COMPARISON OF BONE REGENERATION IN THE EXTRACTION …

COMPARISON OF BONE REGENERATION IN THE EXTRACTION SOCKETS WITH

AUTOLOGOUS PLATELET RICH FIBRIN GEL- AN IN VIVO STUDY

Dissertation submitted to

The Tamil Nadu Dr. M.G.R. Medical University

In partial fulfilment of the degree of

MASTER OF DENTAL SURGERY

BRANCH III

ORAL AND MAXILLOFACIAL SURGERY

2013-2016

ACKNOWLEDGEMENT

I extend my sincere and heartfelt gratitude to my teacher Dr. Mathew

Jose, Prof and HOD, under his able guidance and encouragement, I had the

opportunity of being taught and explained the surgical techniques, tips and

tricks of being an efficient surgeon. Vast experience of him has attributed in

unwinding the complex nature of many questions that took me through different

but difficult situation in my post graduate studies. I could not have asked for a

better mentor or guide for my post graduate studies.

I thank Dr. Dhineksh Kumar for the able guidance and support he has

provided me during the research and also post graduate studies. He with his

friendly talks, critical questions and sometimes being strict has made sure that I

am travelling in correct path of being a good surgeon. Best part of him always

approachable and ready to share the vast knowledge and experience he has

acquired in years.

I take this opportunity to thank our principal Dr. Elizabeth Koshi for

her help, support & patient guidance for finishing this work on the time bound

limits.

I express my sincere gratitude to my teacher Dr. Sajesh continually and

persuasively conveyed a spirit of adventure in regard to research and

scholarships, and an excitement in regards to teaching. He has always supported

me and his helpful nature has one of been the driving force for the fulfillment

of my research work.

I extend my deepest gratitude and thanks to Dr. Jomy Varghese who

has always been a great teacher and for us. He has taught us to have the right

kind of attitude a fellow surgeon need to have, he has tried to mold us into a

well behaved and classy professional with etiquette and moral and ethical

responsibilities.

I am indebted to my teacher Dr. Achuthan Nair who has tried to bring

the best out of me. He has motivated me to a mass knowledge and taught us

how learning process could be an enjoyable moments. He has been one of the

serious critics of my post graduate studies.

I extend my sincere thanks to Dr.Nandagopan for his contributions in

my thesis work. His humbleness and dedication to profession coupled with

tremendous skill had always been a fascination to me.

I am highly obliged Dr.Rajeev for being there always to clear my

doubts and guide me through the right path.

I would like to thank Dr. Murugan for supporting me through the hard

times and motivating me to study harder.

I would like to thank my super seniors Dr.Sivalinga Raja and Dr.Deepu

for teaching me the intimate skills of surgery.

I would like to thank my seniors Dr.Prem Anand and Dr.Thinakar

Babu for timely support and guidance.

I would like to thank my juniors Dr.Swaminathan, Dr.Shameem Jamal,

Dr.Harinee and Dr.Abirami for the support and enjoyable moments we had

during the post graduate life.

Above all I thank Almighty God, My parents Mr.A.Wilson and Mrs.A.Jansi

and my wife Dr.S.Shani Carmela for walking with me in each and every step of my

life and making this study to successful one.

SPECIAL ACKNOWLEDGEMENT

I take this opportunity to thank our chairman Dr.C.K.Velayudhan Nair

MS & Dr.Rema.V.Nair MD Director, Sree Mookambika Institute Of Dental

Sciences, Kulasekharam, TamilNadu for giving me an opportunity to utilize

the facilities available in this institution for conducting this study.

CONTENTS

SI.NO INDEX PAGE NO.

1 List of Abbreviations i

2 List of Tables ii

3 List of Graphs iii

4 List of Figures iv

5 Abstract v-vi

6 Introduction 1-5

7 Aims and Objectives 6

8 Review of literature 7-20

9 Platelet Rich Fibrin 21-22

10 Materials and Methods 23-33

11 Figures vii-xi

12 Results 34-36

13 Tables and Graphs 37-45

14 Discussion 46-52

15 Summary and Conclusion 53-54

16 Bibliography

17 Annexures

LISTS OF ABBREVIATIONS

PRF- Platelet Rich Fibrin

PRP- Platelet Rich Plasma

PPP- Platelet Poor Plasma

PDGF- Platelet Derived Growth Factor

TGF- Transforming Growth Factor

IGF- Insulin Derived Growth Factor

PDWHF- Platelet Derived Wound Healing Factor

AFA- Autologous Fibrin Adhesive

BMP- Bone Morphogenic Protein

bFGF- basic Fibroblast Growth Factor

EGF- Epidermal Growth Factor

PRGF- Platelet Rich Growth Factor

NBR- Natural Bone Regeneration

NTR- Natural Tissue Regeneration.

i

LISTS OF TABLES

Table 1 Demographic data of study population

Table 2 Mean bone density values of different groups at different post operative

periods

Table 3 Comparison of mean bone density values immediate between the

groups

Table 4 Comparison of mean bone density values 1 month between the groups



Table 7 Comparison of mean bone density values between the groups at

different time periods

Table 8 Comparison of mean bone density values within the groups at

immediate

Table 9 Comparison of mean bone density values within the groups at 1month

Table 10 Comparison of mean bone density values within the groups at 3 month

Table 11 Comparison of mean bone density values within the groups at 6 month

Table 12 Multiple comparison of mean bone density values within the groups at


ii

LISTS OF GRAPHS

Graph 1

Demographic data of study population

Graph 2

Comparison of mean bone density values between the groups at


Graph 3

Multiple comparison of mean bone density values within the

groups at different time periods

iii

LISTS OF FIGURES

Figure 1 Armamentarium for the preparation of platelet rich fibrin

Figure 2 Medico centrifuge

Figure 3 Withdrawal of blood

Figure 4 After centrifugation

Figure 5 Platelet rich fibrin gel

Figure 6 Extracted socket

Figure 7 Suture done after platelet rich fibrin placed in the extraction

socket

Figure 8 Grey level histogram value evaluation from Intraoral Periapical

radiograph

Figure 9 Intraoral periapical radiograph of postoperative periods

iv

Introduction

Page 1

Maxillofacial reconstruction, regenerative procedures and oral implants

etc. are depends on successful healing and regeneration . According to the clinical

research it is the most greatest challenge faced in development of bioactive surgical

additives which regulates inflammation and healing is increased. To improve the

quality of bone in Bone regenerative procedures graft materials, protein and barrier

membrane are mostly used. Tissue healing is mediated by variety of signalling

proteins. Platelets plays a vital role in wound healing.16

Repairing the bony tissue is a complex process which involves

mineralization of the defect and a number of cellular functions which is followed by

an inevitable remodelling the defects to achieve original structure1. In the platelet gel

therapeutic concept Platelet-rich fibrin (PRF) represents a new step with lacking of

artificial biochemical modification in a simplified process. 2 The platelet derived

growth factors are released abundantly by platelet concentrate which are essential

regulators for survival of mesenchymal cell lineages, migration and proliferation.3

Platelet concentrates used as the tools of regenerative medicine which is

specially designed for the local release of platelet growth factors into a wounded or

surgical site, in order to regeneration or stimulate tissue healing. Platelets are rich in

protein molecules like cytoskeleton regulatory proteins, signalling, cytokines,

membrane proteins, and other bioactive peptides which plays an essential role not

only in hemostasis but also in regulating and initiating which is the basic aspect in

wound healing.4 One of the greatest challenge in clinical research is the development

of bioactive surgical additives in regulating, enhancing healing and regeneration of

Introduction

Page 2

bone and tissues.5 Combination PRF along with fat grafts is more effective in

lipostructure surgery. 13

Platelet rich plasma (PRP), is the first generation autologous platelet

concentrate. The PRP is prepared by adding citrate to whole blood which binds the

ionized calcium and inhibit the clotting cascade. This is followed by two steps of

centrifugation in which the first step separates the white and red blood cells from

platelets and plasma and in the second step of centrifugation it further concentrates

the platelets and ultimately produces the PRP separate from poor plasma and platelet.

But the real efficacy of PRP is debatable. It is hypothesized that, just before the cell

outgrowth from the surrounding tissue, PRP releases growth factors quickly.

Platelet rich fibrin (PRF), is a second generation platelet concentrate is

a elementary process which represents a original measures in therapeutic concept and

absence of artificial biochemical modification such as the use of bovine thrombin6.

PRF is prepared by the immediate centrifugation of whole blood without any

anticoagulant with the formation of three biological phases like a bottom layer

consists of coagulated red cell, a elastic and rigid PRF gel as intermediate layer and a

supernatant serum. During centrifugation a rapid activation of coagulation cascade

and synthesis of thrombin take place this results in inducing the fibrin formation and

the platelet activation. The essential point of PRF synthesis is to accumulate platelets

and release the cytokines in a fibrin clot recompense by a natural polymerization

process during the centrifugation with release of growth factors and matrix

glycoproteins for more than 7 days.7

Introduction

Page 3

Recently, the platelet concentrates has suggested that it is an aid for

epithelial tissues and osseous regeneration in oral surgery.24 In 1974 the platelets

regenerative potential which shows better result in regenerative dentistry by Ross

et al.25

Platelet rich plasma is derived from concentrated human platelets which

is taken from plasma which have been explained to induce healing. PRF along bovine

porous it is a bone mineral which promotes bone regeneration during periodontal

defects.23

The process of wound healing is a complex mechanism which is

characterized by four phases; hemostasis, inflammation, proliferation and

remodelling they are coordinated by cell-cell interactions and by a soluble growth

factors which is released by various types of cells. The Platelets includes high

concentration of different types of growth factors with relatively low molecular

weight ranging from 6 to 45 kDa in their alpha granules which is extremely important

in regenerative process and the platelet cell membranes also play a important role in

wound healing through their growth factor receptor sites. The PRF have anti-

infectious activities.8 The platelet poor plasma have growth cells helpful to induce

