COMPARISON OF POST OPERATIVE HEALING OF EXTRACTION …

COMPARISON OF POST OPERATIVE HEALING OF EXTRACTION SOCKET

WITH AND WITHOUT CALCIUM CHLORIDE ACTIVATED PLATELET

RICH PLASMA- 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

2017-2020

ACKNOWLEDGEMENT

“At times our own light goes out and is rekindled by a spark from another person.

Each of us has cause to think with deep gratitude of those who have lighted the flame

within us.”

I extend my sincere and heart felt 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. Sajesh 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. Nandagopan 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. Marban joe 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.Sindhuja Devi for her contributions in my thesis

work. Her humbleness and dedication to profession coupled with tremendous skill

had always been a fascination to me.

I am highly obliged Dr.Meglin for being there always to clear my doubts and

guide me through the right path.

I would like to thank Dr. Yazhini for supporting me through the hard times and

motivating me to study harder.

I would like to thank my super seniors Dr.Abirami and Dr.Harine

for teaching me the intimate skills of surgery.

I would like to thank my seniors Dr.Ruban and Dr.Subin for timely support and

guidance.

I would like to thank my juniors Dr.saravanan, Dr.Tamil, Dr.Saumya and

Dr.Venkidesh for the support and enjoyable moments we had during the post

graduate life.

Above all I thank Almighty God, My parents and my husband Mr.Aneesh 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 specially our Chairman Dr.C.K.VELAYUTHAN NAIR

MS, Sree Mookambika Institute of Dental Sciences, our Director Dr.REMA.V.NAIR

MD, Sree Mookambika Institute of Dental Sciences and our Trustees

Dr.R.V.MOOKAMBIKA MD, DM, Dr.VINU GOPINATH MS, MCH and

Mr.J.S.PRASAD, Adminstrative officer for giving me the 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-

7 Aims and Objectives

8 Review of literature

9 Platelet Rich Plasma

10 Materials and Methods

11 Figures vii-

12 Results

13 Tables and Graphs

14 Discussion

15 Summary and Conclusion

16 Bibliography

17 Annexures

LISTS OF ABBREVIATIONS

PRP Platelet Rich Plasma

PRF Platelet Rich Fibrin

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

i

LISTS OF TABLES

Table 1 Demographic data of study population

Table 2 Comparison of mean soft tissue healing values post operative

3rd day between the groups.


7th day between the groups.


14th day between the groups

Table 5 Comparison of mean soft tissue values between the groups at

different time periods

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

immediate

Table 7 Comparison of mean bone density values at 4th week

between the groups

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

3 month

Table 9 Comparison of mean values between the groups at different

time periods

ii

LISTS OF GRAPHS

Graph 1

Demographic data of study population

Graph 2

Comparison of mean soft tissue healing values between

the groups at different time periods

Graph 3

Comparison of mean bone density values between the

groups at different time periods

Graph 4

Multiple comparison of mean soft tissue healing values

within the groups at different time periods

Graph 5

Multiple comparison of mean bone density values within

the groups at different time periods

iii

LISTS OF FIGURES

Figure 1 Armamentarium

Figure 2 Medico centrifuge

Figure 3 Withdrawal of blood

Figure 4 After first centrifugation

Figure 5 After second centrifugation

Figure 6 Platelet rich plasma activated with 10% calcium

chloride

Figure 7 Calcium chloride activated PRP gel

Figure 8 Extracted socket

Figure 9 Suture done after placement of calcium chloride

activated PRP in the extraction socket

Figure 10 Post-operative clinical evaluation of extraction

socket healing

Figure 11 Grey level histogram value evaluation from

digital radiograph

Figure 12 Postoperative radiograph evaluation of bone

density of extraction socket.

iv

ABSTRACT

ABSTRACT

The study was conducted to evaluate the efficiency of calcium chloride activated platelet-

rich plasma in bone regeneration and soft tissue healing in the extraction socket.

