerned. 2
CHEMICAL COMPOSITION OF THE SURFACE

Studies on implant surfces with similar roughness characteristic showd difference
in the composition of microflora, which can be explained bythe antibacterial properties of
some material. Titanium for example has a bacteriostatic effect on oral bacteria(Bundy et
al.1980:Leonhardt & Dahlen 1995), although some others failed to prove such an effect. 2
52
Modifications made to increase the antimicrobial capacity of titanium:
 Photocatalytic bactericidal effect of anastase titanium dioxide (TiO 2) under

ultraviolet A illumination, added onto commercially pure titanium surfaces via
plasma source ion implantation (Suketa et al. 2005) a photocatalyst can decompose
various organic compounds by generating active-oxygen species such as OH, OH 2,
HO2, and H2O2.
 Dry ion implantation of F+ on titanium (Yoshinari et al. 2001)
 Anodized titanium being discharged in NaCl resulting in Ti–Cl, which exhibit high
antibacterial activity.
 Antibiotics incorporated into the titanium surface (Parvizi et al. 2004).
In vitro these modification look very promising, but their efficiency in clinic still has
to be proven. When antibiotics are concerned, other side-effects such as microbial
resistance have to be considered.
THE IMPLANT–ABUTMENT FIT
The interstitia between implant components, especially those located

subgingivally, offer an ideal environment for de novo plaque formation and/or for plaque
retention during cleaning. The size of the gap between implant and abutment of nine
different systems, including those with conical interfaces, was found to range between 1
and 10 μm (Jansen et al. 1997) and 49 μm (Binon et al. 1992) depending on whether or
not the rounded edges of the abutment margin were included. Although the discrepancies
of these prefabricated parts are significantly smaller compared with those of dental
restorations (ranging from 50 to 150 μm), it still allows microbial leakage (Wahl et al.
1992; Quirynen & van Steenberghe 1993; Quirynen et al. 1994; Jansen et al. 1997). This
microleakage is comparable for different implant systems and decreases significantly
when the closing torque is increased (Gross et al. 1999). As observed by Ericsson et
53
al.1995 in the Labrador dog model, bacterial leakage results in an inflammatory cell
infiltrate (called abutment ICT) in the peri-implant mucosa at the borderline between
abutment and implant, irrespective of the oral hygiene. The connection between the
abutment and the prosthetic supra-structure (sometimes located subgingivally in order to
improve aesthetics) shows even larger discrepancies (Binon et al. 1992), especially for
cemented restorations (Keith et al. 1999). 2
FUTURE DIRECTIONS
The future of prosthodontics research will be in development of rational systems

to replace lost tissues by use of scientific methods to evaluate both biomaterials and
treatment designs based on desired biological outcomes. The profession should be
cognizant that physical and chemical properties of a material are not endpoints but
beginning in the evaluation of a biocompatible material. Like the specialty of
periodontics, where current therapies are founded on scientific knowledge of the disease
process, prosthodontics must base treatment on scientific evidence of cellular responses
to proposed replacement materials. To achieve these goals, a further understanding of
key areas, including pellicle formation as a function of surface composition, microbial
adhesion to biomaterials, and cell reaction to implant biomaterials is not only necessary
but essential. 9
SURFACE AND PELLICLE STUDIES
Identification of the macromolecules that adsorb onto specific surfaces will permit
assessment of the molecules that affect subsequent functions of the restorative material.
The agenda of research may include the following:
 Determine compositional profiles of pellicles formed on metal, ceramic,

polymer, and enamel or dentin surfaces, and determine the relationship of
specific pellicle molecules on cell adhesion.
 Establish relationships between surface composition or microstructure and
adsorption of specific proteins.
54
 Determine how pellicle molecules adsorbed by restorative materials affect

dimensional change, color stability, or chemical solubility of the substrate.
 Develop materials or coatings that can be used as controlled-release devices for

therapeutic agents.
 Develop polymeric coatings that resist colonization by pathogenic

microorganisms.
DENTAL IMPLANT STUDIES
The major objective of dental implant studies is to achieve a better

understanding of the processes occurring at the implant-tissue interface and the
implant-osseous interface so that biomaterials can be designed at the molecular level.
The implant materials should have surface characteristics that promote adhesion of
host cells and tissues in preference to bacteria. Studies should include the following
program.
 Develop biocompatibility determination tests for currently used materials that

are based on cell responses at a tissue culture level.
 Assess materials bioactivity in the stimulation and determination of the implant
tissue interface on the basis of the composition of the surface and nature of the
initially adsorbed surface layers.
 Determine optimal implant designs based on implant stresses that are