differentiation of periodontal ligament and osteoblast mainly for periodontal

regeneration and bone regeneration.47 PRF shows more advantages such as surgical

time is less, good healing properties, few techniques elimination, resorption is very

less in healing.10

Introduction

Page 4

The production protocol of PRF clot and membrane often varies in

different literature. Choukroun et al2 advocated 3000 RPM for 10 minutes while kiran

et al 20119 used 2700 RPM for 12 minutes. Platelets plays an important role in

process of wound healing because platelets contain many growth factors like Insulin-

like Growth factor(IGF), platelet derived growth factors (PDGF) and also

transforming growth factor-ß (TGF-ß) which when secreted are responsible for

increasing cell mitosis, collagen production, initiating vascular in-growth and it

induce cell differentiation12. In degree of grade II furcation PRF plays important role

in healing.11

Fibrin matrix of PRF is elastic, flexible and very strong. Which consists

of weak thrombin which require equilateral junction. These junctions are connected

and permits the establishment of flexible and fine network which is capable of

supporting cellular migration and cytokines results in the increase of life spans of

cytokines. The concentration of platelet and growth factors differs in distinct

preparation protocol like disparity of tubes, gel inducing enzyme and centrifugation

force30. The topical application of PRF helpful for epithelialisation in donar site and

autografts of meshed-split thickness graft.48

A platelet proteome project reveals that more than 300 proteins which is

released by human platelets are responsible for thrombin activation and 190

membrane-associated, 262 phosphorylated proteins when identified via independent

Introduction

Page 5

proteomic and phosphoproteomic profiling.14 A progressive slow polymerization

mode may raised incorporation of circulating cytokines in fibrin meshes are released

in a relatively long-term and controllable way which helpful in soft tissue healing and

accelerates bone regeneration.15

This prospective clinical trail is designed to compare bone regeneration

with autologus platelet rich fibrin in bilateral extraction socket.

Aims & Objectives

Page 6

The aim of the study is to evaluate the comparison of bone regeneration in the

extraction socket with autologous platelet rich fibrin gel.

Review of Literature

Page 7

Matras et al 198517 described about fibrin glue which is derived from

blood product mainly used to stimulate healing and to seal wounds. It contains

concentrated fibrinogen polymerization induced by calcium and thrombin. By using

donar plasma it was prepared and used to improve skin wound healing in a rat model.

He who first described fibrin glue used as surgical adhesive which helpful for healing

process.

Knighton et al 198618 examined the efficacy of platelet derived

wound healing factors(PDWHF) which applied topically prepared with 2- step

centrifugation procedures. It was applied in 41 patients which left in place for 12

hours in 71 chronic non-healing cutaneous ulcers. He explained that PDWHF

stimulate chronically non-healing human wounds by granulation tissue formation and

epithelialization. There is no evidence of over healing like keloid formation or

hypertrophic scar.

Lynch et al 198719 suggested that at the site of injury platelet derived

growth factors (PDGF) plays an major role in the initiation of the wound healing

process.

Gibble et al 199020 found that fibrin glue is commercially available

products like beriplast, tisseel provided a consistent source of fibrinogen. The

preparation of fibrin glue contained aprotinin and bovine derived protease inhibitor

with anti-fibrinolytic activity.


Page 8

Knighton et al 199021 in his study chronic non-healing wounds in 32

patients were treated with platelet-derived wound healing formula (PDWHF). This

study shows a statistically highly significant effect of topically applied PDWHF for

the repair of chronic non-healing ulcers with epithelialisation of the wound.

Tayapongsak et al 199422 introduced mandibular reconstruction

using autologous fibrin adhesive (AFA) is the novel idea of reconstructive options.

Slater et al 199527 conducted a study to examine the efficacity of

concentrated human platelet as a eked out to basic medium in the functional and

proliferative activity of human osteoblastic like cells. The results shows that eked out

platelet medium stimulates the proliferation and maintains the activity of

differentiated function of human fetal osteoblast like cells.

Heldin et al 199733 presented a report on intracellular signal

transduction pathways which involves PDGF induced cell motility and growth also

discussed the fact that the inhibitory stimulatory pathways are parallely induced. He

also reported that the PDGF exert their effect by the binding of two protein tyrosine

kinase receptor which is structurally related.

Whitman et al 199726 used “platelet gel” derived from platelet

concentrate produced by a gradient density cell separator shown their clinical results

in reconstruction of oral and maxillofacial surgery. The authors considered the platelet

gel as a fibrin glue helpful for mandibular reconstruction, alveolar cleft procedure,


Page 9

closure of oro antral and oro nasal communication and also show greater effect in

implant placement.

Green et al 199834 reported that platelet gel was used as wound

sealant. The presence of leukocytes and platelets in the formulation of antimicrobial

and hemostatic support which bring the growth factors and cytokines to the surgical

site.

Marx et al 199828 first introduced platelet rich plasma in dental studies

clinically. The platelet rich plasma is used to improve the graft incorporation in the

mandibular reconstruction to the patients who have received cancellous bone marrow

grafts after the tumor removal. In their study it have been showed that PRP contains a

concentrated platelets and growth factors. The PRP which was used with grafts

showed a evidence of radiographic maturation rate 1.6 to 2.16 times faster than the

grafts which was used without PRP. This data suggested strongly that by adding PRP

to bony grafts accelerates the degree and rate of bone formation.

Anitua et al 199931 performed a study in 20 patients for whom

extraction was done and subsequent implant placement was done. A biopsy in that

area was obtained without any additional discomfort, macroscopically there was a

evident of epitheliazation microscopically the bone was compact and mature and well

organized trabeculae was evident with normal physiology.


Page 10

Heldin et al 199932 reported that the first growth factor was the PDGF

which shows chemotactic for migration of cells in the healing skin wound like

fibrolast, monocytes and neutrophils. He also proved that PDGF enhances not only

the proliferation of fibroblast and extracellular matrix production but also it stimulates

the fibroblast to contract collagen matrices and also induces the myofibroblast

phenotype which suggest a major role in healing of wound. Also he demonstrated that

the series of clinical and experimental studies about the useful effect of PGDF which

can be used in wound healing disorders.

Schuldiner et al 200035 examined the ability of eight growth factors

such as basic fibroblast growth factor(bFGF), activin-A, bone morphogenic protein 4

( BMP-4), ), transforming growth factor ß1 (TGF-ß1), epidermal growth factor(EGF),

hepatocyte growth factor(HGF), b nerve growth factor (bNGF) and retinoic acid(RA).

Anitua E (2001)36 conducted various studies to see the effect of

platelet rich in growth factors (PRGF) and proved it is a conventional methods to

accelerates soft tissue and bone regeneration in post extraction sockets.

Man et al 200129 described a new technique by preparing autologous

fibrin glue and platelet gel. They examined the effective stopping of capillary

bleeding in surgical flaps for the patients undergoing cosmetic surgery. Their finding

showed both products have effect in maintaining hemostasis and sealing of capillary

bleeding.


Page 11

Soffler et al 200338 suggested that the fibrin sealants may enhance

overall outcome of the surgical intervention due to adhesive, hemostatic and healing

properties.

Lacoste et al 200339 quantified that the concentration of TGF-ß1,

bFGF, VEGF and PDGF-BB from the platelet concentrates and the whole blood

before and after there is addition of various concentrations such as calcium and

thrombin and also have been assessed that physiological importance of growth

factors on angiogenesis.

Weibrich et al 200337 studied the level of growth factors in PRP by

dispersion of cell separation method and also point of care method. It is also called as

‘buffy coat method’. From 115 healthy donars whole blood was drawn . The

increase in thrombocyte count was obtained in both of the methods which results in

higher TGF-ß1 levels while increase in the leukocyte count in curasan PRP shows

more PDGF-AB levels.

Anitua et al 2004 40showed that the platelets contains the storage

pools of growth factors such as VEGF, TGF-ß and PDGF and cytokines which

includes proteins like PF4 and CD40L. He also reported that the growth factors

attached to the platelets and/ or fibrin which results in enhanced activity over the

recombinant proteins, also he reviewed the various situation in the muscle/tendon

repair, orthopaedic surgery, hole repair in the eye surgery , reversal of skin ulcers and

cosmetic surgery where platelet accelerates the healing mechanism.


Page 12

Dohan et al 200641 quantified the level of cytokine within PRF clot

exudates serum and platelet poor plasma supernatant : IL-1b, IL-4 , IL-6,VEGF and

TNF-alpha that was obtained with in plasma and in serum. He proved that PRF

shows a complex tridemsional architecture it makes a real platelet and also a

leukocyte rich fibrin biomaterial.

Li et al 200642 explained the pleiotropic function of TGF-ß which

signalling in T cells. It shown that T cell specific deletion in mice with of TGF-ß

receptor II which developed lethal inflammation and it is associated with T Cell

activation and differentiation.

Thor et al 200743 calculated that effect of platelet rich plasma on the

early stage and late stage bone healing after maxillary sinus grafting. There was

significantly more new bone were formed at platelet rich plasma treated sites when

compared to control group in 3 months of healing. He suggest that platlet rich plasma

have low regenerative capacity which influenced the bone healing in the early phase.

Diss et al 20085 studied that the radiographic changes occurs in the

apical portion the bone levels on the microthreaded implants when it is placed in

subsinus residual bone height it is according to a bone added osteotome the sinus floor

elevation technique with PRF is used as grafting material in 20 patients.


Page 13

Lundquist et al 200846 platelet rich fibrin gives a sustained release

and also protection against the proteolytic degradation of the endogenous fibrogenic

factors which is most important for wound healing.

Su et al 200844 analyzed that growth factors release from the platelet

gels from 10 volunteers where the whole blood was collected along with

anticoagulant citrate dextrose(ACD). The estimation of growth factor from platelet

gel with releasate, platelet gel, releasate with thrombin at 5 , 60, 120, 300 minute

time points. TGF-ß1, PDGF-AB, EGF and VEGF increase was identified in platelet

gel with releasate of the PDGF-AB of 0.6x10-16 and also the TGF-ß1 of 0.9x10-16 per

PLT.

