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DIPLOMARBEIT

Systematische Übersichtsarbeit für neuartige Papilla-


augmentationstechniken
Systematic review for novel papilla augmentation techniques

Zur Erlangung des akademischen Grades

Doktorin der Zahnheilkunde

(Dr. med. dent.)

an der

Medizinischen Universität Wien

ausgeführt an der

Parodontologische Abteilung der Universitätszahnklinik Wien

unter der Anleitung von

Assoc. Prof. in Univ.-Doz. DDr.Xiaohui Rausch-Fan

OA Univ.Ass.Dr.Michael Müller

und

eingereicht von

Sanja Milosavljevic

Matrikelnummer: n 1442842

ͳ

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ʹ

Danksagung

Es ist mir ein persönliches Anliegen, folgenden Personen meinen Dank

auszusprechen:

Frau Assoc. Prof.in Univ.-Doz.in DDr.in Xiaohui Rausch-Fan und Herr Univ.

Asis. Dr. Michael Müller danke ich für die Betreuung und Unterstützung bei der

Verfassung der vorliegenden Arbeit.

Meiner Kollegin Frau Dr. Jelena Djordjevic danke ich für die Unterstützung und

große Hilfe, um diese Arbeit zum Abschluss zu bringen.

Meine Eltern, Boza und Dusica Milosavljevic, haben mich zeitlebens unterstützt.

Ihnen gilt mein großer Dank. Auch meiner Schwester Nevena Milosavljevic

möchte ich danken. Sie war und ist mir eine Quelle der Inspiration.

Ich bedanke mich auch bei allen meinen Freunden und Freundinnen, die mir

während des Studiums ihre Zeit widmeten, mir zuhörten, meine Sorgen mit mir

teilten und mich ermutigten.

͵

Table of Content


1. ZUSSAMENFASSUNG.....................................................................................................7

2. ABSTRACT ........................................................................................................................8

3. INTRODUCTION .............................................................................................................9
3.1. Definition .......................................................................................................................9
3.2. Historical background of platelet aggregation............................................................10
3.3. Old methods in the reconstruction of papilla.....................................................................11

4. FUNDAMENTALS ..........................................................................................................14
4.1.Papillary anatomy and morphology ...................................................................................14
4.2.Factors influencing the presence of papilla .......................................................................15
4.2.1. Crestal bone height......................................................................................................16
4.2.2. Periodontal biotype .....................................................................................................17
4.2.3. Tooth form and shape .....................................................................................................17
4.2.4. Interproximal distance of roots ...................................................................................18
4.3.Classification of the interdental papilla loss ......................................................................18

5. STRATEGIES OF TISSUE ENGINEERING ..............................................................20


5.1.Cell source ..........................................................................................................................21
5.1.1. Dental pulp stem cells .................................................................................................22
5.1.2. Stem cells from exfoliated deciduous teeth......................................................................22

5.1.3. Stem cells from apical papilla....................................................................................23

5.1.4. Periodontal ligament stem cells..................................................................................23

5.1.5. Dental follicle precursor cells.........................................................................................24

5.2. Scaffold or supporting matrix............................................................................................25

5.2.1. Biomaterials used as scaffolds...................................................................................26

5.2.2. Ceramics.....................................................................................................................26

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5.2.3. Polymers.....................................................................................................................26

5.3. Signalling molecules...........................................................................................................29

5.3.1. Platelet derived growth factor (PDGF).................................................................... .30

5.3.2. Fibroblast growth factor (FGF).................................................................................30

5.3.3. Bone morphogenetic protein (BMP)...............................................................................31

5.3.4. Insulin-like growth factor (IGF).......................................................................................31

5.3.5. Transforming growth factor-ȕ 7*) .............................32

5.3.6.Periodontal ligament derived growth factor.....................................................................32

6. CLASSIFICATION OF PLATELET CONCENTRATE .............................................34


7. AVAILABLE PRF.............................................................................................................35

7.1. Principles of PRP preparation........................................................................................36

7.2. Principles of PRF preparation.....................................................................................37


7.3. Principles of CGF preparation...........................................................................................38

7.4. Physiological working principle of growth factors........................................................40

7.5. Steps for obtaining the final platelet products................................................................41

8. STUDY PURPOSE .............................................................................................................47

9. MATERIAL AND METHODS...........................................................................................47

10. RESULTS...........................................................................................................................52

10.1. HY as monotherapy or as adjunct to non-surgical/surgical periodontal treatment.........52

10.2. HY as monotherapy by implant supported crowns............................................................54

10.3. Fibroblast injection......................................................................................................55

10.4. TEP injection...................................................................................................................56

10.5. PRF in periodontal treatment..........................................................................................56

11.DISCUSSION......................................................................................................................60

12.FUTURE DIRECTIONS/CONSIDERATIONS..............................................................64

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13. CONCLUSION ........................................................................................................... .65
14. REFERENCE..................................................................................................................66

15.SUPPLEMENT................................................................................................................76
15.1. List of abbreviations...............................................................................................75
15.2. List of figures..........................................................................................................77
15.3. List of Tables...........................................................................................................78

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1. Zussamenfassung
Einer der komplexesten Prozesse in unserem Körper ist die Regeneration von parodontalem
Gewebe. Dies stellt immer noch eine große wissenschaftliche Herausforderung dar. Die
Forschung sucht nach effektiven Methoden zur Behandlung von Parodontitis und
Rekonstruktion verloren gegangener Strukturen, die vorhersagbar und langfristig sind.

Ziel: Diese Literaturübersicht beschäftigt sich mit dem Problem der Rekonstruktion interdentaler
Papillen in der ästhetischen Zone. Frühere wissenschaftliche Studien und Methoden haben auf
lange Sicht Schwierigkeiten gezeigt Leistung und Effizienz dieser Therapie zu objektivieren. Der
Zweck hinter dem Schreiben dieser Arbeit ist, die Anwendung von Stammzellen in
verschiedenen Bereichen der Zahnmedizin aufzuzeigen, mit dem Schwerpunkt auf
Rekonstruktion der Interdentalpapille durch Verwendung von Thrombozyten-reichen
Blutderivaten und den Einfluss von Wachstumsfaktoren (GF) auf das Verhalten der
Stammzellen.
Methodeǣ Diese Studie führt eine systematische Überprüfung (SR) von randomisierten
kontrollierten Studien durch (RCT s). Die Frage, ob Thrombozytenwachstumsfaktoren für die
Behandlung von verloren gegangenen interdentaler Papillen eingesetzt werden können und
gleichzeitig eine Verbesserung der Ästhetik beim Patienten bewirken können und somit
langfristig für zufriedenstellende Ergebnisse sorgen. Für die Datensammlung wurde Pubmed,
Cochrane-Bibliotheken, EMBASE und ausgewählte Zeitschriften verwendet.

Ergebnisse: Die Ergebnisse der vorliegenden Untersuchung unter Verwendung von


Thrombozytenderivaten zeigten, dass Tissue Engineering in der parodontalen Behandlung für die
ästhetische Verbesserung des "schwarzen Dreiecks" nützlich sein können, aber Studien mit einer
größeren Stichprobengröße notwendig wären, um die Nützlichkeit dieser Methoden in der
Zukunft zu bestätigen.

Fazit: In der Literatur wurde über mehrere Ansätze zur Behandlung von "schwarzen Dreiecken"
berichtet, aber die meisten dieser Studien betrachten Fallserien mit kurzen Follow-up- und
unvorhersehbaren Ergebnissen. Aufgrund der großen Heterogenität der Studien kann keine
Empfehlung über die Art der Anwendung getroffen werden. Es zeigte sich aber eine verbesserte
Wundheilung durch Einsatz dieser Materialien.

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2. Abstract


One of the most complex processes in our body is regeneration of periodontal tissue. To find the
most effective method in the treatment of periodontal disease which has predictable and long-
term successful results still represents a major scientific challenge.

Aim: This literature review deals with the problem of reconstruction of inter-dental papilla in the
aesthetic area. Previous scientific studies and methods have shown difficulties in the long-term
performance and efficiency of this therapy. The purpose behind writing this thesis is to highlight
the application of stem cells in various fields of dentistry, with the main emphasis on
reconstruction of inter-dental papilla by using platelet-rich blood derivatives and the influence of
growth factors (GF) on the behaviour of stem cells.
Methods: This study represents a systematic review (SR) of randomized controlled trials (RCT
s) to inspect if platelet derived growth factors used in treatments of lost inter-dental papilla may
upgrade aesthetics at patient and provide a long-term satisfactory results.

For data collection Pub Med, Cochrane libraries, EMBASE and selected journals were used.
Results: Results of the presented studies using platelet derivatives indicated that tissue
engineering in periodontal treatment could be useful for the aesthetic improvement of Äblack
triangle´ but a study with a larger sample size would be necessary to confirm usefulness of these
methods in the future.

Conclusion: Several approaches for treating Äblack triangle´ have been reported in literature but
most of these studies considered case series with short follow-up and unpredictable outcomes.
Due to a large heterogeneity of the studies, a recommendation about the mode of application
cannot be made. However, improved wound healing through a use of these materials has been
shown.

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3. Introduction

3.1. Definition
One of the important factors in the field of restorative dentistry is to achieve a satisfactory
gingival aesthetic. One of the consequences of periodontal disease is the loss of inter-dental
papillae, which leads not only to aesthetic and phonetic problems, but also to the difficulty in
maintaining oral hygiene which affects the health of the teeth and surrounding tissues.
Therapeutic methods of augmentation inter-dental papillae are intended to fill a black triangle
but still represent one of the complicated cosmetic procedures today [1].

One of the possible techniques in periodontal tissue regeneration proposed Langer in 1993 by
tissue engineering. Tissue engineering is a multidisciplinary field that, with the help of stem
cells, bioactive molecules or synthetic product obtained in the laboratory, returns the function of
tissues and organs [2].

Progenitor stem cells represent a major breakthrough in medicine because of their two major
properties: they are capable of self-renewal and, upon division they can give rise to cells that
have the potential to differentiate [3].

On the basis of their ability to differentiate (cell potency), three types of stem cells have been
constituted: totipotent stem cells ± each cell has the capability of evolving into an whole
organism, (2) pluripotent stem cells ± embryonic stem cells that were grown in vivo under
induced requirement and are able to differentiate into all types of tissue, and (3) multipotent stem
cells ±postnatal stem cells or adult stem cells with the ability of multiline age differentiation. The
presence of postnatal pluripotent cells is determined in many organs including the dental tissue
[4].

Except progenitor cells, tissue engineering includes two other important elements such as
scaffold or supporting matrix and bioactive molecules (growth factors) [5].

Platelets, a significant reservoir of growth factors in the body, have a major part in many
processes such as coagulation, immune response, angiogenesis and the healing of damaged

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tissues. In the alpha-granules of platelets is located a number of important proteins such as :
platelet-derived growth factor (PDGF), transforming growth factor (TGF), platelet factor
interleukin (IL), platelet-derived angiogenesis factor (PDAF), vascular endothelial growth factor
(VEGF), epidermal growth factor (EGF) and insulin-like growth factor IGF [6].

With support of the latest methods and apparatus, blood products such as platelet-rich plasma
(PRP) and platelet-rich fibrin (PRF) which represent autologous cocktail of growth factors, can
be successfully isolated from venous blood sample of the patient [5].

Depending on the technique of obtaining platelet aggregates from the blood that has been
modified over time, these products can be divided into two generations and the first attempt to
isolate them from blood was described in 1999.

Platelet-rich blood derivatives, such as platelet-rich plasma (PRP) and platelet-rich fibrin (PRF)
as a second-generation platelet concentrate have consistently shown to potentiate stem cell
proliferation, migration, and differentiation [7].

Stem cells are cells that have the ability to re-establish themselves through mitosis and can
differentiate into a several specialized cell lineages [8].

3.2. Historical background of platelet aggregation

In promoting periodontal wound repair in clinical studies as first generation of platelet


concentrate were described platelet-rich plasma (PRP) and platelet-rich growth factor (PRGF).

The first methods were based on adding a clotting substance to a blood sample to achieve a gel
consistency suitable for application.

Marx et al.1999 first introduced PRP but did not bring great results in practice because of
addition of animal-derived thrombin for clotting. PRGF was introduced by Anitua et al.2001
ͳͲ

who modified the procedure for replacing the animal-derived thrombin with calcium chloride to
obtain a gel condition. Because of the failure to apply these preparations with pipetting methods,
Choukroun et al. 2006 presented a second generation of platelet concentrates platelet- rich fibrin
(PRF) [9] [10] [11].