Twenty-five patients needing bilateral similar tooth extractions were selected, and one side

is taken as a control group and another side as the case group. PRP was prepared with blood

drawn from individuals who came for extraction using standard double centrifugation

technique, and 10 % calcium chloride was added to activate PRP. The formed PRP gel was

placed in the extraction socket of the case group and secured with sutures. An immediate

digital radiograph was taken after extraction for both groups. Moreover, for soft tissue

evaluation, patients reviewed at postoperative day 3rd, seventh, and 14th day and soft tissue

healing index taken. For radiographic analysis, socket surface area and density were

performed using a computer graphics software program. The clinical follow-up

assessments were performed in the fourth week and third month, and the collected data

were assessed using ANOVA and multiple comparison tests.

RESULTS:-Patients were aged between 18 to 32 (Mean age 24 years), including 17

females and 8 males. The mean soft tissue healing for the case group were 3.46, 4.42,5 at

3rd 7th and 14th day, respectively. The soft tissue healing was significantly higher among

the test group as compared to the control group.

The mean radiographic density in the case group was 57.752, 76.458 in 4th week and 3rd

month, respectively. The mean bone density was significantly higher in the test group than

the control group at all-time intervals.

v

CONCLUSION:-The study outcomes demonstrate that 10 % calcium chloride activated

PRP is biocompatible and accelerates socket wound healing after tooth extraction and

increased bone density.

KEYWORDS:-Platelet Rich Plasma, 10 % Calcium Chloride, Soft Tissue healing, Bone

Density, Bilateral Extraction Socket.

vi

INTRODUCTION

Introduction

1 | P a g e

Platelet-rich plasma (PRP) is a modern and potentially

valuable adjunct in the area of oral and maxillofacial surgery1. Platelet-rich plasma (PRP),

an autologous concentrate of platelets in a proportionately meager quantity of plasma,

expedites the delivery of growth factors in risen amounts to surgical sites to enhance wound

healing. Platelets contain many growth factors. Some of the growth factors are, platelet-

derived growth factor (PDGF) (including αα, αβ, and ββ isomers), transforming growth

factor-β (TGF-β) (including β1 and β2 isomers), fibroblast growth factor, insulin-like

growth factor-I (IGF-I), epithelial growth factor, vascular endothelial growth factor. The

platelets also contain numerous other secretory proteins3, 4.

In their article labeled ‘‘Platelet Gel: An Autologous Alternative to Fibrin Glue with

Applications in Oral and Maxillofacial Surgery.’’ Written in 1997, Whitman et al.

proposed that platelet-rich plasma (PRP) can be used widely by the Oral Surgery society.

As per this article, it was recommended that through activation of the platelets, within the

gel, there will be a release of growth factors, and by this, there will be an enhanced wound

healing..’’ In 1998 Max et al. conducted a study that indicated that a combination of PRP

with autogenous bone in mandibular continuity deficiencies resulted in considerably faster

radiographic maturation and histo-morphometrically denser bone regeneration. After the

disclosure of this Benchmarking article, there was a significant increase in then notoriety

in the oral and maxillofacial surgery community. It certainly seemed as though a new age

in bone grafting had begun1, 2, 3. PRP can be expressed as a platelets concentrate, in which

concentrated platelets are suspended in a small volume of plasma. Cell adhesion molecules

for osteoconduction and as a mold for bone, connective tissue, and epithelial migration are

Introduction

2 | P a g e

distinguished as the three proteins in the blood involved in PRP. These cell adhesion

molecules are fibrin, fibronectin, and vitronectin5.

By activating Platelet rich plasma it will impact the availability of bioactive molecules

and consequently influences more accelerated tissue healing. The approach is preferred to

initiate PRP to influence both its physical form and discharged in terms of GF amount

and release kinetic9.