Sunitha et al 200845 concluded that the combination of growth factors

and bone grafts contained in platelet rich fibrin and platelet rich plasma may be

suitable to increase bone density. In experimental study the content such as growth

factor in platelet rich fibrin and platelet rich plasma was measured by using Elisa Kit.

Dohan et al 20097 estimated the growth factors from PRF initial

serum exudates and which was gently flushed out. The membrane was placed in an

significant volume of the culture medium (4ml) which protect the cell integrity of

leukocytes in the clot. He display that when these membranes were prepared with in

the PRF Box, PDGF-AB amounts were significantly higher at each experimental

time and the TGF-1 and VEGF amounts were significantly higher during the first 4

hours.


Page 14

Gassling et al 200916 generated PRF and PRP from the whole blood

samples in ten patients. Human fibroblasts, human osteoblasts and human osteoblast-

derived osteosarcome cells which were used for the cell culture and shown the growth

factors IGF-1and TGF-ß1 and ß2 ,PDGF-AB and BB found that the cytokine

concentration were higher for PRP and PRF in the osteoblasts and also in Saos-2

cultures.

Mazor et al 200950 used platelet-rich fibrin as a sole filling material

during sinus lift and implantation which is stabilized a high volume of regenerated

natural bone in subsinus cavity till the tip of implants. According to Choukroun’s

platelet rich fibrin is an simple and inexpensive biomaterial used during a sinus lift ,

particularly it will protect the schneiderian membrane.

Su et al 200949 observed that the amount of growrh factors into

supernatant serum (SS) and PRF releastate (PRFR) after the preparation of PRF.

Growth Factor release was determined for 300 minutes over total period of time. It

covers the maximum time which needed for surgical procedures and also higher

quantity of transforming growth factor (TGF)-1 and PDGF-AB was released. He

concluded that the PRFR and SS should be mixed along with bone grafts the use of

patients blood helpful for tissue regeneration.

Simonperi et al 200951 showed platelet rich fibrin membranes were

particularly helpful for healing and maturation of periosteum. Thus the thick Peri-


Page 15

Implant in gingival region show the result of several healing phases of platelet rich

fibrin membrane layer.

Gassling et al 201054 platelet rich fibrin is superior to collagen(Bio-

Gide) used as a scaffold membrane for human periosteal cellular proliferation.

Periosteal cell cultivation in bone tissue engineering Platelet Rich Fibrin membranes

plays a major role.

Gurbuzer et al 201055 platelet rich fibrin force not lead to lift bone

healing in the soft tissue impaction of mandibular third molar removal sockets after

4 weeks of surgery.

Lucarelli et al 201052 evaluated the growth factors present in PRFM

in vitro and shown their ability to accelerates the growth of mesenchymal stem cells

plays a major role in tissue repair. There mechanical properties and also the platelet

distribution were evaluated. Platelets found to be localized in one side of membrane.

In the first day the kinetics of release were more for VEGF, and in bFGF more

gradual release was seen. The concentration of the PDGF-AA, PDGF-AB,

VEGF,TGF-ß1,bFGF, EGF in PRFM conditioned media were greater in a day 1

when compared to other days.

Senger et al 201053 showed that both bFGF and VEGF will stimulate

the endothelial proliferation and also angiogenesis but VEGF transcripts encode and

directs active secretion of VEGF from the cell, and it is considered to be the


Page 16

endothelial specific mitogen. The VEGF stimulates nitric oxide production it will

inhibits platelet activation and suggesting that the role of VEGF is to prevent

undesirable thrombosis.

Ahmed et al 201156 described about the autocrine pool between FGF

and PDGF-BB. It shown that the FGF transcriptionally activates PDGF receptors . It

is then reinforced to the membrane. PDGF and FGF trigger expression of PDGFR

and FGFR respectively and resulted in decrease in number of receptors residing in the

plasma membrane after internalization. There is a crosstalk about the proteins to

switch the expression of receptors to sensitize the cells to the respective ligands this

shows how both these factors regulates the neoplasticity metastasis and angiogenesis.

Kiran et al 20119 described method how to prepare PRP and their

clinical application and also the safety concerns of PRP. The evolution of PRF the

second generation platelet concentrate which is prepared by the blood is centrifuged

in centrifuge machine for 12 minutes at 2700 RPM. Then he reported PRF eliminates

adding anticoagulant as well as it needs to neutralize.

Lee et al 201161 did experiments in the animal model with peri-

implant of defect sized 3.0x5.0 mm (width x length) repaired successfully by

applying Platelet- Rich Fibrin alone.

Prakash et al 201158 explained with platelet-rich fibrin, though the

biomaterial appears to accelerate the physiologic healing, there is a numerous point

of view about Platelet Rich Fibrin have to be tested clinically.


Page 17

Ruga et al 201159 combined the action of piezoelectric surgery and

Platelet Rich Fibrin which can be considered a fine and safe technique for alveolar

socket healing and third molar surgery.

Sammartino et al 201157 described L-Platelet Rich Fibrin is a safe

filling and also the hemostatic material it is a reliable therapeutic option which avoid

significant bleeding after extractions in dentistry without any suspension of

continuous anticoagulant therapy in cardiac patients.

Simonperi et al 201160 L-Platelet Rich Fibrin used as a sole filling

material during sinus-lift and implantation which is suitable surgical option for

promoting regeneration of natural bone.

Bambal et al73 2012 he describes the successful rate of platelet rich

fibrin while treating the periapical lesion which shows a better healing when

compared to the other.

Del corso et al 201266 showed that L-Platelet Rich Fibrin also

defining a new therapeutic principles: Natural tissue regeneration (NTR) for intra

bony lesion treatment in periodontal problem and the natural bone

regeneration(NBR)to reconstruct the alveolar ridges.

Dohan et al 201262 estimated that the slow release of growth factors

PDGF-AB, TGF-ß1, VEGF and matrix proteins from P-PRP gel membrane and L-


Page 18

PRF in culture medium. He explained that the less than 5 days P-PRP gel

membranes dissolves completely in the culture medium, mean while the L-PRF

membranes are still intact after 7 days this implies that polymerization and the final

architecture of fibrin matrix influence the strength and the growth factor trapping/

release potential of the membrane.

Jankovic et al 201263 for 6 months he did randomized controlled

clinical study and compared the results with the use of a connective tissue grafts

(CTG) or platelet rich fibrin(PRF) membrane in the

gingival recession and clinically evaluated the impact of PRF on subjective patient

discomfort and early wound healing.

Ozdemir et al 201272 while used along with titanium barriers rate of

bone regeneration is increased due to growth factors present in the Platelet Rich

Fibrin(PRF).

Peck et al 201267 L-Platelet Rich Fibrin is newly developed platelet

concentrate and it is successfully used in the number of surgical procedures to induce

wound healing and also used to stimulate bone formation for ideal and successful

implants placement.

Tunah et al 201265 T-Platelet Rich Fibrin induce the formation of new

bone along with the new connective tissue in the rabbit model helpful for wound

healing in 30 days of treatment.


Page 19

Vinayakumar et al 201264 platelet rich fibrin is a second generation

platelet concentrate mainly used widely used to increase soft and hard tissue healing.

Gassling et al 201370 due to the limits of study, lateral sinus window

were covered by two different absorbable membranes such as a conventional collagen

membrane or platelet rich fibrin.

Khiste et al 201368 described that the PRF starts the healing process

to increase predictability. It contains the thrombin concentrations is weak with

equilateral functions it permits to form a fine and flexible network which supports

the cytokines.,

Yosif et al 201369 illustrated the use of PRFM in the implant

osseointegration site. In this study thirty two adult rats, where the titanium implants

were inserted in the femur.PRF place on right side and left side taken as control

group along with screw implants only.

Mc Lellan et al 201471 in 12 healthy patients the PRF is evaluated

thoroughbred geldings and it is compared with the temporal release of the growth

factors to PRP. For five days four groups were analysed for slow and immediate

release PRP and PRF.

Kumar et al 2015 82 Platelet concentrates shown to have great scope

in reconstructive and regenerative medicine as well in dentistry. And PRF being the


Page 20

recent of the Platelet Derivatives which is safer and simpler when compared to

previous PRP concentrates they are used clinically.

Reddy et al 2015 84 observed the healing of PRF membrane site

supports to accelerates the soft tissue healing. PRF membrane act as a bandage is an

efficacious approach which protect the open wound in palate helps to reduce healing

time and discomfort of the patient.

Raaj et al 2015 83 Platelet rich fibrin (PRF), is a second generation

platelet concentrate and shown to promote soft tissue healing because PRF contains

autologous growth factors and cytokines which responsible factors for the

regeneration of bone and also for the maturation of soft tissue.

Platelet Rich Fibrin

Page 21

Platelet rich fibrin is a platelet concentrates. It is single fibrin mesh which

contains all constituents of blood products helpful for healing and immunity. The recent

biomaterial is autologous matrix which has cicatricial properties, like fibrin glue or like

platelet concentrate.59 Fibrin is activated from fibrinogen. This molecule is present in plasma

and in platelet alpha granules plays a major role in hemostatis by the aggregation of

platelets.25,38

Platelets is a major constituents obtained from blood. The structure is

discoidal and a nuclear structure. The diameter is 2-4µm. Megacaryocytes of bone marrow is

the main source for platelet production. The lifespan become 8-10days.11 There are so many

growth factors seen in platelets. They are listed below,

Transforming growth factor ß-1 it is a fibrosis agent. It is considered as inflammatory

regulator which induce fibrous cicatrisation.

Platelet derived growth factor it is a stimulant of mesenchymal lineages. It is a

essential regulators helpful for migration, survival of mesenchymal cell lineages and

proliferation.