PRF was prepared from the blood sample in the absence of anti-coagulants and immediately
centrifuged to fibrin form. Choukroun et al. modified further PRF in advance form (A-PRF)
which was supposed to contain white blood cells (WBC).The procedure used low speed
centrifugation which resulted in obtaining a softer fibrin sample. This method has been changed
by Sacco L. Lecture 2006 who introduced a second modified form of PRF, concentrated growth
factors (CGF).With rolled-speed centrifugation rigidly form of fibrin clot was obtained which
showed a better regenerative capacity [12].

Growth factors have an important role to repair or generate damaged tissue. Most of the growth
factors are in blood plasma and platelets. In the field of dentistry and maxillofacial surgery such
as sinus augmentation, ridge augmentation, periodontal regeneration and soft tissue healing PRP
has been widely used in order to overcome all the disadvantages in the application of already
known methods in the reconstruction of the interdental papilla.

3.3. Old methods in the reconstruction of papilla

Known methods used to reconstruct interdental papilla in periodontal literature have been
described as surgical and non-surgical methods.

In the non-surgical methods, modification of oral hygiene fails in cases of papillae loss due to
inadequate techniques brushing. Eliminating the traumatic effects on papilla is possible to
achieve complete re-epithelization.

ͳͳ

In 1985, Shapiro presented one of the non-invasive techniques and it had shown that repeated
Äscaling and root planning´ and curettage lead to a gingival hyperplasia, followed by gingival
tissue repair [13].

Among the surgical techniques, pedicle flaps, free gingival and connective tissue graft may be
used. Currently there is no predictable surgical procedure to retrieve the interdental papilla. Field
of reconstructive surgery has not found yet a method for a successful and long-term
reconstruction of papilla.

In 1985 by Han and Takei and in 1986 by Tarnow, first free soft tissue graft had been introduced.
They have described a technique in which the interdental papilla was moved and placed
coronally and a connective tissue graft was placed below the papilla. Using half-moon shaped
incision which simultaneously follows the labial free gingival margin, flap is dissected coronary
and moved to cover an exposed root. To fill the interdental space graft is positioned below the
deficient region. In this procedure no sutures are required, there is no tension on the flap, there is
no shortening of the vestibule, and the existing papillae are not interfered with flap.
Unfortunately, surgical procedures must be repeated one to two times after the period of three
months after the first operation. This technique is called papilla preservation flap (PPF) [1].

In 1995, Cortellini et al. proposed a modification in the PPF and named it as Modified Papilla
preservation flap (MPPF) and in 1999, he proposed a simplified papilla preservation flap (SPPF)
that requires a releasing incision in the papillary area and placement of a barrier membrane under
the surgical site [14].

A little earlier, Beagle (1992) presented a method to reconstruct interdental papilla using the
principles of Abram¶s roll technique and Evian¶s papilla preservation technique. This technique
has disadvantages because there is a risk of damage to the incisive nerves and vessels and
incorporation of fat in the undersurface of the flap. The aim of this method was to avoid another
surgical intervention but this procedure has not found wide resonance and application [15].

ͳʹ

In 1998, Azzi et al. applied a papilla reconstruction using subepithelial graft jointed with a partial
flap which is lifted in the labial and palatal side, to leave the space for the conjunctive graft
which is displayed from the area of the tuberosity. The labial and palatal flaps are stitched up
jointly and subepithelial graft is placed below them. To enlarge the volume of interdental tissue,
in 2001 Azzi et al. introduced a modified method in which he used autogenous bone graft from
maxillary tuberosity together with a connective tissue graft from palatum [1].

Wu et al. 2003 described that flap surgery has shown better results than the free gingival graft
[16].

Multiform techniques have been given in the past to reconstruct the papilla by surgical methods,
but very less data are accessible about the long-term success and predictability in these methods.

The major limiting factor for all surgical techniques is inadequate blood supply because
interdental papilla is a small tissue with a low blood supply, which originates from multiple
sources [15].

Most surgical interventions lead to tissue necrosis and therefore hinder the success of surgical
therapy. In addition to a low blood amount which limits the success of surgical procedures, the
success of therapy also depends on the shape and biotype of the gingiva, as well as crestal bone
height and interproximal distance.

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4. Fundamentals

4.1. Papillary anatomy and morphology

Inter-dental papilla is the gingival part between two contiguous teeth and plays a protective role
on the periodontium, including the alveolar bone crest. It is also acting as a biological barrier
against outside aggressors and inhibiting food accumulation. Height of the alveolar bone,
distance between the teeth and interdental contact point (Fig.1) have an impact on the form of
interdental papilla [1] [17].

)LJ$QWHULRUYLHZRIWKHORZHUSRUWLRQRIDQDGXOWVNXOO 0RGLILHGIURP=HLV]5&1XFNROOV-'HQWDO
$QDWRP\6W/RXLV0RVE\ 

First who described the morphology of the papilla was Cohen in 1959, as a part of the gingiva
and called it interdental gingiva. Interdental papilla in the incisal region has the shape of a
pyramid with the tip located just below the contact points while in posterior region it has a
concave cole of bridge shape [18].

ͳͶ

Due to the different morphology and position of teeth, contact point between the upper central
incisors is placed in the incisal third of labial surface, between upper central and lateral incisors
is placed in the middle third, while between the lateral incisors and canine teeth the contact point
is located at the apical third [18]. With other words between upper central incisors, there is the
largest area that should be filled with papilla and it is the most visible aesthetic zone [1].

4.2. Factors influencing the presence of papilla

The appearance and presence of papilla could be affected by many factors such as crestal bone
height, interproximal distance, tooth shape and gingival biotype (Fig.2) [19].

Ž˜‡‘Žƒ”
„‘‡Ȁ‹–‡”’”‘š‹ƒŽ
…‘–ƒ…–
‡”‹‘†‘–ƒŽ„‘‡

),*-Etiology pyramid of the gingival black space

Source: Sharma and Park

ͳͷ

4.2.1. Crestal bone height

Crestal bone height as one of the influencing factors on the presence of interdental papilla means
that osseous crest follows the shape of the cementum-enamel junctions and the position of the
interproximal bone is more coronal than the radicular bone. When this distance (Fig.3) is 5.0 mm
or less between two teeth, the papilla filled 100% of the interproximal space while at 6 mm it
dropped to 56% and at 7 mm it only filled 27% , according to Tarnow et al. in 1992[18].

This measures show that if the distance between alveolar crest and the contact point equal to or
less than 5 mm is, the periodontal tissue is considered as healthy tissue. However the distance
between 5 and 6 mm is the most critical and leads to the formation of a black triangle [1].

Periodontal biotype is also beside the crestal bone height one of the essential factors that can
affect the gingival recession after intervention.

)LJ-(1) Distancefrom the interdental contact point to the bone crest and the presence or
absence of "black gingival triangles". (2) The height of the interdental papilla should be 4.5 mm.
Tarnow et al. (1992)

ͳ͸

4.2.2. Periodontal biotype

Several classifications have been proposed for gingival/periodontal biotypes and there is no
universally accepted classification for gingival/periodontal biotypes. There is also a lack of
agreement between authors. However, gingival biotypes were initially classified in two groups
by Oschenbein and Ross as Äscalloped and thin´ and Äflat and thick´. Other authors, Seibert and
Lindhe have proposed classification into a WKLFN• 2mm DQGWKLQELRW\SH” 1mm. Based on the
distance between the interproximal and mid-facial level of the alveolar bone, Becker et al. made
a third division on a flat (2.1mm), scalloped (2.8mm) and pronounced scalloped biotype
(4.1mm). Over the time, several more classifications of gingival biotypes have been proposed.

The thin and scalloped periodontal biotype is present in small incisal contact areas in the teeth
and triangular anatomic crowns and is prone to decompose. A minimal zone attached gingiva
which is present in this biotype, increases a risk of recession after the crown preparation and
leads to higher incidence of fenestration and dehiscence after periodontal or implants surgery.

The thick and flat biotype is fibrotic and resilient with a flat gingival contour, apical contact
areas in the teeth and square anatomic crowns. Followed by a wide zone of attached gingiva and
underlying thick osseous form, making it resistant to surgical procedure. Therefore, thick papilla
biotype is morphologically much more convenient [18] [21].

4.2.3. Tooth form and shape

Periodontal biotype is mainly followed by a specific form and shape of the teeth. There are three
types of gingival scallop: circular, square or triangular. Square teeth provide a better
interproximal papilla arch and they have more favorable esthetic outcome than ovoid or
triangular-shaped crowns because a longer interproximal contact requires less papilla to fill in
the interproximal space [17] [18].

ͳ͹

4.2.4. Interproximal distance of roots

In 1984, it has been reported by Tal that the interproximal distance between each two adjacent
root surfaces also influences the appearance of infrabony defects and the loss of interdental
papilla. Measurements were taken between the mesial mid-vertical line of the distal root surface
and the distal mid-vertical line of the mesial root surface. These were taken at the most coronal
level of the interproximal alveolar bone crest. Distance, which is equal to, or greater than 3 mm
leads to infrabony defects and causes loss of interdental papilla [22].

All of these factors affect the presence as well as the absence of interdental papilla and may
reduce effects in periodontal therapy. The extension of the interdental papilla in the
interproximal space is of great importance and there are many classifications by various authors.

4.3. Classification of the interdental papilla loss

The most widely used classification and from which other modified classification emerged, was
suggested by Nordland and Tarnow in 1998, who used three reference points and those are: the
interdental contact point, the most coronal point of the enamelcementum junction (ECJ) at the
interproximal surface and the most apical point of the ECJ at the labial surface. Based on the loss
of papillary height they proposed the following classification (Fig.4):

‡1RUPDO WKHLQWerdental papilla fills up the space up to the apical extension of the interdental
contact point;

‡&ODVV, the tip of the interdental papilla is placed between the interdental contact point and the
most coronal point of the ECJ at the interproximal surface;

‡ &ODVV ,, WKH WLS RI WKH SDSLOOD LV SODFHG EHWZHHQ WKH PRVW FRURQDO SRLQW RI WKH (&- DW WKH
interproximal surface and the most apical point of ECJ at the labial surface;

‡Class III: The tip of the interdental papilla lies in the level with or apical to the facial CEJ [23].

ͳͺ



)LJ -Schematic illustration, Nordland and Tarnow classification

The loss of interdental papilla is a major challenge in periodontal therapy due to the specificity of
the tissue itself, as well as the factors that have previously been described, what affect the
outcome of periodontal treatment. Up to date, there are no techniques which lead to the
regeneration of interdental papilla. In all treatments so far, the flap method has been used for the
reconstruction but not for the regeneration of the interdental tissue. Although there are surgical
and non-surgical techniques for reconstruction of interdental papilla, there are no treatments to
assure predictable success. In 1993, Langer and colleagues proposed tissue engineering as a
possible technique for regenerating lost periodontal tissues [24].

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5. Strategies of tissue engineering
In tissue engineering, the most important elements for tissue regeneration (Fig.5) are stem
(progenitor) cells, biomaterial scaffolds (supporting matrix) and growth and differentiation
factors (signaling molecules).




Fig.5- The concept of tissue engineering

ʹͲ

Interface between materials science and biocompatibility, and integrates cells, natural or
synthetic scaffolds promising an effective novel approach to reconstruct and engineer the
periodontal apparatus. The tissue engineering, combining three key elements enhances bone and
periodontal regeneration and the role of each of them is very important [2].

The basic idea in the application of tissue engineering is to deliver a certain number of
responsive cells to the defective tissue. These stem cells are capable of self-regeneration as well
as to induce the surrounding environments to regenerate the lost tissue structures.  order to
demonstrate their regenerative ability, stem cell should be transported to a defective tissue,
which should be protected by scaffold matrix from an overlying tissue under. In order to have
adequate mechanical properties, it is necessary that stem cells should be hold together by one
construct, extracellular (scaffold) matrix. As the third element in tissue engineering, growth
factors are necessary to regulate the growth of cells and their distribution, the required gene
expression and cell differentiation in the desired tissue type [25].

