DEPARTMENT OF PROSTHODONTICS

SRM DENTAL COLLEGE AND HOSPITAL

RAMAPURAM, CHENNAI

SEMINAR

ROLE OF PLATELET RICH FIBRIN IN PROSTHODONTICS

PRESENTED BY

AKSHAYAA.B

PG I- YEAR

GUIDED BY

DR.K. MURUGESAN, MDS DR .MUTHUKUMAR MDS

PROFESSOR PROFESSOR & HOD

DR.S.SUGANYA, MDS

READER
CONTENTS:

1. INTRODUCTION
2. EVLOUTION OF PLATELET CONCENTRATES
3. PROPERTIES OF PLATELET CONCENTRATES
4. PLATELET RICH FIBRIN
5. ROLE IN TISSUE ENGINEERING
6. CLINICAL APPLICATION
7. DRAWBACKS
INTRODUCTION:

Regenerative medicine holds promise for the restoration of tissues and organs
damaged by disease, trauma, cancer, or congenital deformity. Regenerative
medicine can perhaps be best defined as the use of a combination of cells,
engineering materials, and suitable biochemical factors to improve or replace
biological functions in an effort to effect the advancement of medicine.
The basis for regenerative medicine is the utilization of tissue engineering
therapies. Probably the first definition of tissue engineering was by Langer and
Vacanti who stated it was “an interdisciplinary field that applies the principles
of engineering and life sciences toward the development of biological
substitutes that restore, maintain, or improve tissue function.”1 MacArthur and
Oreffo defined tissue engineering as “understanding the principles of tissue
growth, and applying this to produce functional replacement tissue for clinical
use.”
The term tissue engineering was originally coined to denote the construction in
the laboratory of a device containing viable cells and biologic mediators (e.g.,
growth factors and adhesins) in a synthetic or biologic matrix, which could be
implanted in patients to facilitate regeneration of particular tissues.
The role of tissue oxygenation in wound healing became the focal point in the
1980s. Tissue oxygenation enhances phagocytic and bactericidal ability of host
immune cells and supports collagen as well as other protein synthetic events.
The importance of growth factors in enhancing wound healing has become the
focus of research in the present day. In addition, a link has been established
between tissue oxygenation and growth factors.

Macrophage stimulation causes the release of angiogenic and other growth

factors that support wound healing and resist infection. In general, tissue
engineering combines three key elements, namely scaffolds (Collagen, bone
mineral), signaling molecules (Growth factors), and cells (Osteoblasts,
fibroblasts). Tissue engineering has been redefined presently as the relatively
new, highly promising field of reconstructive biology, which draws on the
recent advances in medicine and surgery, molecular and cellular biology,
polymer chemistry, and physiology.
Bone graft materials commonly used are demineralized freeze-dried bone
allograft (DFDBA) and freeze-dried bone allograft (FDBA). The osteoinductive
properties of DFDBA have made it the grafting material of choice as compared
to FDBA, xenografts, and alloplasts. However, the osteoinductive potential of
DFDBA procured from different bone banks or from different batches of the
same bank may vary highly. The bioactivity of DFDBA seems to be dependent
on the age of the donor; the younger the donor the more osteoinductive the graft
material.6 This controversy as well as concerns about disease transmission has
pushed clinicians toward using xenografts and alloplastic materials. Although
these materials are biocompatible and are osteoconductive in nature, clinical
outcomes are unpredictable. The problem that arises next is how to improve
clinical outcomes by improving the properties of these grafts. Some commonly
used materials in regenerative procedures include guided tissue regeneration,
guided bone regeneration, distraction osteogenesis and more recently introduced
emdogain and preclinical trials on use of fibroblast growth factor 2 (FGF2) for
periodontal regeneration.
In the past two decades, an increased understanding of the physiological roles of
platelets in wound healing and after tissue injury has led to the idea of using
platelets as therapeutic tools. Platelet-Rich Plasma (PRP) consists of a limited
volume of plasma enriched with platelets, which is obtained from the patient.
The use of PRP as a potentially ideal scaffold for regenerative therapy and has
been documented in the literature. However, the use of bovine thrombin for the
activation of Platelet Rich Plasma (PRP) has been an issue of controversy which
led to the development of the second generation platelet concentrate known as
Platelet Rich Fibrin (PRF) which is totally autologous in nature. It is very
simple and inexpensive. PRF contains platelets, growth factors, and cytokines
that might enhance the healing potential of both soft and hard tissues.