Activation of PRP gel, thus causing degranulation of α-granules present in the platelets and

releasing the growth factors. The various agents for the activation reported in the literature

include CaCl2 alone, CaCl2 plus bovine thrombin, human thrombin, autologous bone, or

whole blood that contains thrombin2. In our plan, an autologous platelet gel was prepared

in which CaCl2 alone was combined with PRP. This platelet gel was free of extracting any

antigen-antibody reaction, as it was acquired from patients’ blood. The study symbolizes a

marked enhancement in the soft tissue healing and more active reformation of bone after

third molar surgery in cases handled with PRP as corresponded to the control group

postoperatively. This advancement in wound healing, reduction in pain, and improvement

in the bone density signify and highlights the use of PRP, certainly as a proven approach

in influencing and stimulating soft and hard tissue regeneration. An added advantage of

PRP recorded in the present investigation is its capacity to compose a biologic gel that

produced clot endurance and functions as an adhesive, CaCl2 induced a gradual release of

GFs from 15 minutes and progressing up to 24 hours.

The system of PRP preparation is modest, cost-effective and has proved beneficial

outcomes. Several studies have suggested that platelet concentrates, especially PRP, may

Introduction

3 | P a g e

stimulate osseous and soft tissue regeneration while also reducing inflammation, pain and

unwanted side effects5, 6.

This prospective clinical trial was designed to compare bone density and soft tissue healing

with and without 10 % of calcium chloride activated autologous platelet rich plasma gel in

extraction socket.

AIMS AND OBJECTIVES

Aims And Objectives

4 | P a g e

The aim of the study is to evaluate clinically and radiographically, extraction socket

healing using calcium chloride activated platelet rich plasma, compared to normal healing

pattern.

REVIEW OF LITERATURE

Review Of Literature

5 | P a g e

Matras et al. 198514 was the one who first described fibrin glue used

as surgical adhesive which helpful for healing process. Fibrin glue which is derived from

blood, is used to stimulate healing and also to seal wounds. This contains concentrated

fibrinogen polymerization induced by calcium and thrombin. This is prepared using donor

plasma and used to improve skin wound healing in a rat model.

Knighton et al. 198615 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 and kept 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. 198716 suggested that at the site of injury, platelet

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

process.

Knighton et al. 199017 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 increase in epithelialisation of the wound.

Slater et al. 199518 conducted a study to examine the efficacy of

concentrated human platelet as a basic medium in the functional and proliferative activity

of human osteoblastic like cells. The results shows that eked out platelet medium stimulates


6 | P a g e

the proliferation and maintains the activity of differentiated function of human fetal

osteoblast like cells.

Whitman et al. 199719 used “platelet gel” derived from platelet

concentrate which shown their clinical results in reconstruction of oral and maxillofacial

surgery. The ‘Platelet Gel’ was produced by a gradient density cell separator. The authors

considered the platelet gel as a fibrin glue helpful for mandibular reconstruction, alveolar

cleft procedure, and closure of oro-antral and oro-nasal communication and also show

greater effect in implant placement.

Heldin et al. 199720 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 parallelly induced. He also

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

receptor which is structurally related.

Marx et al. 199811 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 tumour removal. In their study it has been showed that, PRP contains a

concentrated platelets and growth factors. The PRP,

Which was used with grafts showed an 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.


7 | P a g e

Green et al. 199821 reported that platelet gel can be used as

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

and haemostatic support which bring the growth factors and cytokines to the surgical site

and induce wound healing process.

Heldin et al. 199922 reported that PDGF was the first growth

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

fibroblast, 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. He also demonstrated a series of clinical

and experimental studies about the useful effects of PGDF which can be used in wound

healing disorders.

Schuldiner et al. 200023 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)

which found in PRP for wound healing.

De Obarrio et al. 200024 states that combined PRP with bone

allograft and guided tissue regeneration for the periodontal therapy of human intra bony

defects and observed significant gain in clinical attachment and bone formation, as revealed

by 2-year follow-up.


8 | P a g e

Choukroun et al. 200025 developed another form of PC in

France, which was labeled as PRF, based on the robust fibrin gel polymerization found in

this preparation. It was stamped as a “second-generation” platelet concentrate because it

was different from other PRPs. This proved an essential milestone in the evolution of

terminology.

Anitua et al. 200126 conducted various studies to see the effect

of platelet-rich in growth factors (PRGF). She proved it is a conventional method to

accelerate soft tissue and bone regeneration in post-extraction sockets.

Petrungaro 2001 27 published a case series in which collagen

membranes covered PRP, subepithelial connective tissue grafts, and gingival recessions;

the therapy was successful in all cases.