5.1. Cell source

One of the main determining components of tissue engineering are stem cells because of their
multipotent differentiation ability. Post-natal stem cells have been isolated from various tissues
including bone marrow, neural tissue, skin, and retina. The major fault of using bone marrow is
the extremely low yield of mesenchymal stem cells (MSCs) from 0.001% to 0.01%. This
limitation of availability leads to the fact that isolated stem cells from human bone marrow are
difficult. In the oral cavity stem cells can be isolated from many sources and five types of dental
stem cell have been identified: dental pulp stem cells (DPSC), periodontal ligament stem cells
(PDLSC), stem cells from exfoliated deciduous teeth (SHED), stem cells from apical papilla
(SCAP) and dental follicle progenitor cells (DFPC) [25].

ʹͳ

5.1.1. Dental pulp stem cells

First stem cells that were isolated were dental pulp stem cells, isolated from adult human dental
pulp, more precisely from permanent third molars and represent less than 1% of the total cell
population present in dental pulp [4].

DPSC cultures from impacted third molars at the phase of the root growth were able to
differentiate into odontoblast-like cells with a migratory and mineralization potential and they
have ability to differentiate into various cell lineages (neurogenic, osteogenic, dentinogenic, and
myogenic) [25].

In a study presented by Almushayt et al. it was established that two proteins dentin matrix
protein 1 (DMP1), and non-extracellular collagen matrix protein play a role in the differentiation
of DPSC to odontoblasts, and these proteins have been used to treat the exposed pulp in order to
regenerate damaged dentine tissue. This activity can also be reinforced with the presence of
transforming JURZWKIDFWRUȕ 7*)ȕ DORQHRULQa combination with fibroblast growth factor
(FGF2) (see 5.3.) [26] [27].

In addition to their importance in regenerative dentistry, dental pulp stem cells have shown an
active neuronal differentiation and the expression of neural markers which is of importance in
the field of neural disorders [8].

Another type of cell isolated from oral cavity are stem cells from exfoliated deciduous teeth.

5.1.2. Stem cells from exfoliated deciduous teeth

Stem cells from exfoliated deciduous teeth have the ability of inducing bone formation, produce
dentin and differentiate into other non-dental mesenchymal cell derivatives in vitro and
compared to DPSC, SHED show a higher proliferation rate. These cells have the ability to

ʹʹ

differentiate into odontoblast-like cells and they are capable of inducing recipient murine cells to
differentiate into bone-forming cells [4].

Although SHED were showing a large proliferative power, they are not able to completely
regenerate dentin/pulp-like complexes in vivo. Further, due to their ability to secrete neurotrophic
factors and induce the expression of neural markers such as nestin, SHED plays a significant role
in the treatment of neurodegenerative disorders such as Parkinson's disease, as well as in the
repair of motor neurons damaged during injuries [3].

Apical papilla is also one of the sources where the cells could be found.

5.1.3. Stem cells from apical papilla

A population of stem cells isolated from human teeth was found at the tooth root apex. These
cells are called stem cells from apical papilla (SCAP) and show a two to three fold higher
proliferation rate than DPSC. Although stem cells are placed in the pulp and in the apical papilla,
it has been determined that SCAP have a greater proliferative potential. Clinical cases
demonstrate their role in apexogenesis and closing root formation at the apical periodontitis or
abscess. Additionally one of the important factors is the presence of collateral circulation, which
allows SCAP to survive even if the pulp necrosis exists [3] [4] [28].

Beside SCAP, which play a significant role in apexogenesis, other stem cell lineages participate
in the regeneration of periodontal ligaments.

5.1.4. Periodontal ligament stem cells

Periodontal ligament stem cells are applicable as useful sources for the regeneration of
periodontal tissues containing bone, cementum and periodontal ligament. Both, PDLSC and

ʹ͵

DPSC showed similar characteristics. Previous studies demonstrated that PDLSC that were
transplanted into immune-compromised mice formed cementum/PDL-like structures and also
Nakahara et al. have shown that PDLSC implanted into a periodontal fenestration defect in dogs
jaw and showed alveolar bone and cementum regeneration [2][3].

Finally, it is also found that precursor stem cells are responsible for the differentiation of other
cells, but no positive effect on the formation of cement and bone was demonstrated.

5.1.5. Dental follicle precursor cells

Dental follicle precursor cells are localized in the dental follicle, a mesenchymal tissue that
surrounds the tooth germ during third molars eruption between 15 and 28 years of age and can be
isolated after third molars extraction. Human DFPC have the ability to differentiate into alveolar
osteoblasts, fibroblasts, cementoblasts, adipocytes, and neuron-like cell. Although DFPC express
osteocalcin and bone sialoprotein, as it was shown in the studies after transplantation in immune-
compromised mice, they cannot make cementum or bone formation [4].

In previous studies which have been performed on dogs, third molars have been extracted and all
of the above types of stem cells were ex vivo isolated. These studies demonstrated that
autologous PDLSC have the best regeneration capacity of PDL, cementum and alveolar bone as
well a peripheral nerve and blood vessel [3].

ʹͶ

5.2. Scaffold or supporting matrix

Beside stem cells, the second component very important in tissue engineering is the supporting
construct or scaffold. Its role is to allow cell attachment and migration, deliver and retain cells
and biochemical factors, enable diffusion of vital cell nutrients and expressed products and exert
certain mechanical and biological properties to modify cellular behavior. One of the most
derivative and the described method based on the principle of material-based regenerative
techniques in the treatment of periodontal diseases is guided tissue regeneration (GTR). It
involves the usage of bio membranes such as resorbable collagen (BioMend1: Calcitek, Colla-
Tec Inc., USA) and polylactic-co-glycolic acid (PLGA: GC membrane, GC Corporation, Japan)
membranes or non-resorbable expanded polytetrafluoroethylene (EPTFE GORE-TEX
Regenerative Membrane1,W. L. Gore & Associates, Inc., USA) and titanium
(TiJeilmesh,ProSeed, Japan) membranes to cover the bone defect. These membranes are bioinert
materials and unfortunately cannot stimulate bone or soft tissue proliferation. Therefore, for GTR
and socket preservation bioactive materials called scaffolds are necessary, that can ensure
delivery of stem cells, signal molecules in the damaged area, and provide mechanical support for
the formation of new tissue. Except that scaffold molecules should be bioresorbable material,
they must also possess sufficient porosity to allow a blood supply and therefore the better
delivery of bioactive molecules [25] [29].

Over time, scaffolds in the human body have to degrade and they must remain in the organ until
all the cells that are delivered become fully integrated. They should not be retained too long so
that they hinder organ function. There are many different materials used as scaffolds and only
some of them are mentioned.

ʹͷ

5.2.1. Biomaterials used as scaffolds

5.2.1.1. Ceramics

Ceramic scaffold includes ceramic materials such as natural and synthetic hydroxyapatite and
beta tri-calcium phosphate. Synthetic hydroxyapatite can be found under the name (HA:
NEOBONE, Covalent Materials, Japan), natural as well as bovine bone mineral (BBM: Bio-Oss
Switzerland) a beta tri-calcium phosphate under the name (b-TCP: OSferion, Olympus, Japan)
[29].

HA (hydroxyapatite) was one of the first biomaterial used as a ceramic bone substitute, derived
from bovine bone or coralline or made as a synthetic material. CAP- based biomaterials have
bioactive and osteoconductive character but they do not have osteoinductive power to facilitate
new bone formation in nonosseous sites. This is very important in cases of installation of
titanium dental implants where the key for success after surgery depends on the process of
osseointegration. With the aim to improve the properties of CAP-biomaterials in newer methods,
they are combined with bioactive osteogenic growth factors, and thus showed greater success in
treatment with dental implants [29][30].

5.2.1.2. Polymers

Polymers category, as the second large group of supporting matrix, includes synthetic polyesters
(Fig.6) such as polyglicolicacid, polylacticacid, polycaprolactone and natural polymers like
collagen, albumin, hyaluronicacid, cellulose, chitosan, alginate, agarose and polyamino acid[2].

Collagen was fabricated by freeze-drying a solution of collagen and placed in a matrix of desired
form. Collagen can induce proliferation and differentiation of cells through direct binding
interactions and acting like a cistern for growth factors and signaling molecule [25] [31].

ʹ͸

Synthetic polymers have been used also in tissue engineering and these materials are
hydrophobic so they could be used as suture materials and as implants in cases of bone fractures
[2].

As an alternative for bone graft/scaffold synthesis, fibrous silk protein (fibroin) biomaterials
from silkworms were used in tissue engineering because of their porosity but these materials are
still not applicable on humans [32].

ʹ͹

COMMERCIALLY AVAILABLE SCAFFOLD MATERIALS
 Scaffold materials Commercial Name  

1. NATURAL POLYMERS   
Collagen ±based Collaplug®,Collacote®,Gelfoam®
Fibrin ±based Bioseed୶୯
Hyaluronic acid-based Hyaff-11®
Chondroitin-based Integra®
Fibroblast- populated skin Dermagraft୶୯,Apligraf୶୯,Orcel୶୯,Hyalograf
substitutes 3D୶୯,Polyactive୶୯
Acellular skin substitutes AlloDerm®
2. SYNTHETIC POLYMER   
Polyamide-based(Nylon) 
Polylactid acid-based TransCyte୶୯
Polyglicolic acid-based
Poly(lactide-copolyglycolide)-
based
3. ALLOGRAFTS   
Demineralized freeze- dried Available from tissue banks
bone allograft
Freeze-dried bone allograft Available from tissue banks

4. XENOGRAFTS   
Anorganic bovine bone Bio-Oss®,PepGen P- 15®

5. ALLOPLASTS   
Hydroxyapatite OsteoGraf®,PerioGraf®
ȕ- Tricalcium phosphate OsteoGen®,Cerasorb®
SynthoGraft୶୯,GEM 21 S®

Bioactive glass PerioGlas®,BioGran®


Hard tissue replacement
polymer
6. ALLOPLASTS+POLYMERS   
Hydroxyapatite+bovine Healos®
collagen
Hydroxyapatite+bovine
FROODJHQȕ-tricalcium Collagraft®
phosphate
+\GUR[\DSDWLWHȕ-tricalcium
phosphate+Fibrin Tricos®

Fig.6-Commercially available scaffold materials, (it is not claimed that this list is complete)

ʹͺ

5.3. Signaling molecules (growth factors)

The third component in addition to the supporting matrix and cell stem are the signaling
molecules or platelets (Fig.7) and they have shown an active role in periodontal wound repair.
Platelets also called thrombocytes are a component of blood whose function is to react to
bleeding from blood vessel injury and to form a blood clot. They are derived from the
megakaryocytes from the bone marrow and they possess large cell nucleus. The organic zone
that exists is rich in granules. Platelets contain dense, alpha, lambda and gamma granules. Delta
granules contain ADP, calcium and serotonin which are platelet-activating mediators. Gamma
granules are similar to lysosomes and contain several hydrolytic enzymes. Lambda granules are
involved in resorption and alpha granules have an effect in wound healing through several types
of growth factors (Fig.8). These factors recruit and activate stem cells and have influence on
their mitogenesis and differentiation [33].

Fig.7-Components of blood-Source (Encyclopedia Britannica 2006)

ʹͻ

5.3.1. Platelet derived growth factor (PDGF)

Platelet derived growth factor is a dimeric molecule composed of A (AA) or two B (-BB) chains
or a combination of the two (-AB). One of the roles for which PDGF is credited is in the process
of angiogenesis, more precisely in the growth and spread of the already existing network of
blood vessels. Unfortunately excessive effect of this factor leads to uncontrolled angiogenesis
and the development of some diseases such as arteriosclerosis, fibrosis or malignancy. It is also a
potent mitogen for stem cells, fibroblasts, smooth muscle cells as well as glial cells.
Recombinant PDGF has been used in the treatment of chronic ulcers, orthopedics and
periodontal therapy in cases with bone loss. There are 5 isoforms of PDGF and those are A, B, C,
D, AB heterodimer and two types of receptors alpha and beta. PDGF triggers cellular responses
by binding its own receptors at the specific receptor sites on the cell. It was discovered as a
growth factor in serum for fibroblasts, smooth muscle cells and glia cells (Kohler and Lipton
1974; Ross et al. 1974; Westermark and Wasteson 1976). It has been found that PDGF
stimulates synthesis of fibroblasts, which have the major ability to make proteoglycan grids
known as the base of the extracellular matrix. It also affects the activity PDGF on alkaline
phosphatase and osteocalcin, mediators in the creation of bone and cement tissue [2] [34] [35]
[36] [37].