transmitted to natural load-bearing tissues. These goals require development of
models of biomaterial-cell interfaces that accurately reflect stress distribution
by including new instrumentation for load sensing.
 Explore the use of local bone growth and tissue attachment factors as
biomaterials coatings to stimulate tissue growth and regeneration.
SOFT TISSUE STUDIES
55
The response of soft tissues in close apposition to biomaterials at a molecular

level remains largely unknown. In particular, the mechanisms of epithelial
differentiation and attachment to surfaces associated with wound healing are critical
to success of implants and tissue augmentation. Soft tissue studies should include the
following goals. 9
 Examine patterns of epithelial cell differentiation in response to various implant

materials, and identify structural and regulatory genes related to the expression
of these tissues.
 Explore the use of bioadhesive polymers for use in peptide drug delivery
systems (buccal patches) that attach the dosage form of the drug to the
adsorbing mucosa.
 Define mechanisms of soft tissue colonization by oral pathogens such as Candida

albicans in which prostheses in apposition to tissue surfaces may be a cofactor.
In summary, these research areas are very broad and are not intended to be inclusive
of the many directions in which science in prosthodontics may become involved.9
The central concept is and will continue to be that provides a new biologically
based rationale for arch and fundamental treatment modalities in prosthodontics.
56
CONCLUSION
Osseointegrated dental implant play an important role in field of dentistry. Biofilm

formation may cause inflammatory reactions around the implant (peri-implantitis)
leading to implant failure. Hence it is very important to develop implant surfaces (around
transmucosal portion) that reduce the number of initially adhering bacteria, which
minimizes biofilm formation and subsequent inflammation of the soft tissues.
Implant material surface characteristic play a vital role in effecting the biofilm
formation and maturation.
Micro roughness has been suggested to be appropriate for dental implants. (Bollen et
al 1997)Because surfaces below Ra 0.2 μm doesn’t promote bacterial adherence due to
larger size of most micro organisms. Vise-versa surface roughness above 150 nm didn’t
enhance adherence of microorganisms. Hence importantly commercially available
Branemark-type dental implants display a range of surface roughness(350-2500nm) to
reduce bacterial adhesion. Materials with surface energy of 20-30 dynes cm exhibit
minimal biologic adhesiveness, where as higher surface energy support bioadhersion.
Hence, smooth surface with low surface energy is demanded to minimize biofilm
formation. Modifications are also made chemically changing the surface to increase the
antimicrobial capacity of titanium. Bacterial leakage influenced by implant-abutment fit
results in an inflammatory cell infiltration(called abutment ICT) in periimplant mucosa at
borderline between abutment and implant.
Future of prosthodontics research will be in development of scientific

methods to evaluate both biomaterials and treatment designs based on desired biological
outcomes. Physical and chemical composition are not end points but begins in the
evaluvation of biocompatible material. Future understanding of pellicle formation as a
function of surface composition, microbial adhesion to biomaterials, and cell reaction to
implant biomaterials is not only necessary but essential.
57
REFERENCE
1. Anne.D.Haffajee and Sigmund.S.Socransky introduction to microbial aspects of

periodontal biofilm, communities, development and treatment. Perio 2000;2006
42:7.
2. Birte Groessner-Schreiber, Matthias Hannig, Alexander Dück, Michael
Griepentrog, DirkF.Wenderoth Do different implant surfaces exposed in the oral
cavityof humans show different biofilm compositions and activities? European
Journal of Oral Sciences 2004, 112 (6) 516–522.
3. Carl.E.Misch Dental Implant Prosthetics, first edition, Elsevier Mosby

publication, 2005
4. Carl.E.Misch, Contemporary imlpant dentistry second edition mosby publication

1999:
5. Carranza, Newman, Takei Clinical Periodontology Tenth edition, Harcout (India)

private limited publication, 2007.
6. Jan Lindhe, Clinical Periodontology and implant dentistry, fourth edition, Jaypee
brothers medical publishers (p) LTD, New Delhi, 2003.
7. Jeanne Gerber, Doris Wenaweser, Lisa Heitz-Mayfield, Niklaus P. Lang, G.