Lacoste et al. 200328 quantified that concentration of TGF-ß1,

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

after there is an 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. 200329 studied the level of growth factors in PRP.

Dispersion of cell separation method and also point of care method. It is also called a ‘buffy

coat method’. Whole blood is drawn from 115 healthy donors. The increase in

thrombocyte count was achieved in both of the methods, which result in higher TGF-ß1

levels, while an increase in the leukocyte count in curasan PRP shows more PDGF-AB

levels. Quantified that concentration of TGF-ß1, bFGF, VEGF, and PDGF-BB from


9 | P a g e

platelet concentrates and the whole blood before and after there is an increase of various

concentrations such as calcium and thrombin and also have been assessed that

physiological significance of growth factors on angiogenesis.

Anitua et al. 200430 showed that the platelets contain the

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

include proteins like PF4 and CD40L. He also reported that the growth factors attached to

the platelets or fibrin resulted in enhanced activity over the recombinant proteins. He also

reviewed various situations in the muscle/tendon repair, orthopaedic surgery, whole repair

in the eye surgery, the reversal of skin ulcers, and cosmetic surgery, where platelet

accelerates the healing mechanism.

Barry L. Eppley et al. 200431 suggests Platelet-rich plasma,

and the associated fibrin clot, can conceivably support in wound reconstruction and back

to complete and maintain hemostasis, or can be blended with other tissues as an adjunct to

their transplantation healing.

Hanna et al. 200432 suggested that the use of PRP in Intra-

bony defect resolution will result in an additional mean probing pocket reduction of 1.0mm.

Oyama et al. 200433 suggested the use of PRP enhances the

osteogenesis of alveolar bone grafting in cleft lip and palate patients. It may be beneficial

for succeeding in orthodontic therapy.

Dohan et al. 200634 quantified the level of cytokine within

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


10 | P a g e

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

a complex three-dimensional architecture. It produces a real platelet and also a leukocyte

rich fibrin biomaterial.

Li et al. 200635 explained the pleiotropic function of TGF-ß

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

receptor II developed a lethal inflammation and is associated with T cell activation and

differentiation.

Thor et al. 200736 calculated the impact of platelet-rich plasma on

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

significantly more new bone developed at platelet-rich plasma-treated sites when compared

to the control group in 3 months of healing. He recommended that platelet-rich plasma has

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

Su et al. 200837 interpreted that growth factors released from the

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

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

with the release, platelet gel, released with thrombin at 5, 60, 120, 300-minute time points.

TGF-ß1, PDGF-AB, EGF, and VEGF increase was recognized in platelet gel with the

release of the PDGF-AB of 0.6x10-16 and also the TGF-ß1 of 0.9x10-16 per PLT.

Sunitha et al. 200838 concluded that the combination of

growth factors and bone grafts contained in platelet rich fibrin and platelet rich plasma may

be suitable for the increase in bone density. In experimental study the content such as


11 | P a g e

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

Kit.

Kajikawa et al. 200839 suggested that PRP can both inhibit

excess inflammation and also augment stem cell proliferation and maturation, as

demonstrated in vitro studies.

Gassling et al. 200940 generated PRF and PRP from the whole

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

derived osteosarcoma 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.

Dohan Ehrenfest et al. 200941 proposed the first

classification of platelet concentrate. This classification defined four leading families

based on the separation of the products using two fundamental parameters. The cellular

content (primarily leukocytes) and fibrin architecture. The first one being Pure platelet-rich

plasma (P-PRP) - or leukocyte-poor platelet-rich plasma (LP-PRP); the second one is the

Leukocyte-and platelet-rich plasma (L-PRP); the third one being the Pure PRF (P-PRF) -

or leukocyte-poor PRF; and the last and the fourth one is Leukocyte- and platelet-rich fibrin

(L-PRF).

James L. Rutkowski et al. 20109 study suggests that the use

of a simple, cost-effective BC-PRP method to enhance the rate of bone formation and

reduce healing time in the initial two weeks following oral surgery may be advantageous.


12 | P a g e

Treatment with PRP may not induce pain, bleeding, and/or numbness, but may decrease

inflammation.