5.3.2. Fibroblast growth factor (FGF)

Fibroblast growth factor is the member of polypeptide growth factor family and it has a high
affinity for heparin sulfate proteoglycan. Thereby it can activate cell surface FGF receptors.
There are two forms of this factor isolated from a normal tissue: bovine acidic FGF and human
basic FGF. FGF plays diverse roles in regulation of cell proliferation, migration and
differentiation. In addition, it stimulates angiogenesis, DNA synthesis and cell replication. Thus,
it has effects on periodontal soft tissue and bone healing process. Over-exposure can contribute
to the pathogenesis of cancer. Terranova (1989) et al. SURSRVHGWKDWȕ-FGF in low concentration

͵Ͳ

of  ȝJ SHU GHQWLQ EORFN VWLPXODWHG 3'/ FHOO FKHPRWD[LV DQG LQ FRQFHQWUDWLRQ RI  ȝJ
stimulated endothelial cell migration and proliferation[2][38][39].

5.3.3. Bone morphogenetic proteins (BMP)

Bone morphogenetic proteins are a group of growth factors also known as cytokines and
metabologens. They have high ability to induce the bone and cartilage formation. There are 15
different types of these proteins as a part of transforming growth factor superfamily except of
BMP1, which does not belong to the TGF-ȕ IDPLO\. It is a metalloproteasa which has an
influence on procollagen I, II and III. BMP2 is an important protein because it has a significant
role in osteoblasts differentiation and promote periodontal healthy. It also stimulates activity of
alkaline phosphatase and thereby promotes bone formation. Combination of rh-BMP2 on a
collagen basis is often used as a bone grafts for sinus lift and implant dentistry [2] [40].

5.3.4. Insulin like growth factor (IGF)

Insulin like growth factor is a protein with high sequence simililarly to insulin and belongs to the
group of somatomedins. This system consists of two types of ligands: IGF-1 and IGF-2. IGF-1 is
a chemotactic agent for vascular cells and has influence in process of neovascularization. IGF-2
promotes bone formation but it is not potent as IGF-1. IGF plays an important regulatory role
during the first inflammatory phase and appears to assists in the proliferation and migration of
fibroblasts and in increased collagen production. Studies have demonstrated that IGF exerts a
mitotic effect on fibroblasts and cell-lines, effects cellular morphology, growth pattern and DNA
synthesis. Matsuda (1992) et al. promotes that IGF in concentration higher than ȝJPOexerts
the maximum mitogenic effect. In addition, it has been shown that it slightly enhanced synthesis
of proteins but has no effect on collagen synthesis by PDL fibroblastic cells. Because of that,
combination of PDGF-AB and IGF-1 give synergistic effect results because PDGF-AB

͵ͳ

stimulates collagen synthesis and PDGF-BB and IGF-1 stimulate proliferation and chemotaxis of
PDL fibroblast cells[41][42][43].

5.3.5. Transforming growth factor-ȕ 7*)

Transforming growth factor-ȕ superfamily is associated with inflammatory response, apoptosis,


angiogenesis, wound repair and fibrosis. Until now, three isoforms were isolated: TGF1, TGF2
and TGF3. The highest concentration was found in bones and platelets. TGF stimulates PDL
fibroblast activity, collagen type I, fibronectin and osteocalcin synthesis and osteoblast
chemotaxis. This family was also involved in production of extracellular matrix. On the other
hand, TGF reduces the activity of tissue mediators involved in connective tissue destruction,
LQKLELWVDFWLYLW\RIଟRVWHRFODVWOLNH‫ފ‬FHOOV and therefore participates in bone remodeling. Pisoschi
(2012) et al. confirmed that gingival inflammation is associated with high levels of TGF-ȕ, but
there is not a clear evidence for its role in the periodontal disease. It has been proved that TGF-ȕ
is an important mediator of fibroblast proliferation and extracellular matrix synthesis [2] [44].

5.3.6. Periodontal ligament derived growth factor

Nishimura and Terranova (1996) promoted a novel polypeptide isolated from human periodontal
cells, which is more potent than all other growth factors, but its role is still being examined and it
is called periodontal ligament derived growth factor [45].

͵ʹ

BIOLOGIC  APPLICATIONS IN TISSUE

MEDIATOR FUNCTIONS ENGINEERING

PDGF Mesenchymal cells-potent Neoangiogenesis, bone and extracellular


mitogen, Inflammatory matrix formation, growth and division of
cells-chemoattractant cells

IGF Cell proliferation and bone Increasing cells numbers and matrix
matrix formation formation

FGF Mitogenesis, angiogenesis Increasing cell numbers and improve


blood perfusion

EGF Proliferation, Increasing cell number


differentitation and cells
growth

TGF Inflammatory mediator, Increasing cell number, wound healing


proliferation and and matrix formation
differentiation of osteoblasts
and extracellular matrix
formation, stimulating
expression of BMP s

VEGF Proliferation of endothelial Accessory blood vessels


cells and increasing of
permeability of blood
vessels

BMP s Stimulating differentiation Bone and cartilage formation


of mesenchymal cells into
chondroblasts and
osteoblasts

EMD Cell growth and Angiogenesis and cement formation, cell


differentiation of growth and differentiation
mesenchymal cells

Fig.8 - The role of biologic mediators in tissue engineering applications

͵͵

Platelet cells, which are containing growth factors, could be used in the form of platelet
concentrates but a protocol for their preparation is not standardized yet. There are several
available techniques but the ways of their application are various, because of what each method
leads to different products with different potential uses. Here, we present classification of platelet
concentrates depending on their leucocytes and fibrin content [46].

6. Classification of platelet concentrates

Based on the process of preparation, pharmacological properties and characteristics of the


obtained materials, Dohan Ehrenfest et al. made the classification of platelet concentrates and
divided them into 4 groups:

1. Pure platelet-rich plasma (P-PRP)

2. Leukocyte - and platelet-rich plasma (L-PRP)

3. Pure platelet-rich fibrin (P-PRF)

4. Leukocyte- and platelet-rich fibrin (L-PRF)

Pure platelet-rich plasma (P-PRP) is a product without leukocytes and with low compactness

of fibrin reticule after activation.

Leukocyte and platelet-rich plasma (L-PRP) is a leukocytes-rich product and also with low

density of fibrin reticule.

Pure platelet-rich fibrins (P-PRF) is leukocytes-poor preparation but with a high

compactness of fibrin network. This product can be used only in the form of a gel and cannot be

administrated by injection.

Leukocyte and platelet-rich fibrin (L-PRF) or second generations of PRP is a material with

leukocytes and with high density of fibrin network [46].

͵Ͷ

For the exact classification of the platelets, three main parameters are required: preparation kits
and centrifuges (size, duration, cost of kits and device), content of the concentrates (volume and
efficiency in collecting platelets and leucocytes) and the fibrin network (density and
polymerization process). These products are commonly called PRP, which is wrong, because this
name refers as the original platelet concentrate in transfusion. This causes confusion in the
nomenclature and obscures differences between the various types of systems and methods for
obtaining these concentrates [46].

7. Available PRP techniques

All the available methods of obtaining PRP have something in common, like that blood is
collected immediately before or during surgery and processed in a centrifuge for further
separation. It takes about 1 hour to obtain a platelet concentrate and centrifugation is performed
in two steps. The first step of centrifugation is necessary for the blood to be separated into three
layers, which are red blood cells (RBCs) at the bottom, acellular plasma (PPP, poor plasma
platelet) at the top and between them, a middle layer called the buffy coat layer (BC), in which
the platelets are concentrated. At the next centrifugation step, the RBCs layer and PPP plasma
have being removed. The middle layer of the platelet concentrate remained and administrated by
syringe with thrombin and /or the calcium chloride, which is necessary for the platelet activation,
applied to the surgical site. The second generation of these thrombocytes concentrates,
Choukroun¶s PRF (platelet-rich fibrin), has an altered mode of administration because the blood
was collected without the addition of anticoagulants and immediately distributed for
centrifugation. This method is quick and simpler, certainly less expensive, and the natural way of
coagulation helps to easily obtain leukocytes and thrombocytes rich fibrin layer (L-PRF) [46].
PRP belongs to a first-generation of platelet concentrates used in a regenerative therapy and the
way of obtaining this concentration was done in two steps (see 7.1.).

͵ͷ

7.1. Principles of PRP preparation

The concept behind platelet-rich plasma (PRP) is to concentrate growth factors. Marx and
Carlson purposed that one million platelets per micro liter is the concentration necessary to have
clinically increased tissue healing [47].
PRP is prepared in two steps by centrifugal separation (Fig.10). WB (whole blood) was collected
in a tube with anticoagulant and during the first centrifugation step separated into three layers:
plasma cells in the upper layer, platelets and leukocytes in the middle, and the red blood cells in
the bottom one. For obtaining the pure PRP (P-PRP), PPP and superficial ଟEXII\ FRDW‫ ފ‬BC are
transferred to another tube. After spin centrifugation (at high centrifugal force), most of the PPP
layer is discarded. The final P-PRP concentrate consists of an undetermined fraction of BC
(containing a large number of platelets) suspended in some fibrin-rich plasma. Most leucocytes
are not collected. For production of leukocyte-rich PRP (L-PRP), PPP, the entire BC layer and
some residual RBCs are transferred to another tube. After spin centrifugation, the PPP is
discarded. The final L-PRP consists of the entire BC, which contains most of the platelets and
leucocytes, and residual RBCs. Derivation of L-PRP in relation to the PRP varies in speed and
the number of centrifugation. PRP is obtained by double centrifugation at a lower speed and for
L-PRP high speed is used and the whole blood is immediately centrifuged in one-step
centrifugation [7] [46] [48].

There are numerous and diverse protocols in the current literature which describe the optimal
conditions for the centrifugation. Amable al. suggested that the most optimal results were
obtained in centrifugation at 300 g for 5min at 12°C or 240g for 8 min at 16°C for the first spin
and 700 g for 17 min at 12 °C for the second spin. Longer centrifugal periods decreased the
concentration of white blood cells and low temperatures delayed platelet activation [46] [48]
[49].

Second-generation platelet concentrates, platelet-rich fibrin (PRF) appeared later, whose


preparation did not require the addition of anticoagulants and used only one-step of
centrifugation.

͵͸

7.2. Principles of PRF preparation

Second-generation platelets, platelet-rich fibrin (PRF), are a product, which did not require the
presence of anticoagulants. Centrifugation is performed at 700 g for 12 min to obtain standard
PRF (S-PRF) or at 200 g for 14 min to obtain activated PRF (A-PRF). The difference between a
standard and active PRF lies in the fact that active one contains more platelets and standard one
more neutrophils. With the help of neutrophils, S-PRF has an effect on matrix metalloproteinase
enzymes and therefore its role in angiogenesis cannot be diminished [48].
PRF is different from PRP in the appearance of fibrin networks and because of that, he can
release high quantities of multiple growth factors. Since it does not require anticoagulants, PRF
acts as a network for the accumulation of platelets and leukocytes. It can be used in an injection
form by compressing PRF membranes between metallic sheets.
After centrifugation, fibrinogen, which is concentrated in the higher layers of the tube, reacts
with thrombin from the blood and forms fibrin as a middle layer of the tube. This protocol
requires speed in the performance, because without the presence of anticoagulant, blood almost
immediately begins to coagulate in contact with the glass tube. It is necessary immediately after
taking the blood from the patient to insert a tube into the centrifuge. In summarized clinical data,
PRF has proved to be a better product because it does not require anticoagulants and its
physiological architecture it¶s more suitable for healing processes because of more efficient
cellular migration and proliferation. Because of that, slow polymerization is better for the
incorporation of platelet cytokines in the fibrin network, which were released in the process of
remodeling the fibrin matrix. Unlike, PRP has limited potential in regeneration because it starts
to release growth factors before the start of cell growth of the surrounding tissue. And finally
anticoagulants used for PRP like bovine thrombin could exert toxic effects on body cells [50].

Beside platelets, leukocytes also play an important role due to the release of anti-inflammatory
F\WRNLQHVVXFKDVLQWHUOHXNLQ ȕDQG71)Į 7XPRUQHFURVLV IDFWRU ZKLFKSOD\D UROHLQ
wound inflammation, vascularization and regeneration [7].

͵͹

7.3. Principles of CGF preparation

Concentrated growth factor (CGF) is the modification of second generation platelet concentrates
(PRF) and was prepared following the same procedure as for the PRF preparation but, with
difference in centrifugation speed. CGF uses variable rpm (revolution per minute) from 2400-
2700 rpm to separate cells. After the centrifugation, a fibrin block layer was obtained, which is
denser and richer with growth factors than the one obtained after the centrifugation of PRP and
PRF. Because of higher tensile strength, higher viscosity and adhesivity it is often used as a
barrier membrane to accelerate soft tissue healing or is mixed together with bone graft to
accelerate new bone formation [51].