Rutger Persson Comparison of bacterial plaque samples from titanium implant
and tooth surfaces by different methods Clinical Oral Implants Research 2006, 17
(1) 1–7.
8. Moroso Pier-Francesco, Robert J. Adams, Mark G. J. Waters, David W. Williams

Titanium surface modification and its effect on the adherence of Porphyromonas
gingivalis: an in vitrostudy Clinical Oral Implants Research Dec 2006, 17 (6)
633–637.
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9. Mia Edgerton, Michael J. Levine, Biocompatability it’s future in prosthodontic

research Journal of Peosthodontics 69, (4) 1993.
10. Steven.K.Nelson, Kent.L.Knoernschid, Fonda.G, Robinson and

George.S.Schuster Lipopolycaccharide affinity fot titanium implant biomaterials
Journal of Prosthodontics 77, (1) 76-82
11. W. Heuer, c. Elter, a. Demling, a. Neumann, s. Suerbaum, m. Hannig, t.

Heidenblut, f. W. Bach, m. Stiesch-scholz Analysis of early biofilm formation on
oral implants in man Journal of Oral Rehabilitation 34 (5) 377–382.
12. Wim Teughels, Nele Van Assche, Isabelle Sliepen, Marc Quirynen
Effect of material characteristics and/or surface topography on biofilm
development Clinical Oral Implants Research 2006, 17 (2) 68–81.
59

Bio Film in Implantology

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Bio Film in Implantology

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Original article of Dr.

0 Original article of Dr. Komagal |

Biofilm: nature and properties 4

Factors affecting biofilm development 9

Material interaction with biological systems 17

Designing Biocompatable material 20

Instrumentation for analysis of biomaterial surfaces 22

Formation of biofilm in implant surface 26

Dental plaque in implant vs tooth surface 31

Biofilm an Surface roughness 35

Surface modifications- effecting P.gingivalis adherence 40

Titanium implant biomaterial: effecting LipopolySachharide(LPS) adherence 43

Surface free energy 48

Chemical composition of surface 50

Implant abutment fit 51

Periimplantitis is an inflammatory process affecting the tissues around an

Hence the importance of biofilm in implant prosthetics is a field to facilitate the

THE NATURE OF BIOFILMS

A crude analogy to the development of a biofilm might be the development of a

interrelated activities. Inhabitants of cities or biofilms are capable of "metabolic

Biofilms are composed of microcolonies of bacterial cells (15–20% by volume)

Exopolysaccharides – the backbone of the biofilm

Exopolysaccharides can exist in both ordered or disordered forms. At high

The chemical composition and tertiary structure of the exopolysaccharides will

enhancing substrate utilization by bacterial cells. Exopolysaccharides are effective in

Physiological heterogeneity within biofilms

FACTORS AFFECTING BIOFILM DEVELOPMENT AND

Biofilms can also be affected by changes in nutrient concentration. Stoodley et al.

Detachment of cells from biofilms

The detachment of cells from biofilms is essential to allow colonization of new

Related to the phenomenon of detachment of biofilm cells in groups is the

As will be discussed later, A. naeslundii is one of the most important colonizing

the presence of fimbrial-like structures on Peptostreptococcus micros, although their role

 An increased metabolic diversity and efficiency (molecules that are normally

 An enhanced resistance to environmental stress, antimicrobial agents, and the host

Recent studies suggest that the environmental heterogeneity generated within

The consensus definition of biocompatibility is “the ability of a material to

The biomaterial is “a nonviable material used in a medical device, intended to

Materials designed with an emphasis on biologic response centered on coating

3 interactive and dynamic components are involved in designing material or biologic

 Chemical nature of biomaterial surface

 Microbial and host cellular response

MATERIAL INTETRACTION WITH BIOLOGICAL

CHEMICAL NATURE OF THE BIOMATERIAL SURFACE

Synthetic material placed in mouth is exposed to salivary protein rich solution

Molecules are absorbed to a surface by physical forces which includes hydrogen

Selective adsorption of protein at interfaces is uniquely determined both by

MEDIATING PELLICLE LAYER

A dental implant placement can be surrounded by mixture of plasma and cell

Selective macromolecules also show a pattern of selective adsorption to metal

Albumin, prealbumin, immunoglobuling have been identified as proteins

Albumin and fibrinogen have been reported to adsorb to titanium alloys.

MICROBIAL AND HOST CELL RESPONSE

critical in bacterial adhesion to oral pellicles. Bacteria possess protein-like surface

DESIGNING BIOCOMPATIBLE MATERIAL

SURFACES CAN BE MODIFIED TO ALTER PELLICLES

Schemes for surface modification.

After recognition of surface changes that may have occurred as a result of

Chemical alteration of surface layers to optimize adsorption of desired

Physical immobilization in which molecules remain at surfaces by electrostatic or

Modification of metal surfaces

New materials and tissue analogs

INSTRUMENTATION FOR ANALYSIS OF