Senger et al. 201042 revealed that both bFGF and VEGF

would stimulate endothelial proliferation and also angiogenesis. However, VEGF

transcripts encode and direct active secretion of VEGF from the cell, and it is considered

to be the endothelial-specific mitogen. The VEGF stimulates nitric oxide production. It

will restrain platelet activation and proposing that the role of VEGF is to prevent

undesirable thrombosis.

Ahmed et al. 201143 described the autocrine pool between

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

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

FGFR, sequentially, and ended in a reduction in the number of receptors staying in the

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

expression of receptors to sensitize the cells to the respective ligands; this shows how both

these factors regulate the neoplasticity metastasis and angiogenesis.

Kiran et al. 201144 described method of 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 a centrifuge machine for 12 minutes at 2700 RPM. Then he reported that PRF eliminates

adding anticoagulant as well as it needs to neutralize.


13 | P a g e

Stacie G et al. 201245 suggests PRP is a useful regenerative

therapy to address many musculoskeletal injuries. It is important to understand that PRP is

more than just platelets and that it contains many bioactive factors that act in anabolic,

catabolic, proinflammatory, and anti-inflammatory pathways.

Nathan E Carlson et al. 201246 suggests the development

of an autologous PRP has been shown to be relatively easy, and to be effective as a surgical

adjunct, to retain high levels of the desired growth factors after preparation and to be

clinically useful in expediting postsurgical healing in both periodontal and oral surgery

administrations.

Dohan et al. 201247 estimated that the slow release of

growth factors PDGF-AB, TGF-ß1, VEGF, and matrix proteins from the P-PRP gel

membrane and L-PRF in a culture medium. He explained that the less than five days, P-

PRP gel membranes dissolve entirely in the culture medium. Meanwhile, the L-PRF

membranes are still intact after seven days. This implies that polymerization and the final

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

release potential of the membrane.

Tomasz Bielecki et al. 201248 The study grants an in-depth

understanding concerning the application of stem cells and PRP in vitro, in vivo, and their

utilization in clinical studies in the tomorrow.

Ruchi Pathak Kaul et al. 20123 This study attempted to use

autologous PRP to encourage wound healing and osseous reconstruction in human third


14 | P a g e

molar extraction sites. It designates a visible advancement in soft tissue healing and faster

reformation of bone after third molar surgery in cases handled with PRP as analyzed to

control group postoperatively

Antonino Albanese et al. 20138 this review of the literature

implies that the use of PRP in the alveolar socket after tooth removals is unquestionably

capable of improving soft tissue healing and positively impacting bone regeneration.

Nevertheless, following the outcome, as mentioned above, appears to reduce a few days

after the extraction. PRP has manifested more reliable conclusions in periodontal therapy

in comparison with distinct elements than when it is used solely, implying that the

particular preference of agents/procedures mixed with PRP could be vital.

Amable et al. 201350 investigated fluctuations in relativistic

centrifugal force (RCF), temperature, and time for optimizing circumstances for platelet

isolation and quantification of cytokines and extension factors in PRP before and

subsequent platelet activation.

Mc Lellan et al. 201451 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

of PRP and PRF.

Kumar et al. 201552 Platelet concentrates shown to have great

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


15 | P a g e

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

PRP concentrates they are used clinically.

Mourão et al. 201553 suggests that the use of PRP in the

alveolar socket subsequent tooth uprootings is unquestionably capable of enhancing soft

tissue healing. It assuredly induces bone reformation, but the following consequence seems

to reduce a few days after the extraction.

Carola Cavallo et al. 201614 Platelet-Rich Plasma (PRP) is a

low-cost method to produce high concentrations of autologous growth factors (GFs).

Platelet activation is a requisite level that forces impact the availability of bioactive

molecules and, therefore, tissue healing. Activation of PRP from ten intentional healthy

males was conducted by adding 10% of CaCl2, 10% of autologous thrombin, 10% of a

mixture of CaCl2 + thrombin, and 10% of collagen type I. CaCl2 induced a gradual release

of GFs from 15 minutes and progressing up to 24 hours. The approach is practiced to

initiate PRP impacts both its physical form and the discharge rate in terms of GF amount

and deliver kinetic.