CGF is a autologous concentrate and it is a non-toxic and immunologically neutral product.


Its role in stimulating the proliferation of MSC cells, neo-angiogenesis was demonstrated. It re-
builds the blood vessels after 24 h, promotes the proliferation of fibroblasts and collagen. For its
preparation anticoagulant is not necessary and viral dissemination diseases is avoided [52].

The CGF is characterized by 4 layers:

x The first top layer makes the serum, blood plasma without fibrinogen and factors of
coagulations. Serum is the brightest layer and is capable to amalgamate all the types of
grafts. It is a straw yellow color and it consists of water, proteins, minerals and carbon
dioxide.
x A middle layer called the buffy coat consists of a polymerized fibrin dense block in the
form of a gel. It has a role in a plasma and platelet cytokines repair, in anti-inflammation,
in the transmission of the signals and the release of GFs. It could be used like a cavity
filler, membrane support, autologous membrane or mixed with other filing materials.
x Layer just below the fibrin block is the third layer and it is in the liquid form. It contains
growth factors, white cells and stem cells. This layer could be aspirated with a pipette and
mixed with autologous bone in order to obtain activated graft.

͵ͺ

x Layer at the very bottom which is still called the red phase has a viscous consistency and
is rich in platelets, red blood cells, white blood cells and clotting factors. – can be used in
a pure form or mixed with fibrinogen or autologous bones for large cavities [47].

Fig.9-Steps for PRP preparation-source TRENDS in Biotechnology [46].

Step 1: Whole blood is collected with anticoagulants and briefly centrifuged with low forces
(soft spin). Three layers are obtained: red blood cells (RBCs), µbuffy coat¶ (BC) layer and
platelet-poor plasma (PPP). BC is typically whitish color and contains the major proportion of
the platelets and leucocytes. Step 2A: For production of pure PRP (P-PRP), PPP and superficial
BC are transferred to another tube. After hard spin centrifugation (at high centrifugal force),
most of the PPP layer is discarded. The final P-PRP concentrate consists of an undetermined
fraction of BC (containing a large number of platelets) suspended in some fibrin-rich plasma.
Most leucocytes are not collected. Step 2B: For production of leukocyte-rich PRP (L-PRP), PPP,
the entire BC layer and some residual RBCs are transferred to another tube. After hard spin
centrifugation, the PPP is discarded. The final L-PRP consists of the entire BC, which contains
most of the platelets and leucocytes, and residual RBCs suspended in some fibrin-rich plasma.
Therefore, the final product greatly depends on the means of BC collection. The transfer step is
often performed with a syringe or pipette, with only visual estimation as a measuring tool.
Because the manual PRP process is not clearly defined, this protocol might randomly lead to P-
PRP or L-PRP.

͵ͻ

Centrifugation is one of the most widely used processes based on the application of a centrifugal
force that is much higher than gravity. In this way, the growth factors have been released from
alpha granules, which are part of the platelets.

Growth factors after releasing from the alpha granules have been bound to a specific cell surface
and transmit their growth signal to other intracellular components. This process is called signal
transduction.

7.4. Physiological working principle of growth factors

Platelets have not cell nucleus but contain an organic zone composed of 4 types of granules:
alpha, gamma, lambda and delta. In alpha granules growths factors are concentrated, which after
releasing from the granules bind to a specific cell surface called Ägrowth factor receptor´ (GF
receptor). Their attachment to the extracellular part of the growth receptor activates the
intracellular signal molecules, which subsequently trigger a series of chain reactions leading to
the transcription of DNA using a DNA polymerase II. Thereby the gene sequences for the cell
proliferation, matrix formation, bone production and synthesis of collagen are expressed [48].
In transmitting of growth signals, proteins of phosphorylation cascades play a very important
role. Enzymes with kinase activity or phosphatase activity are important because kinase adds a
phosphate group on a protein, and phosphatase removes a phosphate group. Many growth factors
are peptides/proteins, which bind with a high affinity to a specific surface receptor,  ƒ† the
receptor-binding site for peptide/protein growth factors is extracellular. Most growth factors
receptors show tyrosine kinase activity, meaning they add a phosphate group on a tyrosine
residue located in intracellular part of the receptor. Exceptions are surface receptors for the
transforming growth factor-beta (TGF-beta) cytokine family. This receptor can phosphorylate
downstream serine and threonine residues. Phosphorylation of the cell receptor leads to the
activation of signaling molecules within the cell. They transmit growth signals to the nucleus and
activate transcription factors that lead to the expression of genes responsible for cell
differentiation, proliferation, and apoptosis [53].

ͶͲ

Fig.10-Shematic illustration of physiological working growth factors

7.5. Steps for obtaining the final platelet products

The procedure starts by taking a blood sample from the patient (Fig.12) and about 9 ml of blood
is need to be collected in 2 to 8 vacuettes (tube). Tubes have to be placed quickly and gently into
the rotor of the medifuge (centrifuge accelerator) (Fig.13)Ǥ After approximately 13 minutes of
spinning, the serum is separated from the other phases of the CGF and stored in a specific sterile
dappen dish. The fibrin phase is separated and stored in diluted antibiotic solution ( Lincocin 600
mg). The initial portion of the coagulation containing the GFs and the stem cells were
immediately stored in the dappen dish provided. The coagulum, which is rich in red blood cells
and platelets, as well as iron, calcium and other fundamental components, was prepared to be

Ͷͳ

used for the preparation of fillers, for mixtures of biomaterials or autologous bones taken for
osteotomy.
The fibrin block, divided from the red phase, could be applied directly into the graft cavity in the
shape of a membrane with the use of the specific forceps provided. Another possibility is that it
can be used as a graft particle for mixing with biomaterial or living autologous bone. Layer on
the bottom after the first centrifugation is centrifuged again in order to discard poor plasma from
layer on the bottom purred with platelets, red blood cells, leukocytes and clotting factors, what is
needed for PRP. It can be used in a pure form or mixed with fibrinogen or autologous bone for
large cavities.
The resulting PRP fraction was collected using syringes equipped with long needles and stored at
- 80°C until determination of GF levels [47].

Fig.11- Blood sample procedure- Retrieved from National Cancer Institute, ©2007Terese
Winslow, U.S.Govt. has certain right

Ͷʹ

Fig12. - Schematic representation of PRP therapy

Ͷ͵

For obtaining platelets derivatives, manufacturers on the market were presented system of tubes
for blood collection. Tubes or Vacutainers are marked with different colors (Fig.14) to be
distinguished by substances they contain.

There are three main types Vacutainers tube for receiving different forms of CGF:

x Z serum clot activator with RED CAP-consist silicon crystals as clotting and separation is
done at room temperature;
x Z no additives with WHITE CAP-No additives or anticoagulants, 20 min after separation
at room temperature gives the product;
x Sodium heparin with GREEN CAP- consist sodium heparin to prevent coagulation. Rise
into liquid at 20°C for 8h or at 4°C FOR 40h.

Fig.13Schematic representation of Vacutainer colors-source Greiner Bio-One

ͶͶ

x Purple tube containing EDTA as an anticoagulant, binding the calcium ions and
interrupting the clotting cascade. – ‹• used in hematology, molecular diagnostics and viral
determination.
x Black tube containing sodium fluoride as a glycolysis inhibitor, in order to determine
glucose level.
x Blue tubes are available with sodium heparin and are used for determining trace elementsǤ
A trace element is a chemical element whose concentration (or other measure of amount)
is very low.

In a test tube with RED CAP, CGF was obtained in the form of fibrin clot platelet gel. That has
been used in the soluble phase as subepithelial gingival graft and in sinus lift procedures, ridge
augmentation, socket preservation, infrabony defects, periimplantitis, filling of cystic cavity,
furcation defects and TMJ regeneration. In implantology, CGF have been used as a bone ring
around the implant and together with implant embedded into the bone. In a test tube with
WHITE CAP, CGF is prepared as a liquid phase for infiltration treatments and can be used 20-30
minutes after separation. – ‹• used mainly for aesthetic treatments and with the treatment of
alopecia, skin pigmentation problems, stretch marks, large pores on the face, burn scars, and in
orthopedics for the treatment of osteoporosis and other bone diseases. Tube with GREEN CAP,
provides CGF in a liquid phase with sodium heparin for different infiltration treatments. The
application is found in the treatment of ulceration, trigeminal neuralgia, TMJ regeneration,
tendinopathies, intervertebrale diseases, injuries of ligaments, nerves and muscles, as well as in
oral and dental surgery [54].

Ͷͷ

Fig.14- Medifuge CGF

Source Silfradent®

Ͷ͸

8. Study purpose

This literature review deals with the problem of reconstruction of inter-dental papilla in the
esthetic area of teeth or implants. Previous scientific studies and methods have shown difficulties
in the long-term performance and efficiency of this therapy. The advantage of this study is to
show that new methods have been found and there are strong evidences in conducted studies that
in the future effective solution for the therapy of lost inter-dental papilla will be reproducible and
less traumatic as possible.

The purpose behind writing this review is to highlight the application of stem cells in various
fields of dentistry, with the main emphasis on reconstruction of inter-dental papilla by using
platelet-rich blood derivatives and the influence of growth factors on the behavior of the stem
cells. The ultimate goal is to investigate new studies and methods, which have shown objective
and predictable improvement in this therapy.

9. Material and methods

One of the first methods as a minimally invasive treatment in augmentation of interdentally


papilla in the aesthetic zone was hyaluronic acid injection (HY). After administration of local
anesthetic, 0.2 mL of a commercially available and Food and Drug Administration-approved gel
of hyaluronic acid was injected 2±3 mm apical to the coronal tip of the involved papillae. After
the application it was necessary to perform the massage of the region for about 1 minute and the
treatment was repeated for 21 days and then for 42 days. The method of hyaluronic injection was
used on patients with natural teeth as well as in patients with implants supported by anterior
maxilla. High heterogeneity in the results and monitoring for only 6 months, cannot recommend
this method as safe and long-term therapy in the regeneration of the interdental papilla. Injection
of HY adjacent to maxillary anterior implant-supported crowns did not result in any clinical
conspicuous volume augmentation of deficient papillae [55] [56] [57] [58].

Ͷ͹


Another method introduced McGuire et al. (2007) in a randomized double blind placebo study as
the use of autologous fibroblast injections, following a minimally invasive papilla priming
procedures to augment open interproximal spaces. The procedure started with taking of
keratinized tissue from maxillary tuberosity with 3mm biopsy instrument, followed by local
anesthetic. The biopsy sample is then transferred in a sterile bottle and sent to the laboratory and
the donor site has been treated with cyano acrylate dressing. The biopsy sample is conducted
with standard tissue culture techniques in the laboratory. Most cells are transplanted within 12
hours of receiving them. On the other side, 5 to 7 days prior to the initial injection papilla should
receive a control surgical content. In this way, an acute inflammatory response is induced and
enabling the papilla to increase its volume and to receive a large cell suspension. After that, a
small space could be made using the Orban knife from the facial aspect into the base of papilla
avoiding the extension into the mesial and distal sulcus. Then, the 12 B knife is placed into the
created space and this leads to the apex of papilla perpendicular to the incisal incision. It is
necessary to be careful to avoid cutting through the tip of the papilla and the lingual perforation.
After 5 to 7 days, the first initial treatment is injected with a suspension of the concentrated cells
20-106 cells/millisecond. Injection treatment has to be performed 7 to 14 days after the first one,
and the third treatment 7 to 14 days after the second application.  the first treatment of the
papilla, the needle has to be applied coronally from the alveolar crest from facial aspect, another
application is in the same region but only from the lingual aspect and the third application has to
be performed by pulling the needle slowly from the tip of the papilla to the crest of the bone.
Monitoring results of the last six months noticeable improvement has been found in the closure
of the interdentally space. Clean injected fibroblasts are capable of production of collagen and
thus play an important role in wound repair. Unfortunately, there is not much data on long-term
success of this method because it has been followed up only for a short period of time. Still it
represents a good turning point for future scientists, to find the most efficient methods in the
augmentation of interdentally papilla [59].

A third method, described in many studies, is the use of platelet-rich concentrate as a source of
growth factors for improving the healing process.