EVOLUTION OF PLATELET CONCENTRATES:

Platelets are small, irregularly shaped anuclear cells, 2-4 μm in diameter, which
are derived from fragmentation of precursor megakaryocytes. The average life
span of a platelet is between 8 and 12 days. Platelets play a fundamental role in
hemostasis and are a natural source of growth factors. Growth factors stored in
the α-granules of platelets include platelet derived growth factor, insulin-like
growth factor, vascular endothelial growth factor, and transforming growth
factor-β.
The release of growth factors is triggered by the activation of platelets, which
may be initiated by a variety of substances or stimuli, such as thrombin, calcium
chloride, collagen or adenosine 5c-diphosphate. In addition to these growth
factors, PRP contains fibrinogen and a number of adhesive glycoproteins that
support cell migration.
In general, platelet concentrates are blood-derived products used for the
prevention and treatment of hemorrhages due to serious thrombocytopenia of
the central origin. Platelet concentrates have been developed to be used as
bioactive surgical additives that are applied locally to promote wound healing
stems from the use of fibrin adhesives. Since 1990, medical science has
recognized several components in blood, which are a part of the natural healing
process; when added to wounded tissues or surgical sites, they have the
potential to accelerate healing.
Fibrin glue was originally described in 1970 and is formed by polymerizing
fibrinogen with thrombin and calcium. It was originally prepared using donor
plasma; however, because of the low concentration of fibrinogen in plasma, the
stability and quality of fibrin glue were low.
These adhesives can be obtained autologously from the patient or can be
obtained commercially. These products are heat-treated, thus immensely
reducing, but not entirely eliminating, the risk of disease transmission.
Therefore, the commercially available adhesives constitute an infinitely small
risk of disease transmission.

PROPERTIES OF PLATELET CONCENTRATES:

1. Increase tissue vascularity through increased angiogenesis.

2. Enhancing collagen synthesis.
3. Enhancing osteogenesis.
4. Increasing the rate of epithelial, and granulation tissue production.
5. Antimicrobial effect.
6. Reaction with other material: PRP does not react or interfere with any other
restorative material glass ionomer cements or composite resin used as filling
material are not affected by it.
7. Biocompatibility: PRP offers a biologically active substance with the release
of growth factor.
8. Tissue regeneration: PRP allows regeneration of tissue with the release of
growth factors.
Platelet rich plasma gel (PRP gel) is an autologous modification of fibrin glue
obtained from autologous blood used to deliver growth factors in high
concentrations. It is an autologous concentration of human platelets in a small
volume of plasma, mimics coagulation cascade, leading to formation of fibrin
clot, which consolidates and adheres to application site. Its biocompatible and
biodegradable properties prevent tissue necrosis, extensive fibrosis and promote
healing.
PRP contains high concentration of platelets and native concentration of
fibrinogen. The alpha granules of platelets include a high concentration of
factors, which are released on activation of s. PRP obtained from autologous
blood is used to deliver growth factors in high concentrations to the site of bone
defect or a region requiring augmentation.
A blood clot is the center focus of initiating any soft-tissue healing and bone
regeneration. In all natural wounds, a blood clot forms and starts the healing
process. PRP is a simple strategy to concentrate platelets or enrich natural blood
clot, which forms in normal surgical wounds, to initiate a more rapid and
complete healing process. A natural blood clot contains 95% red blood cells,
5% platelets, less than 1% white blood cells, and numerous amounts of fibrin
strands. A PRP blood clot contains 4% red blood cells, 95% platelets, and 1%
white blood cells.9
Sanchez et al. have elaborated on the potential risks associated with the use of
PRP. The preparation of PRP involves the isolation of PRP after which gel
formation is accelerated using calcium chloride and bovine thrombin. It has
been discovered that the use of bovine thrombin may be associated with the
development of antibodies to the factors V, XI and thrombin, resulting in the
risk of life-threatening coagulopathies. Bovine thrombin preparations have been
shown to contain factor V, which could result in the stimulation of the immune
system when challenged with a foreign protein.
Other methods for safer preparation of PRP include the utilization of
recombinant human thrombin, autologous thrombin or perhaps extrapurified
thrombin. Landesberg et al. have suggested that alternative methods of
activating PRP need to be studied and made available to the dental community.
Other drawbacks about the use of PRP include legal restrictions on handling the
blood and also controversies in the literature regarding the benefits and clinical
outcome of use of PRP. All these have led to the generation of a new family of
platelet concentrate called platelet-rich fibrin which overcomes many of the
limitations of PRP.
The purpose of this review article is to describe a novel second-generation
platelet concentrate called PRF, which is an improvement over the traditionally
prepared PRP for use in regenerative dentistry.