Ahmed Abdullah Alzahrani et al. 201710 Outcomes of the study

demonstrate that the use of PRF accelerates socket wound healing after tooth extraction.

As it is noticed by increased bone fill and reduced alveolar bone width resorption using

clinical and radiographic methods10.

Kanae Niimi et al. 201754 The study, the outcomes described the

ubiquity of advanced inflammatory effects and a vibrant fibrin network in the experimental


16 | P a g e

group, which led to the creation of thick, vessel-rich granulation tissue. The conclusions of

day-5 specimens showed the remaining granulation tissue on the full area seen in the

control group. Meantime, the basement membrane regenerated in the full area in control

groups, then in the experimental groups in the study of the day-7 specimens. We revealed

that the utilization of PRP excites wound healing in tooth uprooting bone deficiencies;

however, the method of execution of the growth constituents continues unclear.

Elena Conde-Montero et al. 201755 The study describes the

fundamental postulates of PRP in wound healing. It aims to offer a renewed evaluative

appraisal of the available clinical evidence that confirms the utility of PRP for the

therapeutic challenge that chronic ulcers reproduce in our daily clinical practice.

Francesca Zotti et al. 201856 suggests arthrocentesis with

platelet-rich plasma. The platelet-rich plasma injections in temporomandibular disorders’

management were found to be effective in reducing pain and joint sound as well as in

improving mandibular motion in a maximum follow-up of 24 months.

Gisselle Escobar et al. 201857 suggests P-PRP (Pure Platelet Rich

Plasma) and S-PRP (Supernatant of Calcium Activated Platelet Rich Plasma) do not have

an equivalent biological effect on macrophages, and suggest that they could present a

different clinical tissue-repair potential. And he stated that in some cases clot formed after

PRP activation can be discarded and the S-PRP itself can be used as a platelet product for

tissue repair therapy as it contains the growth factors released by activated platelets.


17 | P a g e

Jihua Chai et al. 201958 regeneration potential of hDPCs when

confronted with conventional PRP. Moreover, liquid PRF also attenuated the inflammatory

situation grasped by LPS and kept a supportive regenerative ability, for the stimulation of

odontoblastic differentiation and reparative dentin in hDPCs.

El-Sayed A. 201959 study aimed to compare platelet-rich plasma

(PRP) versus conventional ordinary dressing in the management of diabetic foot wounds.

On a comparative approach the highest healing rate was observed for both groups at the

fourth week, and it was better for the PRP group.

Platelet Rich Plasma

18 | P a g e

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.

Platelets are essential in the wound healing process. They appear instantly at the

wound position and commence coagulation. They release multiple wound healing growth

factors and cytokines, including platelet-derived growth factor (PDGF), remodeling

growth factors (TGF)/β1 and β2, vascular endothelial growth factor (VEGF), platelet-

derived endothelial cell growth factor (PDGF), interleukin- 1 (IL-1), primary fibroblast

growth factor (bFGF), and platelet Activating factor-4 (PAF-4). These growth factors are

accountable for bone regeneration and progressed vascularity, vital features of a healing

bone graft1 .

The results from the previous studies demonstrated that PRP and PRF, were able to

release growth factors over time from their respective platelet formulations. Interestingly,

PRP demonstrated the ability to release significantly higher levels of growth factors at very

early time points and sudden fibrin polymerization depending on the amount of surgical

additives (thrombin and calcium chloride). Bilateral Junctions (condensed tetra molecular)

are constitute with strong thrombin concentrations and allow the thickening of fibrin

polymers leading to the Constitution of a rigid network, unfavourable to cytokine

Platelet Rich Plasma

19 | P a g e

enmeshment and cellular migration, whereas PRF had a more gradual release of growth

factors up to a 10-day period12. PRP is a platelet concentrate, but how many platelets are

enough for the properties and action of PRP is a critical question. The activity of Haynes

worth et al. explained that the increase of adult mesenchymal stem cells and their

differentiation were undeviatingly linked to the platelet concentration. They explicated a

dose-response curve, which symbolized that a sufficient cellular response to platelet

concentrations first began when a 4- to 5-fold increase over baseline platelet numbers were

accomplished. Most individuals have a baseline blood platelet count of 200,000 -

75,000/uL, a PRP platelet count of 1 million/uL as marked in the standard 6-mL aliquot

has displayed the benchmark for therapeutic PRP1.