Ͷͺ

These factors chemotactically recruit and activate stem cells as well as induce their mitogenesis
and differentiation. Classification of platelet concentrate, which has been described, is based
among other things on the protocol to obtain products as well as on the centrifugation speed.

Because of the PRP properties for rapid transmission of growth factors, treatment success varied
in patients. Second generation of platelets, PRF and CGF, have one-step centrifugation and a
higher speed. The preparation of the PRF was performed before the start of the operation
according to the protocol that was developed by Choukron et al. [6]. 10 milliliter of intravenous
blood (antecubital site) was collected in a sterile 10 ml tubes without the addition of an
DQWLFRDJXODQW DQG FHQWULIXJHG DW  UHYROXWLRQV § î J  SHU PLQXWH IRU  PLQXWHV 35)
settles down between the platelet poor plasma (PPP) at the top and the red blood cells (RBC) at
the bottom of the tube [60].

The receiver site was developed using Han & Takei technique. Intraoral antisepsis was carried
out by rinsing with 0.12% chlorhexidine digluconate for 30 seconds and adequate local
anesthesia was applied with 2% lignocaine hydrochloride (HCL). A split thickness semi -lunar
incision was given about 1 mm coronal to the mucogingival junction in the interdental region
and the split thickness flap was continued through the semi lunar incision toward the interdental
papillae to create a pouch in the interdental area. A curette was used around the neck of teeth to
free the tissue attachment from the root surface, extenuating the displacement of gingivopapillary
unit coronally [61].

The prepared PRF was separated using the sterile tweezers and trimmed with scissors and
transferred onto a sterile gauze. A thick fibrin membrane was obtained by squeezing the serum
out of the PRF clot. This membrane was eased in to the pouch and pushed coronally, enabling to
fill the bulk of the interdental papillae. The incisions were secured using absorbable sutures.
Analgesics and antibiotics should be prescribed and after 10 days sutures should be removed [62]
[63].

Interventions that are conducted with the use of PRF and pedicle flap offer a reliable settlement
as PRF membrane has both mechanical adhesive properties and biologic functions like fibrin

Ͷͻ

glue, it sustains the flap in a stable position, increases neoangiogenesis, decreases the necrosis
and shrinkage of the flap and stabilization of the gingival flap in the highest covering position.
The PRF is easy to purchase, does not much cost and can be prepared in a few minutes. PRF
provides ideal healing properties. This fibrin matrix consists of its platelets, leucocytes, and
cytokines, allows remodeling of interdental papilla to happen. It has been found that PRF
organized as a dense fibrin scaffold with a specific release of growth factors (TGF-ȕ 3'*)-
AB, vascular growth factor (VEGF) and glycoprotein (thrombospondin -  GXULQJ •  GD\V LV
critical for the rejection the grafted PRF membrane [63].

Combination of PRF and the pedicle flap ensure maximum vascularity and minimal scar tissue
because this pouch-like design ensures dual blood supply from the connective tissue below and
overlies and maintains the flap in a steady position [54]. However, because of problems
including donor site morbidity from autologous grafts cell transplantation by the injection should
be simple, relatively pain-free and minimally invasive. Yamada et al. 2015 proposed a new
method as a non-surgical minimally invasive therapy in the regeneration of papillae with the aim
of long-term success and screening results over a period of 5 years. Š‡› made a TEP (tissue
engineering papilla) injection containing mesenchymal cells as isolated cells, PRP as a growth
factor and non-animal HY as a scaffold. One and half month before the intervention 20ml from
the patient iliac crest bone marrow is aspirated for the preparation of BMDSCs (bone marrow-
derived mesenchymal stem cells) and is fed into a control medium which contains 10 percent
patient blood or 10 percent fetal bovine serum, basal medium, low glucose medium, L-glutamine
and penicillin-streptomycin mixture. The Patient is allowed to choose between patient serum and
bovine serum. For the preparation of human serum, 200 ml of blood is taken from the patient by
the transfusion method. Collected blood is centrifuged at 3500 rpm for 10 minute. Cells are
incubated at 37° humid atmosphere and before use, the safety of contamination is controlled. For
preparation, PRP is taken 1 day before intervention. 200ml of blood is also drawn from the
patient and centrifuged in two phases. In the first phase, 10 min at 1500 rpm, the yellow plasma /
buffy coat with platelets and leukocytes is removed and then centrifuged for 5 minutes at 3500
rpm. Platelet poor plasma is removed and the resulting, pellets of platelets and the buffy coat, are
resuspended in the residual 20ml of the plasma before used in the platelet gel. During this time,

ͷͲ

in a separate bottle, human thrombin is prepared with 10% calcium chloride. In the first 2.5 ml
PRP, BMDSCs, HY and air are aspirated with a sterile syringe, and in other 2.5 ml the ȝOof
thrombin-calcium chloride mixture. In the period of 5 to 30 seconds, the contents of the bottle
are converted into the gel due to the thrombin that polymerizes fibrin. Donor site is anesthetized
with 2% of xylocaine adrenaline and TEP injection is administered without incision [64].

Fig.15- Novel methods for the treatment of interdentally papilla augmentation

ͷͳ

10. Results

Using the Meta analysis, a comparison was made of all previous minimal invasive methods in
the treatment of defective interdental papillae. The following databases were included: Ovid
MEDLINE(R) In-Process & Other Non-Indexed citations, Ovid MEDLINE (R) 1999 to present
with daily Update, EMBASE, EBM Reviews-Cochrane Database of Systematic Reviews.
Inclusion criteria: The studies had to contain original data, human teeth, studies with periodontal
diseases, minimal invasive therapy and therapy with growth factors, regenerative methods,
natural dentition or implants, in vivo studies, studies from 1999 to present in English or
German language.

10.1. HY as monotherapy or as adjunct to non-


surgical/surgical periodontal treatment

For the analysis of HY therapy in periodontal treatment (Table 1), 13 publications were included:
3 clinical studies on surgical treatment and 10 clinical trials reported on non-surgical treatment in
periodontitis patients.

Among the studies on non-surgical treatment, mean age of the patients (ranging from 38 to 51
years old) was reported in six studies and other studies informed only on age range. In five
studies missing smoking status, one study reported smoking and non-smoking status and four
included only non-smokers. Follow up period was from 3 weeks to 1 year and sample size
ranged from 9 to 60 patients because discontinuation of follow-up was reported in nine studies.
One study evaluated HY as a monotherapy without any additional treatment and other nine HY
was applied as adjunct to scaling and root planning (SRP). Significantly less BOP (3-13,7%) was
reported in three out of seven studies and in one study also significantly less values for bleeding
index (0,19) were reported. Further, significant superior reduction of PD (0,2-0,9mm) was
reported in five out of ten studies and one study reported significantly lower level of residual PD

ͷʹ

(0,5mm). In addition, one out of seven studies reported a significant superior CAL gain (0,7mm)
(Table 2) [57].

Among the surgical studies, mean of age ranging from 45 to 49 was included and one study did
not report smoking status. Other two studies included only non-smokers. The sample size ranged
from 6 to 40 patients and the follow-up period from 6 to 24 month. There were no patients lost in
the follow-up. Furthermore, one study included treatment with open flap debridement (OFD) or
modified widman flap (MWF) with or without HY application and three studies compared the
outcome of treatment after guided tissue regeneration (GTR) with or without HY application.
Two studies on surgical treatment notified a significant higher CAL gain (0.8-1, 0 mm) and one
also reported significantly higher PD reduction (0,8 mm). Bleeding on probing was in all tree
studies unclear reported (Table 2) [57].

Most of these studies describe positive results after HY application as monotherapy and as
adjunct in non-surgical/surgical treatment with respect to BOP and PD compared to control
groups. All non-surgical studies confirm PD change, some of them CAL gain and BOP values.
All the surgical studies reported PD and BOP changes, but it is unclear whether HY in the
treatment of infrabony defects results in CAL gain compared to surgery alone. HY application as
an adjunct to non surgical/surgical treatment proved to be a safe method with no negative effects.
Due to the high heterogeneity of the included studies, there are great variations in the results both
due to the type of product, application mode (supragingival/subgingival), in office or at home,
frequency (1-60) applications as well as the application period (1-45 days), it is not possible to
make a recommendation on the mode of the application [57][58].

ͷ͵

10.2. HY as monotherapy by implant supported crowns

Based on data from the previously published case series 21 patients (Mean age 30 ± 6, 4 years)
received the allocated treatment and finished the follow-up period of 6 months. The following
parameters were assessed at baseline and after 3 and 6 months:

a) Modified papilla index score (MPIS): Score 0-no papilla is present, score 1- less than half of
the papilla height is present, score 2- half or more of the papilla height is present, score 3- the
papilla fills up the entire proximal space, score 4- hyperplastic papilla

b) PT-CP: distance in mm between the papilla tip (PT) and the contact point (CP)

c) Probing depth (PD) and clinical attachment level (CAL) in mm, bleeding on probing (BOP)
and plaque in percentage (%)

d) Area of the black triangle in mm² using standardized digital photograph and gingival volume
change in mm²

e) Radiographic alveolar bone height (ABH) at the implant next to the injection site in mm

f) Esthetic appearance with VAS- visual analog scale (0-100, where 0 indicated best imaginable
appearance/no defect and 100 indicated worst imaginable appearance/defect.

All included deficient papilla presented a MPIS score 2 and none of them was changed after
treatment. In this study it was noticed that there was no significant change in periodontal
parameters such as PD (WHVWJURXSǻ%/PRQWKV-“PPǻ%/PRQWKV“PP ,

CAL (test JURXSǻ%/PRQWKV-0.10±0.34mm, ǻ%/PRQWKV“0.39mm) and plaque (test


JURXSǻ%/PRQWKV“Əǻ BL 6 months 9.1±49.1).

BOP showed DVLJQLILFDQWLQFUHDVHLQWKHWHVWJURXSIURPEDVHOLQHWRPRQWKV ǻ%/PRQWKV


“ ǻ %/  PRQWKV “  DQG QR GLIIHUHQFHV LQ WKH EODFN WULDQJOH DUHD ZHUH
observed. There are also almost minimal changes in the black triangle area in a several cases in
WKHWHVWJURXS ǻ%/PRQWKV±“ǻ%/PRQWKV“ 

ͷͶ

The height of the alveolar bone on the side of the implant in comparison with the injection site
also showed no change even after 6 months WHVW JURXS ǻ %/  PRQWKV “  It is also
noticed that there is no significant difference in the aesthetic view between control and test
groups (VAS values on average < 30 at all time points) ( Table 2).

Of the negative effects, only two cases of lip swelling have been reported after the second
injection and one case with a painless granuloma that receded after 4 weeks [55][57].

10.3. Fibroblast injection

In this study 21 subjects were enrolled and 20 subjects were treated in the study. All patients
were non-smokers between the age of 18 and 70 years. Percentage change in papillary height
was primary parameter measured by periodontal probe from the base of the contact area to the tip
of the interproximal papilla. A visual analog scale (VAS) was used to confirm clinical
measurements. In the second month, analysis yielded a difference in a raw change between test
and placebo of 0.27±0.6mm and using the dental probe measurements a difference in percentage
change between test and placebo of 4.0±27.8ƏZDV also demonstrated (Table 2).

This method showed a percentage increase in the papillary height from baseline at 2 months, but
the following endpoints failed to show evidence of treatment effect at 4 months. At 2 months, a
significant difference was noted in height of the papilla with periodontal test as well as with the
VAS scale. This shows that the test treatment was superior to placebo.

The complete method of cell transplantation has proven to be safe and painless. Exceptions are
only two cases requiring analgesia. No infection is detected and without a change in color and
texture of the tissue which indicated that the treatment was well tolerated and without adverse
effects [59].

ͷͷ

10.4. TEP injection

In this study, which described the use of tape injection in the treatment of interdental papilla, the
distance from the tip of the papilla to the base of the contact area in each study region was
measured. The mean age of ten cases includes in a study was 39±14.9 (range 20-64), the mean
volume 1.32±0.25ml, operation time 2.2±1.62 times and follow up period 55.3±17.7 months.

The mean IBT ±improved distance from tip of the papilla to the base of the contact area was
2.55±0.89 mm (Table 2) but there was no significant radiographic difference. Clinical view after
3 years has shown that the interdental papilla is stable [64].

10.5. PRF in periodontal treatment

In cases with the usage of PRF has shown significant increase of the interdental papilla height at
3 months and after 6 months complete papillary fill was obtained. The distance from the contact
point to the tip of the papilla was measured and it was 4mm from baseline. It was reduced to 1
mm after 3 months and 0mm after 6 months indicating complete papillary fill (Table 2).