PLATELET RICH FIBRIN

PRF was first developed in France by Choukroun et al. in 2001. This second-
generation platelet concentrate eliminated the risks associated with the use of
bovine thrombin.
Platelet-rich fibrin (PRF) contains platelets and growth factors in the form of
fibrin membranes prepared from the patient’s own blood free of any
anticoagulant or other artificial biochemical modifications.
The PRF clot forms a strong natural fibrin matrix, which concentrates almost all
the platelets and growth factors of the blood harvest and shows a complex
architecture as a healing matrix with unique mechanical properties which makes
it distinct from other platelet concentrates.
PRF is superior to other platelet concentrates like PRP due to its ease and
inexpensive method of preparation and also it does not need any addition of
exogenous compounds like bovine thrombin and calcium chloride. It is
advantageous than autogenous graft also because an autograft requires a second
surgical site and procedure. Thus PRF has emerged as one of the promising
regenerative materials.

PREPARATION OF PRF:

The protocol for PRF preparation is very simple and simulates that of PRP. It
includes collection of whole venous blood (Around 5 ml) in each of the two
sterile tubes (6ml) without anticoagulant and the tubes are then placed in a
centrifugal machine at 3,000 revolutions per minute (rpm) for 10 min, after
which it settles into the following three layers: Upper straw-colored acellular
plasma, red-colored lower fraction containing red blood cells (RBCs), and the
middle fraction containing the fibrin clot. The upper straw-colored layer is then
removed and middle fraction is collected, 2 mm below to the lower dividing
line, which is the PRF. The mechanism involved in this is; the fibrinogen
concentrated in upper part of the tube, combines with circulating thrombin due
to centrifugation to form fibrin.
A fibrin clot is then formed in the middle between the red corpuscles at bottom
and acellular plasma at the top. The middle part is platelets trapped massively in
fibrin meshes. The success of this technique entirely depends on time gap
between the blood collection and its transfer to the centrifuge and it should be
done in less time. The blood sample without anticoagulant, starts to coagulate
almost immediately upon contact with the glass, and it decreases the time of
centrifugation to concentrate fibrinogen. Following proper protocol and quick
handling is the only way to obtain a clinically usable PRF clot charged with
serum and platelets. Resistant autologous fibrin membranes may be available by
driving out the fluids trapped in fibrin matrix.
Because of the absence of an anticoagulant, blood begins to coagulate as soon
as it comes in contact with the glass surface. Therefore, for successful
preparation of PRF, speedy blood collection and immediate centrifugation,
before the clotting cascade is initiated, is absolutely essential. PRF can be
obtained in the form of a membrane by squeezing out the fluids in the fibrin
clot.
PRF also contains physiologically available thrombin that results in slow
polymerization of fibrinogen into fibrin which results in a physiologic
architecture that is favorable to wound healing. The cytokines which are present
in platelet concentrates play an important role in wound healing.
The structural configuration of PRF with respect to cytokine incorporation in
fibrin meshes is different from that present in PRP. The natural polymerization
in PRF results in increased incorporation of the circulating cytokines in the
fibrin meshes (Intrinsic cytokines). These intrinsic cytokines will be having an
increased lifespan and they will be released and used only at the time of initial
cicatricial matrix remodeling which creates a long term effect.
Another added advantage of PRF is the presence of natural fibrin network
which protects the growth factors from proteolysis. PRF also favors the
development of micro-vascularization leading to endothelial growth factor and
glycoproteins such as thrombospondin-1.16 Leukocytes that are concentrated in
PRF scaffold play an important role in growth factor release, immune
regulation, anti-infectious activities and matrix remodeling during wound
healing.
The slow polymerization mode of PRF and cicatricial capacity creates a
physiologic architecture favorable for wound healing