The insulin like growth factor is a cell protective agent. 11

Platelet rich fibrin prepared by centrifugation of blood without add any

anticoagulant. It contain fibrin matrix which is polymerized into tetra molecular structure,

cytokines, platelets, circulating stem cells and leukocytes include into it. Platelet gel can also

Platelet Rich Fibrin

Page 22

used as an membrane. Fibrin gel can also used along with bone grafts which helpful for

healing, bone regeneration and also achieve haemostasis.59

Choukroun et al first developed platelet rich fibrin specifically used in oral

surgery. In this technique no need to require any anticoagulant. Patients own blood is

centrifuged so there is no possible of spreading any blood borne diseases.25

The blood is collected without any anticoagulant and immediately

centrifuged for 10 minutes in 3000rpm. Due to absence of anticoagulant most of the platelets

activated in few minutes in contact with tube walls the coagulation cascades released. Fibrin

clot obtained between red blood corpuscles and a cellular plasma. A cellular plasma is the

top layer, fibrin is the middle layer and the red blood corpuscles is the bottom layer.25

The secret of the success present on faster blood collection and immediately

transfer to centrifuge. Prolonged time make chance of failure in process.

Platelet rich fibrin induce soft tissue healing, bone regeneration and it is

mixed with graft material. Platelet rich fibrin used in treating residual extraction sockets.34 It

can also used to treat gingival recession and protect the root platelet rich fibrin used in sinus

lift and dental implant procedure.72

Materials & Methods

Page 23

The patients with bilateral extraction in dental arch were selected

for the study from the out patient department of oral and maxillofacial surgery, Sree

Mookambika Institute Of Dental Science, Kulasekharam. The patients were divided

into two groups for this study. In Study group Platelet-Rich Fibrin was placed after

extraction in the extracted socket. In Control group patients there is no intervention

after extraction in the extracted socket. Before commerencing the study we got

approval from Institutional research committee and Institutional human ethical

committee, Sree Mookambika Institute Of Dental Science, Kulasekharam. All the

patients who were included in the study agreed for the study protocol and written

informed consent was submitted. All patients were recalled for suture removal in 7th

day and reviewed in 1st, 3rd , 6th month postoperatively.

Total duration of the study: 6 months

Number of groups: Two groups

Description of groups:

30 patients were taken for dental extraction. Total 60 extraction (30 in

case and 30 in control) One is case group and other is control group. Atraumatic

extraction carried in the right and left quadrant of the same jaw and same teeth. One

side taken as case and other side as control.

Materials & Methods

Page 24

Group A- Platelet rich fibrin placed

Group B- No intervention

Sample size of each group:

According to this formula 22

1 22 the sample size of each group may be 30.

Inclusion Criteria:

• Patient of age group 18 to 55 years will be selected irrespective of religion, socio-

economic status, caste and sex.

• Patients in whom bilateral extraction is indicated.

• Patients who were agreed for this study protocol/consent.

Exclusion Criteria:

• Allergic to local anaesthesia.

• Chain smoker

• Uncontrolled systemic illness.[on h/o]

• Immunodeficiency pathology [on h/o]

• Bone disorders

• Patient with psychiatric problem

• Patients who were not willing for post operative follow-up

• Platelet disorders

• hematological disorders.

Materials & Methods

Page 25

• Chemotherapy and radiotherapy

• Stress situations

The subjects were informed of the study after the screening

procedure. They were explained that their selection was made since they met the

inclusion and exclusion criteria. The subjects were ensured that their participation is

voluntary and that they have the right to withdraw at any time during the course of the

study without giving reasons. Informed consent was obtained from the volunteers.

ARMAMENTARIUM

• Medical centrifuges

• Tourniquet

• 5ml disposable syringe with 24 gauge needle

• Surgical gloves

• Cotton gauze

• Spirit swabs

• Blood collecting glass tubes

• Nasal dressing forceps

• Scissors

• Stop watch

Materials & Methods

Page 26

PROCEDURE IN DETAIL:

Patient was prepared for the extraction. One side taken as case and

other side taken as control in same patient with bilateral extraction. Under aseptic

condition instruments were arranged, patients was draped. Intraorally inferior alveolar

nerve, long buccal nerve block, lingual nerve block were given for mandibular

extractions and posterior superior alveolar nerve block, middle superior nerve block,

anterior superior alveolar nerve block, greater palatine nerve block and nasopalatine

nerve block were given for maxillary extractions by injecting 2% solution of

lignocaine hydrochloride along with 1:80000 adrenaline. (LIGNOX 2 %). [3ml

Syringe with 24 gauze needle]

SURGICAL TECHNIQUE:

Full thickness mucoperiosteal flap was reflected to achieve adequate

exposure of the surgical site by periosteal elevator. Tooth was delivered using

appropriate forceps. After extraction bony spicules which is sharp and with rough

edges of extracted socket was trimmed and smoothened with bone file and bone

rounger. The hemostasis were achieved. Now after this procedure the prepared

Platelet rich fibrin was placed in the extracted socket for the side of case group . With

the help of 3-0 silk suture material, the extracted socket were sutured. The opposite

side extraction carried after 1 week taken as control.

Materials & Methods

Page 27

PREPARATION OF PLATELET RICH FIBRIN:

A tourniquet was tied on patient’s arm. In the aseptic conditions, for

about 4ml blood was withdrawn and it was collected in the 5ml capacity blood

collection tube which was pre-sterilized without any anticoagulant and it was

centrifuged immediately for 10 minutes at 3000rpm. After centrifugation following

three layers were formed.

1. Topmost layer consists of a cellular platelet with poor plasma

2. Middle layer contains Platelet rich fibrin clot

3. Bottom layer is Red blood corpuscles

Now from the centrifugation tube platelet rich fibrin clot was

withdrawn using a nasal dressing forceps with long and narrow beak. After

withdrawal of platelet rich fibrin clot, the red blood corpuscles clot which was seen is

attached to the base of platelet rich fibrin clot. Using scissors this was separated. Now

the platelet rich fibrin clot is ready to place in the socket.

ROUTINE POST OPERATIVE CARE:

• The post operative instructions were given to the patients which includes a 30minutes

firm pressure with a sterile gauze pack and to take semisolid or cold liquid diet for

first 24 hours and to avoid rinsing.

• After every meals patients were advised to rinse with warm saline rinse and 0.2%

chlorhexidine rinses twice daily after 24 days for 1 week.

• Postoperatively antibiotics and analgesics were prescribed for 5 days.

• On 7th postoperative day the suture was removed.

Materials & Methods

Page 28

FOLLOW-UP:

• Post-operative follow done in case group after 24 hours to check the stability of

platelet rich fibrin in socket.

• After 7 days suture removal was done. Succeeding follow-up was done at 1st month,

3rd month and 6th month postoperatively for the evaluation of radiographic

parameters.

PARAMETERS EVALUATED

BONE DENSITY ANALYSIS

With the help of HP Scan Jet 7400C scanner all the IOPA images were

digitalized. “Adobe Photoshop 7.0” which is a digital software programme is used to

analyse the Bone density and it enlarges the standard IOPA radiograph for better

resolution.

The grey level histogram value was measured in immediate

postoperative IOPA and the graph was marked.. This interpretation of bone density

appears white for dense bone and it appears black on empty defect in radiograph.

The bone density analysis was done and compared postoperatively by using software

measured by grey level histogram value in 1st month, 3rd month and 6th month post-

operative period and the value were compared.

Materials & Methods

Page 29

STATISTICAL TOOLS EMPLOYED:

The study was analyzed by statistical package for social

sciences(SPSS 16.0) version. Unpaired t test applied to find the significant between

the groups. P value less than 0.05(p<0.05) considered statistically significant at 95%

confidence interval.

The grey level in histogram value is used to evaluate bone density by

using Wilcoxon rank-sum test to compare between two groups. The Friedman test

is used to compare within the group.

Materials & Methods

Page 30

SREE MOOKAMBIKA INSTITUTE OF DENTAL SCIENCE, KULASEKHARAM

DEPARTMENT OF ORAL AND MAXILLOFACIAL SURGERY

CASE RECORD

DATE: OP NO.

PERSONAL DATA:

Name:

Age:

Sex:

Address:

History:

Cheif complaints:

History of present illness.

Materials & Methods

Page 31

Medical history:

Dental history:

Personal history:

Family history

EXAMINATIONS:

Extra oral examination:

Face:

Tmj:

Lymphnodes:

Materials & Methods

Page 32

Intraoral examination:

Soft tissue examination:

Buccal/labial mucosa:

Tongue:

Alveolar mucosa:

Floor of the mouth:

Hard tissue examination:

Teeth present:

PROVISIONAL DIAGNOSIS:

Materials & Methods

Page 33

TREATMENT PLAN:

POSTOPERATIVE EVALUATION:

RADIOGRAPHIC EVALUATION

Findings Review

Grey level histogram values

Immediate post-op Date:

1st month post-op Date:

3rd month post-op Date:

6th month post-op Date:

Guide/ Co-guide signature

Figures

Page vii

FIG: 1 ARMAMENTARIUM FOR THE PREPARATION OF PLATELET

RICH-FIBRIN

FIG:2 MEDICO CENTRIFUGE

Figures

Page viii

FIG:3 WITHDRAWAL OF BLOOD

FIG:4 AFTER CENTRIFUGATION

Figures

Page ix

FIG:5 PLATELET RICH FIBRIN GEL

FIG:6 EXCTRACTED SOCKET

Figures

Page x

FIG:7 SUTURE DONE AFTER PLATELET RICH FIBRIN PLACED IN THE

EXTRACTION SOCKET

FIG:8 GREY LEVEL HISTOGRAM VALUE EVALUATION FROM

INTRAORAL PERIAPICAL RADIOGRAPH

Figures

Page xi

FIG:9 INTRAORAL PERIAPICAL RADIOGRAPH OF POSTOPERATIVE PERIODS

IMMEDIATE POSTOPERATIVE DAY

CONTROL CASE

1 MONTH POSTOPERATIVE PERIOD

CONTROL CASE


CONTROL CASE


CONTROL CASE

Results

Page 34

The present study was designed to evaluate the efficacy of Autologous

platelet rich fibrin in bone regeneration after extraction. The study was undertaken on

30 patients with bilateral extraction from the outpatient department of oral and

maxillofacial surgery in Sree Mookambika Institute Of Dental Science,

Kulasekharam.