The presence of the papilla was observed in almost 100% of the cases in which the distance was
less than or equal to 5 mm, in 56% of cases in which the distance was 6 mm, and only 27% of
cases in which the distance was 7 mm or more.

The wound healing was satisfactory and 3 weeks after operation has shown complete healing.
The use of PRF has also reported good esthetic results and excellent color match after healing
[60] [62] [63].

ͷ͸

Method 1 Method 2 Method 3 Method 4 Method 5 Method 6
HY as monotherapy or as HY as adjunct to surgical HY as monotherapy by Fibroblast injection TEP injection PRF+HanΘTakei
adjunct to non-surgical periodontal treatment implant supported crowns
periodontal treatment

STUDIES SINCE 2001 2001 2014 2007 2013 1999


STUDIES NUMBER OF STUDIES 10 3 2 1 1 ?
CLINICAL APPLICATIONS 2001 2001 2010 2007 2013 1999
AND SINCE
TOTAL NUMBER OF 272 40 21 21 10 4
APPLICATIONS PATIENTS
FOLOW- UP PERIOD 3-52 WEEKS 6-24 MONNTHS 3-6 MON NTHS 4 MONTHS 69 MONTHS 6 MONTHS

100ε͕37&3”PP
SUCCESS PROVED BOP 42й,BL.INDEX 14й BOP ?,CAL 66й͕PD 33й ALL MEASUREMENT 0й 2 MONTH 100й 100% 56% , PT-CP= 6 mm
PD 50й,CAL GAIN 14й 4 MONTH NOT REP. 27й͕PT-CPшϳŵŵ
4
SUCCESS VERIFIED BY MEASUREMENTS¹ MEASUREMENTS ² MEASUREMENTS³ MEASUREMENTS MEASUREMENTS 5 MEASUREMENTS 6

LONG-TERM SUCCESS NOT REPORTED NOT REPORTED / NOT REPORTED YES NOT REPORTED

FAILURE PROVED YES / NO NO YES NO NO NO


38-51 YEARS OF AGE 45-49 YEARS OF AGE 18-70 YEARS 18-78YEARS OF AGE 20-64 YEARS OF AGE > 30 YEARS OF AGE
INCLUSION NS/S/NR NS/NR NON SMOKERS NON SMOKERS NOT REPORTED NON SMOKERS
APPLICABILITY CRITERIA AT LEAST 18 TEETH HEALTHY HEALTHY > 6 MONTHS WITH/WITHOUT BONE HEALTHY
AT LEAST 2 SITES WITH THERAPY PLAN- OFD, PLAQUE CONTROL AT LEAST 1 DEFICIENT RESORPTION NO INFLAMATION
3'$1'&$/•PP MWF or GTR ‫ޒ‬20ε PAPILLA BETWEEN IMPLANT/TOOTH HEALTHY GINGIVA
INFLAMATION NATURAL TOOTH AND NO FACIAL RECESION
RADIOGRAPHIC SINGS IMPLANT SUPPORTED
OF BONE LOSS CROWN
ADEQUATE ATTACHED
GINGIVA

EXCLUSION ‫(<ޒ‬$562)$*( ‫ޒ‬45 YEARS OF AGE PATIENTS WHO NO CONTACT POINT ‫(<ޒ‬$562)$*( ‫ ޒ‬30 YEARS OF AGE

CRITERIA SMOKERS SMOKERS RECEIVED CROWN OR PLAQUE SCORE > 20ε NON HEALTHY
LESS THAN 18 TEETH NO HEALTHY PONTIC ON ONE OR PD > 5 mm SMOKERS
7
LESS THAN 2 SITES WITH WITHOUT NEEDS FOR BOOTH TEETH INVOLVED GR > 3 mm INFLAMATION
8
3'$1'&$/•PP SURGICAL THERAPY IN THE INTERPROXIMAL .7‫ޒ‬PP GINGIVA PATHOLOGY
NO INFLAMATION PERIODONTAL POCKET SPACE UNCONTROLED FACIAL RECESION
NO BONE LOSS ROOTH GROOVES DIABETES
INADEQUATE ATTACHED FURCATION MALIGNANCY
GINGIVA MOBILITY > 1 USE OF STEROIDS
OPEN CONTACTS AFFECTING CONECTIVE
PROBING DEPTH > 3 mm TISSUE METABOLISM
RADIOGRAPHIC
PATHOLOGY

UNDESIRABLE PAIN DURING UNDER LOCAL UNDER LOCAL UNDER LOCAL UNDER LOCAL LOW BY TAKING A LOW BY TAKING A
PROCEDURE ANESTHESIA ANESTHESIA ANESTHESIA ANESTHESIA PATIENT'S BLOOD PATIENT'S BLOOD
EFFECTS SAMPLE SAMPLE

PAIN DURING THE FIRST LOW PRESCRIBED LOW PRESCRIBED NOT REPORTED PRESCRIBED
WEEK AFTER ANALGESIA ANALGESIA ANALGESIA
TREATMENT

TREATMENT YES YES YES YES YES YES


RISK

SIDE EFFECTS EVIDENT NOT REPORTED NOT REPORTED SWELLING ON THE LIP NOT REPORTED NOT REPORTED NOT REPORTED
PAINLESS GRANULOMA

9 9 9 9 9 9
SIDE EFFECTS YES YES YES YES YES YES
POSSIBLE

NUMBER OF 1- 60 APPLICATIONS 1 APPLICATION 2 APPLICATIONS 3 APPLICATIONS 2-5 APPLICATIONS 1 APPLICATION

TREATMENTS

EFFORTS FOR THE NO NO NO TEAM WORK WITH TEAM WORK WITH TEAM WORK WITH
EFFORTS DOCTOR LABORATORY LABORATORY LABORATORY

EFFORTS FOR
PATIENT LOW/SRP OFD,MWF,GTR LOW DONOR SITE LOW LOW

COSTS LOW SURGERY LOW LABORATORY ? LABORATORY ? LABORATORY ?

ͷ͹

TABLE 1- Comparison of all minimal invasive methods in periodontal treatment
1.2. BOP- bleeding on probing, CAL-clinical attachment level, PD-probing pocket depth. 3. PT-CP- distance
between the papilla tip and contact point, MPIS-modified papilla index score, BOP, CAL, PD, Area of black triangle
and gingival volume change(mm²), plaque%, radiographic ABH-alveolar bone height, VAS-visual analog scale
4.The distance from the tip of the papilla to the base of the contact area using a University of North Carolina (UNC)
15 mm periodontal probe, Inflammation score using modified gingival index by Lamster et al.ǡcolor test, VAS
scale, digital photographs (1:1) 5. Volume of TEP (ml), operation time, BT- improved black triangle 6. PT-CP,
wound healing index 7. GR- buccal gingival recession 8. KT- keratinized tissue 9. allergy, infection and granuloma

ͷͺ

RESULTS KW;йͿ W > W>Yh >< , s^ D WW͘ /d
>͘/Ey ;ŵŵͿ ;ŵŵͿ ;йͿ dZ/E'> ;ŵŵͿ W ,/',d ;ŵŵ
;ŵŵ²Ϳ / ;йͿ Ϳ
^
EhDZ ϯ ϱ ϭ ͬ ͬ ͬ ͬ ͬ ͬ ͬ
K&^dhz ϭ
^h^^
,zͬEKE ϯͲϭϯ͘ϳ Ϭ͘ϮͲϬ͘ϵ Ϭ͘ϳ ͬ ͬ ͬ ͬ ͬ ͬ ͬ
^hZ'/> 
dZdDEd Ϭ͘ϭϵ
EhDZ ͍ ϭ Ϯ ͬ ͬ ͬ ͬ ͬ ͬ ͬ
K&^dhz
^h^^
,zͬ ͍ Ϭ͘ϴ Ϭ͘ϴͲϭ͘Ϭ ͬ ͬ ͬ ͬ ͬ ͬ ͬ
^hZ'/>
dZdDEd
EhDZ ϭ ϭ ϭ ϭ ϭ ϭ ϭ ϭ ϭ ͬ
K&^dhz
^h^^
,zͬ/DW>E ȴ>ϯ ȴ>ϯ ȴ>ϯ ȴ>ϯ ȴ>ϯ ȴ>ϲ <ϯϬ Ϯ  
d ϭϱ͘ϵц ͲϬ͘ϬϱцϬ͘ϯϱ ͲϬ͘ϭϬцϬ͘ϯϰ ϲ͘ϴцϯϴ͘ϵ ͲϬ͘ϬϮцϬ͘Ϭϳ Ϭ͘Ϭϲц
^hWWKZd ϭϲ͘ϵ ȴ>ϲ ȴ>ϲ ȴ>ϲ ȴ>ϲ Ϭ͘Ϯϯ
ZKtE^ ȴ>ϲ Ϭ͘ϭϬцϬ͘ϰϳ Ϭ͘ϬϱцϬ͘ϯϵ ϵ͘ϭцϰϵ͘ϭ Ϭ͘ϬϯцϬ͘ϭϬ
ϭϱ͘ϵц  
Ϯϱ͘ϳ 
EhDZ ͬ ͬ ͬ ͬ ͬ ͬ ϭ ͬ ϭ ͬ
K&^dhz
^h^^
&/ZK>^ ͬ ͬ ͬ ͬ ͬ ͬ /ŵŐ͘ ͬ ϰ͘Ϭц ͬ
d Ϯϳ͘ϴ
/E:d/KE 
EhDZ ͬ ͬ ͬ ͬ ͬ ͬ ͬ ͬ ͬ ϭ
K&^dhz
^h^^
dW ͬ ͬ ͬ ͬ ͬ ͬ ͬ ͬ ͬ Ϯ͘ϱϱ
/E:d/KE ц
Ϭ͘ϴϵ
EhDZ ͬ ͬ ͬ ͬ ͬ ͬ ͬ ͬ ͬ ϰ
K&^dhz
^h^^
WZ& ͬ ͬ ͬ ͬ ͬ ͬ ͬ ͬ ͬ ϯ͘ϬͲ
ϰ͘Ϭ

TABLE 2- Summary results of all minimal invasive methods in periodontal treatment


BOP- bleeding on probing, BL. index-bleeding index, PD-probing pocket depth, CAL-clinical attachment level,
plaque, BLACK TRIANGLE-proximal space, ABH-alveolar bone height, VAS-visual analog scale, MPIS-modified
papilla index score, PAP.HEIGHT-percentage change in papilla height ,IBT-improved black triangle

ͷͻ

11. Discussion


Based on the ability of the formation of new tissue and organs from the patient own cells, tissue
engineering represents a banding point and the challenge for the regenerative medicine. This
field opened the door for new methods to come up, in both medicine and dentistry. Many of
these methods have already been described in various clinical and preclinical studies. However, a
long-term effectiveness of these methods still has to be followed up in ଟin vivo conditions´ [25].

Therefore, it is of great interest to study cells and molecular mechanism involved in regeneration
of periodontal tissue. Further, the potency of stem cells and their biocompatibility with host
tissue is also very important part of this therapeutic approach [65].

Potential of growth and differentiations factors to regenerate periodontal tissue is nowadays the
most appealing topic in tissue engineering [66].

Concentrated platelets contain many growth factors such as platelet-derived growth factor
(PDGF), transforming growth factor b (TGF-b), insulin-like growth factor (IGF), epidermal
growth factor (EGF), fibroblast growth factor (FGF), and bone morphogenic protein (BMP).
PDGF has a role in the stimulation of angiogenesis, fibroblast cells proliferation and
differentiation and collagen synthesis by fibroblasts. TGF also stimulates collagen synthesis in
fibroblast, which contributes to the regeneration of bone and gristle. IGF stimulates osteogenesis
and also is in studies stated that PDGF and TGF cause chemotaxis osteoblasts precursor on bone
defect site, which is followed up by proliferation and differentiation of osteoblasts. As a natural
cocktail these growth factors have been isolated-PRP, PRF I CGF. PRP after centrifugation
contains 1 million platelets per 1mm2 and PRF contains the same number of platelets but
performs better features because it is itself a natural fibrin network and shields the growth factors
from proteolysis. For its preparation, no additives are required such as thrombin, which leads to
the formation of the inherent fibrin network. CGF has recently become popular because it
contains a larger amounts of the growth factors. This fibrin clot has greater cohesion because of
the agglutination of fibrinogen, factor XIII and XIII thrombin. Factor XIII is activated with the
assistance of thrombin, which leads to a reaction transition of fibrin to clot. This fibrin clot is
stable and resilient because in this way it is protected from plasmin degradation [67].