ROLE IN TISSUE ENGINEERING

The a-granules present in platelets contain growth factors like platelet derived
factor (PDGF), transforming growth factor-b (TGF-b), vascular endothelial
growth factor (VEGF), and epidermal growth factor (EGF). Platelet derived
growth factor (PDGF) has an important role in periodontal regeneration and
wound healing and receptor for PDGF is present on gingiva, periodontal
ligament and cementum and it activates fibroblasts and osteoblasts promoting
protein synthesis. PDGF also functions as a chemo attractant for fibroblasts and
osteoblasts in gingiva and periodontal ligament resulting in their activation.
PRF promotes angiogenesis because as it has low thrombin level optimal for the
migration of endothelial cells and fibroblasts. PRF entraps circulating stem cells
due to its unique fibrin structure. This property of PRF finds application in
healing of large osseous defects where there is migration of stem cells
differentiating into osteoblast phenotype.
PRF also helps in facilitating adhesion and spreading of cells, regulates gene
expression of growth factors, growth factor receptors, proteins, and determines
the outcome of a cell’s response to growth factors due to the presence of
collagen, fibronectin, elastin, other non-collagenous proteins, and proteoglycan
in the extracellular matrix of PRF.
The use of PRF as a tissue engineering scaffold was investigated by many
researchers for the past few years. In a study by Gassling et al. reported that
PRF appears to be superior to collagen as a scaffold for human periosteal cell
proliferation and PRF membranes can be used for in vitro cultivation of
periosteal cells for bone tissue engineering.
PRF has immune functions like chemotaxis as leukocytes present in PRF
degranulates during activation and releases cytokines like IL-1, IL-4, IL-6 and
TNF-a. PRF also contains anti-inflammatory cytokine such as IL-4 which
requires further research
Thus PRF is a potential tool in tissue engineering but clinical aspects of PRF in
this field requires further investigation.

CLINICAL APPLICATION:

In oral and maxillofacial surgery to improve bone healing in implant dentistry.

The most common encountered problems are lack of adequate bone and
proximity to anatomic structures at the implantation site and recent
advancements of PRF usage in surgical procedures can predictably combat such
difficulties. In combination with freeze-dried bone allograft (FDBA) in sinus
floor elevation to enhance bone regeneration.
In various bone reconstruction procedures PRF could provide a possible new
bone. Mazor et al., stated that use of PRF as the sole filling material during a
simultaneous sinus lift and implantation procedure had stabilized a good amount
of regenerated bone in the subsinus cavity up to the tip of implants in a case
series through a radiological and histological evaluation at after 6 months from
the surgery.
PRF membranes protects the surgical site; promotes soft tissue healing; and
when its fragments mixes with graft material, it functions as a “biological
connector” between the different elements of graft and acts as a matrix which
supports neoangiogenesis, capture of stem cells, and migration of
osteoprogenitor cells to the center of graft.
PRF plugs can also be used in treating the residual extraction sockets. Use of
autologous PRF in extracted socket filling after immediate bone augmentation
using titanium membranes applied to the socket walls and primary closure was
found to be feasible and safe with adequate bone filling after 8 weeks or above
for implant fixation.
Anil kumar et al., has reported PRF as a potential novel root coverage approach
for treating gingival recession in mandibular anterior teeth using combined
laterally positioned flap technique and PRF membrane. Combined use of PRF
and bone graft with good results has also been reported for combined
periodontic-endodontic furcation defect.
Aroca et al., in the 6 month of their randomized clinical trial, concluded that
addition of a PRF membrane positioned under the MCAF (Modified coronally
advanced flap) provided inferior root coverage, but an additional gain in
gingival/mucosal thickness (GTH) at 6 months compared to conventional
therapy.
Revitalization of necrotic infected immature tooth is possible under conditions
of total canal disinfection and PRF is an ideal biomaterial for pulp-dentin
complex regeneration.
PRF membrane has been used as a barrier membrane over a large bony defect to
maintain a confined space for the purpose of guided tissue regeneration. PRF in
conjunction with Hydroxyapatite crystals can accelerates the resorption of the
graft crystals and induce the rapid rate of bone formation.
Combination of PRF as a matrix and MTA as an apical barrier is considered as
a good option for creating artificial root end barrier.
DRAWBACKS:

The main shortcoming of PRF is its preparation and storage.

The clinical benefit of PRF depends on time interval between speed of handling
between blood collection and centrifugation as PRF is prepared without any
addition anticoagulants.
PRF storage after preparation. PRF membranes should be used immediately
after preparation as it will shrink resulting in dehydration altering the structural
integrity of PRF.
Dehydration also results in the decreased growth factor content in PRF and
leukocyte viability will be adversely affected altering its biologic properties.
PRF when stored in refrigerator can result in risk of bacterial contamination of
the membranes. These limitations with the use of PRF can be circumvented by
sticking onto a standard protocol for preparation and preservation.