The age, sex and date of procedure were recorded. Grey level histogram

value recorded from IOPA in immediate postoperative day, 1 month, 3 month and 6

months postoperatively of all patients of the case and control groups has been

tabulated.

By statistical analysis the results obtained were given below,

Total 30 patients were selected for this study, among 30 patients 12 patients

were males(40%) and 18 patients were females (60%). The male to female

ration was 1:1.5.

The mean age of the 30 patients were 29.37 years (Table:1, Graph 1)

The mean bone density values become 47.53 and 47.50 of control and case of

immediate postoperative, 58.73 and 60.85 of control and case in 1st month

postoperative, 66.70 and 69.46 of control and case in 3rd month postoperative,

74.23 and 77.30 of control and case in 6th month postoperative period.

(Table:2).

Results

Page 35

The mean bone density values of immediate postoperative become no

significant between control and case (Table:3)

The mean bone density values of 1st month postoperative period become

significant between control and case(Table:4)

The mean bone density values of 3rd month postoperative become significant

between control and case(Table:5)

The mean bone density values of 6th month postoperative period between

control and case become significant. (Table:6)

The comparison of mean bone density values between the groups at different

time periods become significant.(Table:7, Graph 2)

The mean bone density values within the group at immediate postoperative day

is significant when compared within other time periods.(Table:8)

The mean bone density values within the groups at 1 month postoperative

period is significant within other time periods(Table:9)

There is significant values of mean bone density within the groups at 3rd month

postoperative within other time periods.(Table:10)

Results

Page 36

The mean bone density values within the groups at 6th month postoperative is

significant within other time periods.(Table 11)

The multiple comparison of mean bone density values within the groups at

different time periods is significant compared immediate with other time

periods, 1st month with other time periods and 3rd month with other time

periods.(Table:12, Garph 3)

The bone density value is higher in case treated with Autologous

Platelet Rich Fibrin gel compared with control group which has been not treated with

Autologous Platelet Rich Fibrin gel.

Tables & Graphs

Page 37

Table-1: Demographic data of study population

Demographic data

Age

(MEAN±SD)

Gender

Male

Female

Number Percentage (%)

Number Percentage (%)

Groups

29.37±8.26

12

40.00

18

60.00

Table-2: Mean bone density values of different groups at different post operative periods

Groups

Immediate (MEAN±SD)

1 month (MEAN±SD)

3rd month (MEAN±SD)

6th month (MEAN±SD)

Control

47.53±2.09

58.73±2.47

66.70±2.24

74.23±1.83

Case

47.50±2.09

60.85±2.52

69.46±2.16

77.30±1.70

Tables & Graphs

Page 38

Table-3: Comparison of mean bone density values immediate between the groups

Groups


p value

Control

47.53±2.09

0.95

Case

47.50±2.09

(p>0.05 no significant compared between control and case)

Table-4: Comparison of mean bone density values 1 month between the groups

Groups

1 month (MEAN±SD)

p value

Control

58.73±2.47

0.05

Case

60.85±2.52*

(*p<0.05 significant compared between control and case)

Tables & Graphs

Page 39

Table-5: Comparison of mean bone density values 3rd month between the groups

Groups


p value

Control

66.70±2.24

0.001

Case

69.46±2.16*


Table-6: Comparison of mean bone density values 6th month between the groups

Groups


p value

Control

74.23±1.83

0.001

Case

77.30±1.70*


Tables & Graphs

Page 40

Table-7: Comparison of mean bone density values between the groups at different time periods

Groups

Imme+iate (MEAN±SD)

1 month (MEAN±SD)



Control

47.53±2.09

58.73±2.47

66.70±2.24

74.23±1.83

Case

47.50±2.09

60.85±2.52*

69.46±2.16*

77.30±1.70*

p value

0.95

0.05

0.001

0.001


Table-8: Comparison of mean bone density values within the groups at immediate

Time period Control p value Case p value


47.53±2.09 47.50±2.09

1 month (MEAN±SD)

58.73±2.47* 0.001 60.85±2.52* 0.001


66.70±2.24* 0.001 69.46±2.16* 0.01


74.23±1.83* 0.001 77.30±1.70* 0.001

(*p<0.05 significant compared immediate within other time periods)

Tables & Graphs

Page 41

Table-9: Comparison of mean bone density values within the groups at 1 month


1 month (MEAN±SD)

58.73±2.47 60.85±2.52


47.53±2.09* 0.001 47.50±2.09* 0.001


66.70±2.24* 0.001 69.46±2.16* 0.01


74.23±1.83* 0.001 77.30±1.70* 0.001

(*p<0.05 significant compared 1 month within other time periods)

Table-10: Comparison of mean bone density values within the groups at 3rd month



66.70±2.24 69.46±2.16


47.53±2.09* 0.001 47.50±2.09* 0.001

1 month (MEAN±SD)

58.73±2.47* 0.001 60.85±2.52* 0.01


74.23±1.83* 0.001 77.30±1.70* 0.001

(*p<0.05 significant compared 3rd month within other time periods)

Tables & Graphs

Page 42

Table-11: Comparison of mean bone density values within the groups at 6th month



74.23±1.83 77.30±1.70


47.53±2.09* 0.001 47.50±2.09* 0.001

1 month (MEAN±SD)

58.73±2.47* 0.001 60.85±2.52* 0.01


66.70±2.24* 0.001 69.46±2.16* 0.001

(*p<0.05 significant compared 6th month within other time periods)

Table-12: Multiple comparison of mean bone density values within the groups at different time periods

Time period Control Case


47.53±2.09 47.50±2.09

1 month (MEAN±SD)

58.73±2.47* 60.85±2.52*


66.70±2.24*,# 69.46±2.16*,#


74.23±1.83*,#,$ 77.30±1.70*,#,$

(*p<0.05 significant compared immediate with other time periods, #p<0.05 significant compared 1 month with other time periods, $p<0.05 significant compared 3rd month with other time periods)

Graph-1: D

1

1

1

1

1

2

Num

ber

Demograph

0

2

4

6

8

10

12

14

16

18

20

hic data of

Male

study popu

e

G

ulation

Gender

Tab

Femal

bles & Gr

le

raphs

Page 43

Graph

0

10

20

30

40

50

60

70

80

Mea

n

h-2: Mean

Immed

bone densi

diate 1

ity values ooperative p

1 month

Ti

Control

of different periods

3rd mo

ime

Case

Tab

groups at

nth 6th

bles & Gr

different p

h month

raphs

Page 44

ost

Graph-3: Mdifferent ti

0

10

20

30

40

50

60

70

80

90

Mea

n

Multiple coime period

Immed

omparison s

diate

of mean bo

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Control

one density

3rd m

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Page 45

roups at

th

Discussion

Page 46

Bone tissue repair is a complex process helpful for cellular

functions and mineralization of defects to remodelling the surgical defect to regain

the original structure.74 In future protecting wound and regain the bone will become

standard care for all extractions. Platelet-rich fibrin is new step in platelet therapeutic

concept helpful for artificial biochemical modification.8 Platelet-Rich Fibrin is a

autologous fibrin which contain huge amount of platelet and leukocyte cytokines41.

Wound healing involves a sequence of physiological events which

restore and replace the damaged tissue functions. After extraction have been done the

healing is explained in coagulative phase, proliferative phase and osteogenic-

remodelling phase. The coagulative phase starts from immediately after the extraction

and last until 3days which involves filling the socket with clot and inflammatory

process initiation. In this phase platelets, endothelium and fibroblast begins to release

a numerous growth factor. After this the proliferative phase starts and last until 20

days to 2 months following postoperative period . After this the blood clot become

dissolved, connective tissue matrix is formed, there is a supply of blood to the wound

the osteoblastic activity and osteoclastic activity is initiated, the last and the longest

phase is osteogenic-remodelling phase. The healing of the extracted socket last from

weeks till months. This phase involves mineralization of the matrix, secretion of

osteoid and bone remodelling.

The platelets helpful for blood clotting by hemostatic plug

formation. By using platelet concentrate it helpful for natural clot formation which

induce wound healing and bone regenerative process.34 The PRFM creates a gel like

substance which contain functional, nonactivated, intact platelets present in the fibrin

Discussion

Page 47

matrix which release growth factors over the period of 7 days.75 It mainly used as a

protective layer for scheniderian membrane in sinus lift procedure to fill the material.6

Bone morphogenic was first identified in the year 1965 . the bone

formation is induced when demineralised bone matrix is placed. There is a wide

evidence which supports there role as bone induction regulators repair and

maintenance also being critical determinants of embryological development of

mammalian organisms. In differentiation, growth inhibition, proliferation and arrest of

wide variety of maturation cells BMP plays a major role depending on cellular

microenvironment and interaction with other regulatory factors. Some of the demerits

of BMP are poor distribution, large dose requirement, high cost and short half life. In

order to overcome these demerits alternative methods like promoting bone

regeneration and formation.76

The initial coagulative phase contains a series of physiological

process, they are proliferation, cellular migration and differentiation, initiation of

vascular in-growth and increased collagen production. In order to bring efficient and

timely prepare of wounds many type of cell, other proteins and growth factors interact

with one another.

Several studies made in last 10 years in animal and in vivo

conclude polypeptide growth factors helpful for soft tissue and bony healing.

Transforming growth factor (TGF)ß1 and ß2 helpful to inhibit bone resorption. It

helpful for faster maturation of collagen in wounds. Platelet derived growth

Discussion

Page 48

factor(PDGF) helpful to increase the wound healing cells which helpful for increase

wound healing properties. 34,77

The main advantages in using platelet rich fibrin may be34

Helpful for tissue healing

Regenerate the bone defects

Maintain hemostasis

Simple chair side procedure

With drawl of patients own blood

No chance of cross infection

No need of any anticoagulants.