͸Ͳ



Many surgical methods are already known and described in a large number of studies about the
reconstitution of the interdental papilla. Shapiro et al. proposed repetitive curettage in the
treatment of necrotizing ulcerative gingivitis such as treatment which induces the growth of
papilla. In addition, other methods such as a pedicle flap with a coronary papilla transposition, a
roll technique and a subepithelilal connective tissue graft have also been described in many
studies. All of the mentioned methods are invasive, with long post-operative recovery of patients,
discomfort for the patient, and with the risk of necrotizing and shrinkage of graft, which certainly
reduces the overall effect in regenerative surgery. In order to find less invasive and atraumatic
techniques new methods of therapy have been developed. One of those methods includes PRP
injection, which has shown positive results in patients in the reconstruction of interdental papilla.
Treatment is completely non-invasive, inexpensive, short and has positive effects on patient
recovery. PRP is the first preparation that was used, but because of its variability in the success
of the therapy, more superior CGF were discovered, which Choukran et al. (2006) first described
and belong to the latest generation of platelets. Certainly, PRF and CGF have shown the main
characteristic of a better-organized fibrin matrix and so the better efficacy in cell stimulation for
migration and proliferation. Their preparation does not require the presence of biological
additives and the method of preparation is simple and fast. Dohan et al. (2009) described another
feature of PGF and CGF preparations with their slower release of the growth factor compared to
the PRP, which gives them better reparative ability. PRF behaves like fibrin glue due to a good
mechanical adhesive ability and stimulates angiogenesis, greater flap stability and less possibility
of necrosis and contraction of the same as well. As a preparation that properly covers the
defective region, PRF I CGF that contain leukocytes, platelets and cytokines, responsible for
remodeling the interdental papilla were put in use. Growth factors and cytokines are released in
the first seven days as well as the critical period for graft priming. Results in single cases with
PRF product showed full filling of interdental space. If the distance between the papilla tip and
contact point was equal to or less than 5mm, the success rate was 100 percent, and 56% of cases
in which the distance was 6 mm, and only 27% of cases in which the distance was 7 mm or
more.

͸ͳ

If we compare the obtained products used in the regeneration of interdental papilla, it can be
concluded based on all previous studies that the PRF and CGF advantage over PRP is that the
donor tissue is eliminated and this technique is less invasive and more favorable for the patient.
Additionally, the possibility of postoperative symptoms such as edema and pain is reduced and
the process of healing soft tissue is faster. Products are exclusively autologous obtained from the
patient's venous blood and without the subsequent addition of biological additives. One of the
disadvantages of PRP products is that only 10 % of the patient's blood is used, which can be
considered as a waste of patient¶s blood. PGF and CGF use 30-40 % of patient's blood without
the presence of biological additives. CGF as a more superficial product than PGF, is known for
its higher viscosity and contains more growth factors. It can also be used as a collagen membrane
when it is compressed and mixed with a bone graft in sinus augmentation. Unlike, PRP has
shown poor effects in the treatment of sinus elevations. When we look at all new minimally
invasive methods in the reconstruction of defective papilla, the conclusion can be made for each
method individually based on all previous studies.

Another described method is the use of HY as monotherapy or as adjunct to non-


surgical/surgical methods. Among non-surgical methods, significantly less BOP (3-13.7%) was
reported in 42% cases, bleeding index (0.19) in 14% cases, PD (0.2-0.9mm) in 50% and CAL
gain (0.7mm) in 14% all patients. Among surgical treatments, CAL gain (0.8-1.0mm) was
reported in 66% and PD (0.8mm) in 33% cases. The large heterogeneity of included studies
cannot show the exact effectivity of the HY therapy in periodontal treatment.

The use of HY as a monotherapy by implant-supported crowns has shown no differences


between observed cases, neither at baseline nor at 3 and 6 months post-treatment. PT-CP ranged
between 1.8-2.3mm, area of black triangle between 0.47-0.52 mm², PD between 2.0-2.1mm,
CAL 2.0-2.2mm, BOP 25-32.5 %, ABH 3.3-3.6mm and plaque between 32.5-40.9%.

Although it has founded a great application in cosmetic medicine and anti-aging because of its
ability to absorb water and thus increase the volume of tissue, in the application of papillary
therapy did not cause any noticeable increase in papilla volume. One of the possible assumptions
of failure is that the amount of HY needed in the application, and as another possible cause is
histological difference in the composition of the skin, gingiva and peri-implant mucosa. While
the skin is connected with the muscles underneath, tendons and bones, gingiva and peri-implant

͸ʹ

mucosa are attached with alveolar process and elastic fibers that limit the process of enlargement
and volume increase. Even though peri-implant mucosa contains less fibroblasts and smaller
blood circulation, which additionally complicates the success of therapy, studies on patients
examined in the regeneration of the interdental papilla in the region of implant supported anterior
maxilla showed that there are no positive results in increasing the papilla volume (Bertl et al.
2015) [11][46][55][50][68].

Another method such as injection with autologous fibroblasts expanded in vitro (McGuire et al.
2007) can also be considered, but can be accepted with reserve because the effects of interdental
papilla recovery are only followed within 4 months after intervention and it was not followed up
until the end because of technical limitations. The difference between test and placebo sites in
papillary height at 2 months after treatment showed a significant mean percentage increase from
the baseline of 4.0±27.8% [59].

The latest method proposed by Yamada et al. (2015) is based on the use of TEP injection (tissue
engineering papilla) containing HY, PRP and BMDSCs (bone marrow-derived mesenchymal
stem cells) and could be a bright spot for the ultimate finding of a long-lasting effects and
minimally invasive therapy in the papilla augmentation. With other words, BMDSCs cells can
induce the production of type 1 collagen in patients and its deposition, as well as support for the
regeneration of vascular, dermal and epithelial structures, assisted by growth factors from PRP
and HY as a scaffold. Clinical studies on patients performed by Yamada et al. have reported the
improved BT value of 2.55±0.89 mm in a 5-year follow up, showed significant results in
interdental papilla therapy using TEP injection [64].

Another the most important topic of nowadays issue is the risks and consequences of using cell
lines through tissue engineering. One of the possible risk factor is specificity of the cell type, the
external factors during the derivation and manipulation, culturing and storage of the cells as well
as surgical methods and immunosuppression. The main emphasis is on the potential risk of
tumor formation. Namely, the stem cells have some common features with cancer cells such as

͸͵

long life spam, resistance to apoptosis and the ability to replicate for a long time, thus similar
growth regulators are also involved in cancer maintenance.

The mouse model confirmed that autologous bone marrow stem cells cause Helicobacter-
induced gastritis cancer, osteogenic and Kaposi sarcoma as well as oral squamous carcinoma. It
is certain, that in vitro expansion and culturing change the properties of stem cells, so with the
new methods that do not include donor site this possibility can be reduced. In addition, stem cells
can induce the immune response of the host body or even modify it. One of the dilemmas is also
the distribution of stem cells, where non-engrafted cells go and their migration into unwanted
tissue. The number of cells that is needed for the maximum clinical effect is also not clearly
established, and the application of stem cells through a small injection needle can cause the
formation of cell aggregates that could be a potential risk of developing pulmonary embolism or
tissue infarction. It is also not possible to avoid their unwanted differentiation and re-
differentiation into other cell type. Differentiation of mesenchymal cells into osteocytes and
adipocytes has already been demonstrated in the previous studies, as well as the formation of
unwanted formations. Finally, alteration and secretion of cellular molecules can have a toxic
effect for the host body [69] [70].

12. Future direction-consideration




The ability of the production of new tissue and organs from the patient's own cells changes the
treatment and prognosis of a large number of patients, and therefore tissue engineering is one of
the most exciting challenges of today's regenerative medicine. However, the fact is that the in
vitro environment, where the tissue engineering product has been developed, is different from
the current environment in vivo and thus, speculation goes on what happens when a tissue
product from a stable ex vivo environment is placed in a much more dynamic in vivo
environment. Can this product be adapted to existing conditions or change the environment by
systematic and biological influences and whether the formation that will arise will be product of
the cells themselves or the reaction of the surrounding tissue?

͸Ͷ

The purpose of future studies should be to examine all the cellular performance and capabilities,
optimize the bioactive molecular dose, control their distribution, and understand the kinetics of
biological factors. One of the challenges is to examine all components involved in tissue
engineering completely and in which way they should be integrated, in order to provide
predictable results and rapid vascularization of the construct. To overcome these challenges is
necessary for the survival of the tissue engineer graft in patients. Blood derivatives have been
applied in the field of orthopedics, dermatology and vascular surgery, but the composition of
blood derivatives varies from patient to patient, and this is also one of the challenges to be
addressed. One idea suggests multiple donor pools but these products carry with them certain
risks and additional tests. New studies are exploring the concept of combination of PRP and PRF
with biomaterials and minimally invasive methods and this may open the door for new non-
surgical methods, which should be fully safe and exert long-lasting effects in augmentation of
interdental papilla.

13. Conclusion


Blood derivatives, and in particular PRP, represent a promising source of natural autologous
growth factors that potentiate stem cell proliferation, migration, and differentiation. Moreover,
clinical trials have confirmed PRP¶s safety as well as its ability to improve the clinical outcomes
of stem cell based therapies. Several approaches for treating Äblack triangle´ have been reported
in literature but most of these studies are case series with short follow-up and unpredictable
outcomes. Due to a large heterogeneity of studies recommendation on the mode of application
cannot be made. However, gaining control over blood derivatives, chemical composition remains
a key challenge to achieve reproducible and predictable clinical results.

͸ͷ

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͹ͷ

15. Supplements
15.1. List of abbreviations

x PDGF-platelet derived growth factor


x TGF- transforming growth factor
x IL- platelet factor interleukin
x PDAF-platelet ±derived angiogenesis factor
x VEGF-vascular endothelial growth factor
x EGF-epidermal growth factor
x IGF-insulin-like growth factor
x TGF-transforming growth factor-ȕ
x FGF- fibroblast growth factor
x GF-growth factors
x PRP- platelet-rich plasma
x PPP-platelet poor plasma
x PRF-platelet-rich fibrin
x CGF-concentrated growth factor
x WB-whole blood
x RBC-red blood cells
x P-PRP-pure platelet-rich plasma
x L-PRP-leukocyte and platelet-rich plasma
x P-PRF- pure platelet-rich fibrin
x L-PRF- leukocyte and platelet-rich fibrin
x PRGF-platelet-rich in growth factor
x PPF-papilla preservation flap
x MPPF- modified papilla preservation flap

x SPPF-simplified papilla preservation flap


x EJC- enamel-cementum junction

͹͸

x DPSC-dental pulp stem cells
x PDLSC- periodontal ligament stem cells
x SHED- stem cells from exfoliated deciduous teeth
x SCAP-stem cells from apical papilla
x DFPC- dental follicle progenitor cells
x DMP1- dentin matrix protein 1
x GTR- guided tissue regeneration
x CAP- calcium phosphate
x BMP- bone morphogenetic proteins
x HY- hyaluronic acid
x HCL- lignocaine hydrochloride
x BMDSCs-bone marrow-derived mesenchymal stem cells
x TEP-tissue engineering papilla
x ADP-adenosine diphosphate

15.2. List of figures

Fig.1- anterior view of the lower portion of an adult skull

Fig.2- etiology pyramid of the gingival black space

Fig.3- distance from the interdental contact point to the bone crest and the presence of
absence of black gingival triangles

Fig.4- schematic illustration, Nordland and Tarnow classification

Fig.5- the concept of tissue engineering

Fig.6- commercially available scaffold materials

Fig.7- components of blood

Fig.8- the role of biologic mediators in tissue engineering applications

͹͹

Fig.9- steps for PRP preparation

Fig.10- schematic illustration of physiological working of growth factors

Fig.11-blood sample procedure

Fig.12- schematic representation of PRP therapy

Fig.13- schematic representation of Vacutainer colors

Fig.14-Medifuge CGF

Fig.15- novel methods for the treatment of interdentally papilla augmentation

15.3. List of tables

TABLE 1- Comparison of all minimal invasive methods in periodontal treatment

TABLE 2- Summary results of all minimal invasive methods in periodontal treatment

͹ͺ

͹ͻ