The platelet rich plasma is first generation platelet concentrate

contain various growth factors such as platelet derived growth factor, vascular

endothelial growth factor and transforming growth factor obtained from freshly drawn

venous blood. Fibrin, fibronectin and vitronectin are the three proteins present which

helpful for epithelial migration and osteoconduction.

The second generation platelet concentrate is platelet rich fibrin is

centrifuged without any anticoagulants.25 It contains various growth factors which

helpful for wound healing and bone regeneration process. When compared to platelet

rich plasma no need of add any anticoagulants. Platelet rich fibrin release cytokines in

a fibrin clot. In this study we used platelet rich fibrin to regenerate the bony defects in

the extraction sockets.

Discussion

Page 49

PRF and oral surgery:

To restore the bone loss so many regenerative medicine technique

are applied in dentistry. In Choukroun’s PRF the blood collected without

anticoagulant and centrifuged immediately. The natural coagulation process helpful

for easy collection of PRF clot. To induce bone regeneration in sinus lift procedure to

achieve good implant placement it plays major role to achieve good results. L-PRF

act as sole filling material in sinus lift and implantation is a good option to promote

natural bone regeration.50,60,66

PRF based membrane used to cover the alveolar ridge

augmentation in several in vivo study. PRF appears superior to collagen (Bio-Gide)

used as scaffold membrane. PRF membrane is suitable for cultivation of Periosteal

cells in bone tissue engineering.54

Due to inadequate bone in the alveolar ridge implant failure is the

major problem in implant dentistry. Implant success is based on ideal anatomical

position to maintain functional and aesthetic results. It is not always possible due to

bone tissue loss. L-prf is a platelet concentrate used with success in most of the

surgical procedures to obtain wound healing and new bone formation.59,67 PRF is used

along with Bio-Oss as a graft material in maxillary sinus augmentation in severe bone

atrophy.78

Discussion

Page 50

PRF for facial plastic surgery:

Platelets plays a major role in homeostasis, but recent studies

shows improve in wound healing. Platelet concentrate induce wound healing. Platelet

rich fibrin matrix helpful for fat transfer supplementation and volume augmentation.

The prf produce natural and autologous platelet concentrate releases growth factors

which stimulates regeneration of surrounding tissues for cosmetic application.79

There are several studies regarding Platelet Rich Fibrin were

applied in Oral and Maxillofacial surgery,

Choukroun et al11,12,25 used autologous platelet rich fibrin to

improve bony healing in the implant dentistry. Platelet rich fibrin combined with the

material freeze-dried bone allograft(FDBA) helpful for sinus floor elevation to

induce bone regeneration. Simonpieri et al concluded hat there is good results when

Platlet rich fibrin used along with FDBA.

Jankovic et al80 concluded platelet rich fibrin is potential for root

coverage in gingival recession cases which promote wound healing, graft

stabilization, bone regeneration , wound sealing and to achieve hemostasis.The major

disadvantage of this platelet rich fibrin will be large amount of blood collected for

larger defects.

Discussion

Page 51

In this study, according to Choukroun et al2 platelet rich fibrin

was prepared from 4 ml blood sample from same patient without any anticoagulant

collected in test tubes. It centrifuged immediately for 10 min in 3000 rotations. Su et

al joined with Choukroun et al shown the importance of rapid handling of blood

sample. In this study conventional medico centrifuge machine is used.

Platelet rich fibrin taken out from the tube and placed in the

extracted socket. The remaining PPP and RBCs were discarded. After extraction the

platelet rich fibrin is placed in the intra bony defects in different time intervals the

assessment was made for bone density.

By using “adobe photoshop 7.0” software program bone density

were analysed. The IOPA were taken immediate postoperative period, 1month

postoperative period, 3 month postoperative period, 6month postoperative period to

measure the bone density according to this software. By using grey level histogram

value the bone density were analysed. Grey level for each pixels were increase for

white and decrease for black image. The graph were interpreted for bone density and

the dense bone shows white and empty space appears black in intra oral periapical

radiograph jansen et al81

Bone density analysis of immediate postoperative day was

performed on the day of extraction and values were recorded. The mean value become

equal in case and control group. It is not significant the mean value become(p>0.05).

The mean value of grey level histogram were evaluated by IOPA in case group and

control group in 1st month, 3rd month and 6th month postoperative period. The IOPA

Discussion

Page 52

were digitalized by using HP scanner. It was significant in 1st month, 3rd month and

6th month postoperative period the mean value become(p<o.o5). According to my

study the extracted socket treated with platelet rich fibrin shows more bone density

when compared to the normal extraction socket. The results of these study coincide

with Diss et al5 and Chang et al24 study.

The present study to evaluate the bone regeneration in the

extraction socket by using platelet rich fibrin. Bilateral extraction of same tooth in a

patients is choosen for this study one side taken as control and another it was taken as

case. When comparing the case with control the bone regeneration is higher in case

than control. So the patients treated with Platelet rich fibrin gel shows more bone

regeneration than the patients not treated with platelet rich fibrin gel Rao et al1

The limitations of study were listed below,

Bone width and bone height were not assessed

Longer follow up is needed for better results

Platelets quality, quantity and its growth factor were not assessed.

Summary & Conclusion

Page 53

In this clinical study to evaluate the effect of autologous platelet rich

fibrin used in extraction socket for bone regeneration. This study clearly indicates the

improvement in bone regeneration in the extraction socket compared with extracted

socket not treated with platelet rich fibrin.

Patients with bilateral extraction were chosen for this study. Inclusion

and exclusion criteria of this study followed strictly. The benifits of platelet rich fibrin

were taken to the knowledge of the patients. Bilateral extraction were performed at

different appointments. One side is taken as case and other side taken as control.

Extracted socket treated with platelet rich fibrin were taken as case and other

extraction socket taken as control. Bone density were analysed by using intraoral

periapical radiograph at different time intervals. The immediate bone density were

measured on the day of procedure. Patients advised to review after 1 week for suture

removal. Further bone density analysis done at 1st month, 3rd month and 6th month

postoperative period.

IOPA were digitalized using HP scanner 7400C by using “Adobe

photoshop 7” the grey level histogram value were evaluated for bone generation in

case and control group. Black image denotes less bone density and white image

denotes high bone density in the extraction socket. In statistical analysis there no

significant in immediate postoperative between case and control group. But in later

period of 1st month, 3rd month and 6th month postoperative period shows a significant

between case and control group in bone regeneration.

Summary & Conclusion

Page 54

The procedure for the preparation of platelet rich fibrin is simple chair

side procedure, patients own blood, very low cost, time consuming is very less and

shows good results. In this study bone generation in the extraction socket is analysed

in different time intervals. There is significant changes seen in the extracted socket

treated with platelet rich fibrin when compared to the socket not treated with platelet

rich fibrin.

Concluded that the platelet rich fibrin plays major role in bone

regeneration process in the extracted socket when compared to the non treated platelet

rich fibrin socket. It also helpful for osseous regeneration in other post surgical

defects, implant placement, mandibular reconstruction, ridge augmentation, graft for

bone substance etc.

Bibliography

1. Rao SG, Bhat P , Nagesh KP, Gundu H.R.Rao. Bone regeneration in extraction

socket with autologous platelet rich fibrin gel. J.maxillofac.surg (jan-mar 2013)

12 (1) 11-16

2. Dohan DM, Choukroun J, Diss A, Dohan SL, Dohan AJ, Mouhyi J, Gogly B.

Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part I:

technological concepts and evolution. Oral Surg Oral Med Oral Pathol Oral

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3. Andrae j, gallini r, betsholtz c: role of platelet derived growth factor in

physiology and medicine. Genes dev 2008:22:1276-1312

4. Weyrich AS, Schwertz H, Kraiss LW, Zimmerman GA. Protein synthesis by

platelets: historical and new perspectives. Journal of thrombosis and haemostasis

2009(2): 241-46

5. Diss A, Dohan MD, Mouhyi J, Mahler P. Osteotome sinus floor elevation using

choukroun’s platelet rich fibrin as graft material: A 1-year prospective pilot study

with microthreaded implants. Oral surg oral med oralpathol radiol endod 2008:

105; 572-579

6. Dohan Ehrenfest DM, Marco Del Corso, Antoine Diss, Jaafar Mouhyi, Jean-

Baptiste Charrier. Three Dimensional A rchitecture and cell composition of a

choukroun’s platelet rich fibrin clot and membrane. J Periodontol 2010; 81:546-

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growth factors and thrombospondin-1 in choukroun’s platelet rich fibrin(PRF): a

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trial, plast reconst surg 2008; 122(5):1431-40.

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51. Simonpieri A,Del Corso M, Sammartino G, Dohan Ehrenfest DM. The relevance

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54. Gassling V, Douglas T, Warnke PH, Acil Y, Wiltfang J, Becker ST. Platelet rich

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55. Gurbuzer B, Pikdoken L, Tunali M, Urhan M, Kucukodaci Z, Ercan F.

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57. Sammartino G, Dohan Ehrenfest DM, Carile F, Tia M, Bucci P. Prevention of

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61. Lee JW, Kim SG, Kim JY, Lee YC, Choi JY, Dragos R. Reconstruction of a

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CONSENT FORM

PART 1 OF 2 INFORMATION FOR PARTICIPANTS OF THE STUDY

1. Name of the Principal Investigator:

Dr.A.W.GODWIN SAJI,

Post Graduate student,

Department of Oral and Maxillofacial surgery,

SreeMookambika Institute of Dental Sciences,

Kulasekharam, Kanyakumari District-629161

2. Name of the Guide: Dr. Mathew Jose,

Head of the department,

Department Of Oral and Maxillofacial Surgery.



Dear Volunteers, We welcome you and thank you for your keen interest in participation in this research

project. Before you participate in this study, it is important for you to understand why this research

is being carried out. This form will provide you all the relevant details of this research. It will

explain the nature, the purpose, the benefits, the risks, the discomforts, the precautions and the

information about how this project will be carried out. It is important that you read and understand

the contents of the form carefully. This form may contain certain scientific terms and hence, if you

3. Name of the Co-Guide: Dr.N.Dhineksh Kumar,

Professor,

Department Of Oral and Maxillofacial Surgery,



4. Institute:


V.P.M Hospital complex, Padanilam, Kulasekharam,

Kanyakumari – 629161

Tamil Nadu.

5. Title of the study :

“COMPARISION OF BONE REGENERATION IN THE EXTRACTION

SOCKETS WITH AUTOLOGOUS PLATELET RICH FIBRIN GEL - AN

INVIVO STUDY

6. Background information: Platelet concentrates for surgical use are tools of regenerative medicine designed for the local

release of platelet growth factors into a surgical or wounded site, in order to stimulate tissue

healing or regeneration. PRF preparation is very simple wherein the blood is collected without

any anticoagulant and immediately centrifuged at 3000 rpm for 10 minutes. Growth factors are

proteins or polypeptides capable of initiating the proliferation of cells that are in a quiescent

state by stimulating deoxyribose nucleic acid synthesis and progression of the cell cycle. They

are a class of multifunctional biologic mediators which regulate connective tissue cell migration,

proliferation, synthesis of proteins and capable of affecting all growth, differentiation,

angiogenesis, inflammation, tissue repair and immune response. The present study is aimed at

the comparison of bone regeneration in extraction sockets with autologous platelet rich fibrin gel.

7. Aim and objectives:

To evaluate the effects of autologous platelet rich fibrin gel on bone regeneration in extraction socket

8. Scientific justification of the study:

Platelets are a nucleate cytoplasmic fragments derived from bone marrow

megakaryocytes and measure 2–3 micron in diameter. The alpha granules are spherical or

oval structures with diameters ranging from 200 to 500 nm each enclosed by a unit

membrane. They form an intracellular storage pool of proteins vital to wound healing,

including platelet-derived growth factor (PDGF), transforming growth factor beta (TGFb),

and insulin-like growth factor (IGF-I).7These growth factors promote cell proliferation,

collagen synthesis, osteoid production etc.7 PRF is an autologous platelet concentrate that can

be effectively used in repair and regeneration of the periodontal tissues.

9. Procedure for the study:

• Under aseptic condition local anesthesia given

• Extraction done with appropriate forceps

• 4ml venous blood drawn from patient

• PRF has been prepared by centrifugation with 3000rpm for 10 min.

• PRF has been placed in extraction socket.

• Post-op record has been maintained to measure the bone regeneration.

10. Expected risks for the participants:

The needle stick for drawing the blood might cause pain,bleeding and thrombophlebitis at

injected site.

11. Expected benefits of research for the participants:

The blood will be used only for research which will better our understanding of PRF and

for advancement of medical knowledge. Benefit for prosthetic rehabilitation.

12. Maintenance of confidentiality:

• You have the right to confidentiality regarding the privacy of your medical

information(Personal details, results of physical examinations, investigations, and your

medical history).

• By signing this document, you will be allowing the research team investigators, other

study Personnel, sponsors, institutional ethics committee and any person or agency

required by law to view your data, if required.

• The results of clinical tests and therapy performed as part of this research may be

included in your medical record.

• The information from this study, if published in scientific journals or presented at

scientific meetings, will not reveal your identity.

13. Why have I been chosen to be in this study?

a. Chosen because of grouping under the inclusion and exclusion criteria

b. Need of good sampling size

c. Minimal radiation exposure that will notharm your health and helps inderiving the

normal facial proportions which will be useful as a guide for orthognathic surgery

for the society

d. To improve the aesthetic appearance.

14. How many people will be in the study? 30 individuals

15. Agreement of compensation to the participants (In case of a study related

injury): Patient will be taken care in case of complication and medical treatment will be provided in the

institution at the expense of the principal investigator.

16. Anticipated prorated payment, if any, to the participant(s) of the study: For your cooperation to the study, the expenses for the routine blood investigations for which

you originally visited the clinic will be settled by the principal investigator.

17. Can I withdraw from the study at any time during the study period?

The participation in this research is purely voluntary and you have the right to withdraw

from this study at any time during the course of the study without giving any reasons.

18. If there is any new findings/information, would I be informed? Yes

19. Expected duration of the participant’s participation in the study: 1 day

20. Any other pertinent information : No other information

21. Whom do I contact for further information?

Place: signature of principal investigator

Date:

Signature of the participant

For any study related queries, you are free to contact:

Dr.A.W.GODWIN SAJI

Post Graduate student.

Department of Oral & Maxillofacial Surgery,


Kulasekharam, Kanyakumari District-629161.

Mobile No: 08344841644

[email protected]

CONSENT FORM

PART 2 OF 2

PARTICIPANTS CONSENT FORM

The details of the study have been explained to me in writing and the details have been

fully explained to me. I am aware that the results of the study may not be directly beneficial to

me but will help in the advancement or medical sciences. I confirm that I have understood the

study and had the opportunity to ask questions. I understand that my participation in the study is

voluntary and that I am free to withdraw at any time, without giving any reason, without the

medical care that will normally be provided by the hospital being affected. I agree not to restrict

the use of any data or results that arise from this study provided such a use is only for scientific

purpose(s). I have been given an information sheet giving details of the study. I fully consent to

participate in the study titled

“COMPARISION OF BONE REGENERATION IN THE EXTRACTION

SOCKETS WITH AUTOLOGOUS PLATELET RICH FIBRIN GEL”

Serial no / Reference no :

Name of the participant :

Address of the participant :

Contact number of the participant :

Signature / thumb impression of the participant / Legal guardian

Witnesses:

1.

2.

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ntspaplg;glhJ.

9. ,e;j Ma;tpy; ePq;fs; Nrh;tjw;fhd fhuzk;?

• ePq;fs; ,e;j Ma;tpd; Nrh;g;G kw;Wk; tpLg;G tFg;Gfspd;

cs; mikg;gLtjhy;

• Ma;tpd; Njitahd khjphpf;fhfTk;

• r%fj;jpd; kw;Wk; kUj;Jt Kd;Ndw;wj;jpw;fhf

10. Ma;tpd; khjphp vz;zpf;if : 30

11. e~;laPL :

Ma;tpy; Vw;gLk; khw;wq;fSf;fhd rpfpl;ir Ma;T epiyaj;jpy;

toq;fg;gLk;.

12. e~;;;;laPL gzk; :gzk; vJTk; toq;fglhJ

13. Ma;tpy; ,Ue;J ve;j Neuj;jpYk; ve;j fhuzKk; ,d;wp tpyfyhk;

14. Ma;tpd; Kd;Ndw;wj;jpy; Vw;gLk; khw;wq;fs; njhptpf;fg;gl Ntz;Lk;.

15. Ma;tpd; fhy mtfhrk;: MW khjk;

16. NtW ve;j jfty;fSk; Njit ,y;iy.

17.

,lk;:FyNrfuk; Ma;thsh; ifnahg;gk;

Njjp:

gq;FngWgth; ifnahg;gk;

kU. .m.tp.fhl;tpd; r[p

KJfiy gl;ljhhp



fy;Y}hp

FyNrfuk,; fd;dpahFkhp khtl;lk;

njhiyNgrp vz;:8344841644

kpd; mQ;ry; - [email protected]

Xg;Gjy; gbtk;

gFjp-2

gq;FngWgthpd; xg;Gjy; gbtk;

,e;j Ma;tpd; KO tpguq;fSk; vOj;J %ykhfTk; tphpthfTk;

vd;dplk; $wg;gl;lJ. vdf;F ,e;j Ma;tpd; tpisTfs; ve;j

gaDk; jutpy;iy vd;whYk; kUj;Jt mwptpaypd; Nkk;ghl;bw;fhf

gad;gLj;jg;gLk; vd;W mwpNtd;. ehd; ,e;j Ma;tpy; ve;j

eph;ge;jKk; ,y;yhky; gq;F ngWfpNwd;. NkYk; ,e;j Ma;tpd;

vy;yh tpjpfs; kw;Wk; tpjpKiwfisAk; mwpNtd;. ve;j

re;Njhfj;jpw;Fk; Nfs;tp Nfl;fNth my;yJ Ma;tpy; ,Ue;J

tpyfNth vy;yh chpikfSk; vdf;F cz;L vd;W mwpNtd;. ,e;j

Ma;tpd; tpisTfs; ve;j mwptpay; gad;ghl;bw;Fk; gad;gLj;j

vdf;F jilapy;iy. vdf;F ,jw;F Kd;ghf Ma;tpd; jfty;

gbtk; nfhLf;fg;gl;lJ. vdf;F ,e;j Ma;tpy; gq;Fngw KO

rk;kjk;.

Ma;tpd; ngah;

gy; vLj;j gFjpapy; gp.Mh;.v/ itj;Jk; gp.Mh;.v/ itf;fhkYk;

vYk;G cUthFjiy xg;gpLjy;.

Fwpg;G vz;:

ngah;:

Kfthp:

njhiyNgrp vz;:

rhl;rp 1: ifnahg;gk;

2.

,lk;:FyNrfuk;

Njjp:

k½X ]{Xw

`mKw þ 1

]T\hpambn klIcn¡p¶ hyànIfpsS Adnhntebv¡v

{]nbs¸« k¶² tkhI³ / tkhI,

R§Ä \n§sf kzmKXw sN¿p¶p. AtXmsSm¸w Cu ]T\hpambn klIcn¡m\pÅ k¶²XtbmSv \μn tcJs¸Sp¯p¶p. \n§Ä Cu ]T\¯nÂ ]s¦Sp¡p¶Xn\p ap³]v Cu ]T\w Fń\mWv \S¯s¸Sp¶Xv F¶v Andtb−Xp−v. AXn\mÂ Cu t^md¯nÂ KthjW ]T\¯nsâ hnhc§fpw aäpw hniZambn tcJs¸Sp¯nbncn¡p¶p. Cu ]T\¯nsâ coXn, Dt±iw, {]tbmP\w, A]ISkm²yX, t¢iw, ap³IcpXÂ, F§s\ Cu ]T\w ap³t]m«p sIm−pt]mIp¶p F¶n§s\ FÃm hnhc§fpw t^md¯nÂ tcJs¸Sp¯nbncn¡p¶p. kZbw Cu hnhc§Ä hmbn¨p a\Ênem¡phm³ A`yÀ°n¡p¶p. Cu hnhc§fnÂ imkv{X]camb ]Z§Ä DÅXn\mÂ kwib\nhmcW¯n\p {][m\ ]T\IÀ¯mhnt\mtSm Xmsg tcJs¸Sp¯n bncn¡p¶ hyànItfmtSm t^mdw H¸nSp¶Xn\p ap³t]m AsÃ¦nÂ Cu ]T\¯nsâ Imemh[n Xocp¶Xphtctbm kao]n¡mhp¶XmWv.

1. apJy KthjI³ : tUm. F. hn. KmUvhn³ kPn t]mÌp{KmPpthäv Un¸mÀ«vsaâv Hm^v amIvkntemt^jyÂ kÀPdn {io aqImw_nI C³Ìnäyq«v Hm^v sUâÂ kb³kv, IpetiJcw þ 629 161.

2. amÀ¤ ZÀin : tUm.amXyq tPmkv s{]m^kÀ & slUv Hm^v Zn Un¸mÀ«vsaâv,

Un¸mÀ«vsaâv Hm^v amIvkntemt^jyÂ kÀPdn {io aqImw_nI C³Ìnäyq«v Hm^v sUâÂ skâÀ, IpetiJcw 3 kl amÀ¤ZÀin : tUm. Znt\jvIv IpamÀ

{]m^kÀ Un¸mÀ«vsaâv Hm^v amIvkntemt^jyÂ kÀPdn {io aqImw_nI C³Ìnäyq«v Hm^v sUâÂ kb³kkv, IpetiJcw.

4. C³Ìnäyq«v : {io. aqImw_nI C³Ìnäyq«v Hm^v sUâÂ kb³kkv ]S\new, IpetiJcw, I\ymIpamcn þ 629 161. Xangv\mSv.

5. ]T\¯nsâ ioÀjIw :

]sÃSp¯ `mK¯nÂ ]n.BÀ.F^v. h¨n«pw ]n.BÀ.F^v ssh¡msXbpw FÃm−mIp¶ Afhnsâ XmcXays¸Sp¯Â 6. ]Ým¯e hnhcw ? t¹äpseäv h¨v apdnhp`mK¯v icnbm¡p¶Xpw FÃp−mIp¶ \·Ifpw km[yamWv. Cu ]T\¯nÂ ]n.BÀ.F^vþs\ ]sÃSp¯Øe¯v FÃv hfcm³ sN¿pI. 7. e£y§fpw Dt±i§fpw ]T\¯nÂ ]s¦Sp¯hÀ¡v ]sÃSp¯ Øe¯pÅ FÃnsâ hfÀ¨ Ipdhns\ ]n.BÀ.F^v h¨v icnbm¡p¶Xv.

8. KthjWw \S¯m\pÅ \ymboIcWw CXn\p ap³]pÅ ]T\¯nÂ apdnhpÅ Øe¯pw FÃp IpdhpÅ Øe¯pw ]n.BÀ.F^v. h¨v FÃnsâ hfÀ¨ \nco£n¨n«p−v.

9. KthjWcoXn • Cu KthjWhpambn klIcn¡p¶hÀ¡v Hcp \¼À \ÂIp¶XmWv.

• Cu KthjW coXn t_m[n¸n¨n«v AhcpsS ssI¿nÂ \n¶pw k½X]{Xw hm§p¶XmWv.

• ]sÃSp¯ `mK¯v IpdhpÅ FÃp`mK¯nÂ ]n.Bcv.F^v ssh¡Â.

• FÃv hfÀ¨ FIvkvtd aptJ\ Adnbmhp¶XmWv.

10. {]Xo£n¡p¶ A]IS km²yXIÄ þ CÃ

11. Cu ]T\¯nÂ ]s¦Sp¡p¶XpsIm−v F\n¡v Fs´¦nepw D]tbmKapt−m ? t\cn«v {]tbmP\§Ä H¶pw CÃ. F¶ncp¶mepw IpdhpÅ FÃp`mK¯v ]n.BÀ.F^v hbv¡p¶Xpaqew FÃnsâ Ipdhv \nhÀ¯n sN¿m³ km[n¡p¶p. 12. Xm¦sf ]änbpÅ hnhc§Ä FÃmw clkyambn kq£n¡p¶XmtWm ? XoÀ¨bmbpw kq£n¡p¶XmWv. 13. Fs¶ F´psIm−v Cu ]T\¯nÂ DÄs¸Sp¯n ? Xm¦Ä Cu KthjW¯nÂ ]s¦Sp¡p¶Xn\v tbmKyXbmbXpsIm−v Xm¦sf R§Ä Cu ]co£W¯n\v hnt[bam¡n. 14. Ft¸mÄ thWsa¦nepw F\n¡v Cu ]T\¯nÂ \n¶v ]n³amdmtam Xm¦Ä¡v Cu ]co£W¯nÂ ]¦p sImÅp¶Xn\pw ]n·mdp¶Xn\pw ]qÀ®hImiap−v. ]co£W¯nÂ ]¦psImÅp¶Xn\pw CSbnÂ ]n·mdp¶Xnepw Xm¦fpsS NnInÕbv¡v GXphn[ _p²nap«pIfpw GÀs¸Sp¶XÃ.

15. Cu KthjW¯nsâ êambn ]pXnb Fs´¦nepw Is−¯epIfps−¦nÂ AXv Fs¶ Adnbn¡ptam ? Cu ]T\¯nsâ Is−¯epIÄ ]T\ Ahkm\w XoÀ¨bmbpw \n§sf Adnbn¡p¶Xmbncn¡pw. 16. Cu ]co£W¯nÂ GsX¦nepw ]pXnbXmbn I−p]nSn¡s¸«mÂ Fs¶ Andbn¡ptam ? AsX Cu ]co£W¯nsâ Ahkm\w GsX¦nepw ]pXnbXmbn I−p]nSn¨mÂ AXv XoÀ¨bmbpw Xm¦sf Adnbn¡p¶XmWv. 17. Cu ]T\¯nsâ kab ssZÀLyw F{XbmWv ? Bdp amkw 18. Cu ]T\¯nsâ F{Xt]cmWv ]s¦Sp¡p¶Xv. 30 t]À 19. Cu KthjW¯nÂ ]s¦Sp¡p¶hÀ¡v Fs´¦nepw B\pIqeyw e`n¡ptam ? KthjW sNehv ]qÀ®ambpw KthjI³ sNehp hln¡p¶XmWv. 20. Cu KthjW¯nÂ ]s¦Sp¡p¶hÀ¡v \ã ]cnlmcw e`n¡ptam ? CÃ 18. IqSpXÂ hnhc§Ä¡mbn Xmsg ]dbp¶hsc \n§Ä¡v _Ôs¸Smhp¶XmWv. tUm. F. hn. KmUvhn³ kPn t]mÌp{KmPpthäv Un¸mÀ«vsaâv Hm^v amIvkntemt^jyÂ kÀPdn {io aqImw_nI C³Ìnäyq«v Hm^v sUâÂ kb³kv, IpetiJcw þ 629 161. skÂ:8344841644 C.sabnÂ: [email protected] Øew: IpetiJcw XobXn :

k½X]{Xw

`mKw þ 2

Cu ]T\s¯ ]änbpÅ FÃm Imcy§fpw F\n¡v ]dªv a\Ênem¡n XcnIbpw AXnsâ Hcp ]IÀ¸v F\n¡p \ÂIpIbpw sNbvXn«p−v. Cu ]T\w KthjW¯n\mbn DÅXmsW¶pw F\n¡v CXnÂ \n¶v t\cn«v Hcp êhpw D−mInsÃ¶pw Rm³ a\Ênem¡p¶p. Cu ]T\¯nsâ coXnbpw Dt±ihpw F\n¡v a\Ênem¡n X¶n«p−v. AXp t]mse F\n¡v kwib§Ä tNmZn¡m³ Ahkc§Ä e`n¨n«pap−v. CXnÂ ]s¦Sp¡m\pw ]s¦Sp¡mXncn¡m\pw DÅ AhImiw F\n¡ps−¶pw AXpt]mse ]T\¯nsâ GXp L«¯nepw CXnÂ \n¶v ]n³h§m\pÅ kzmX{ýhpw F\n¡ps−¶v Rm³ a\Ênem¡p¶p. Cu ]T\¯nÂ ]s¦Sp¡p¶XpsImt−m, ]s¦Sp¡m¯XpsImt−m Fsâ aäp NnInÕIsf _m[n¡p¶XsÃ¶v Rm³ Andbp¶p.

“FsÃSp¯ `mK¯nÂ ]n.BÀ.F^v. h¨n«pw ]n.BÀ.F^v ssh¡msXbpw FÃm−mIp¶ Afhnsâ XmcXays¸Sp¯Â .” F¶ KthjW¯nÂ ]s¦Sp¡p¶Xn\pw CXnsâ ê§Ä imkv{XteJ\¯nÂ {]kn²oIcn¡p¶Xn\pw F\n¡v k½XamsW¶v Rm³ CXn\mÂ Andbn¨psImÅp¶p.

kocnbÂ \¼À / d^d³kv \¼À :

]s¦Sp¡p¶ Bfnsâ t]cv :

taÂhnemkw :

t^m¬ \¼À :

H¸v / hnceSbmfw

km£n :

Øew :

XobXn

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