MATERIALS AND METHODS

Materials And Method

20 | P a g e

The patients were selected for the study from the outpatient department of oral and

maxillofacial surgery, Sree Mookambika Institute of Dental Science, Kulasekharam. Total

25 patients who needed bilateral similar tooth extraction was selected for the study. Total

50 extraction (25 in case and 25 in control). Atraumatic extraction of tooth was carried out

in the right and left quadrant of the same jaw. In Study group, calcium chloride activated

Platelet-Rich plasma gel was placed in the extraction socket on one side, and the other side

taken as control group.

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 involved in the study accepted

for the study protocol and written informed consent was tendered. All patients were

reviewed in 3rd day 7th day and 14 th day after extraction for wound healing evaluation and

suture removal in 7th day and reviewed in 1st, 3rd month postoperatively for radiographic

examination for bone formation.

Total duration of the study: 6 months

Number of groups: Two groups

Description of groups:

25 patients were taken for dental extraction. Total 50 extraction (25 in case and 25 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.


21 | P a g e

Group A- Calcium chloride activated platelet rich plasma

Group B- No intervention

Sample size of each group:

According to this formula the sample size of each group may be 25.

Inclusion criteria

• Patient of age group 18 to 50 years will be select.

• Patients who needs bilateral similar tooth extraction

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

• Haematological disorders.

• Chemotherapy and radiotherapy

• Who needs surgical removal of tooth.


22 | P a g e

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 tubes

• Curette

• Lignocaine 2% [LA]

• 3ml syringe with 24 / 26 gauge needle.

• Periosteal elevator

• Extraction forceps

• Bone file/bone rounger

• Needle holder


23 | P a g e

• Scissors

• 3.0 silk suture material

• Stop watch

PROCEDURE IN DETAIL:

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

is taken as control in same patient with bilateral similar tooth extraction. Under aseptic

condition, Intraoral inferior alveolar nerve, long buccal nerve block, lingual nerve block

will be given for mandibular extractions and posterior superior alveolar nerve block,

middle superior nerve block, greater palatine nerve block and nasopalatine nerve block will

be given for maxillary extractions, by injecting 2% solution of lignocaine hydrochloride

along with 1:80000 adrenaline. (LIGNOX 2 %) [3ml Syringe with 24-gauge needle]

SURGICAL TECHNIQUE:

Full thickness mucoperiosteal flap reflected to achieve adequate

exposure of the surgical site by periosteal elevator. Then the Tooth removed using

appropriate forceps. After extraction, bony spicules removed.

Sharp and rough edges of extracted socket trimmed and smoothened with bone file and

bone rounger. The haemostasis achieved by compression. After this procedure, the

prepared autologous calcium chloride activated Platelet rich plasma gel placed in the

extracted socket for the side of case group. With 3-0 silk suture


24 | P a g e

Material figure of eight suture placed on the extraction socket. Post-operative instructions

had given to patients followed by antibiotic and analgesic (Cap.Amoxicillin 500mg tid,

T.Zerodol-P bd) for 3 days. Post-operative follow-up done in case group after 24 hours to

check the stability of Platelet rich plasma gel in socket. The opposite side extraction carried

out after 1 week and taken as control.

After 7 days suture removal had done for case groups and succeeding follow-up had done

at 3rd day, 7th day and 14th day for the clinical evaluation of wound healing and 1st month

and 3rd month postoperatively for the radiographic evaluation of the bone fill in socket.

PREPARATION OF PLATELET RICH PLASMA GEL:

1st STEP: Under all aseptic conditions 5 ml of venous blood was collected from the

antecubital region.

2nd STEP: Preparation of PRP

1. The tube was put in a centrifuge machine and counterbalanced. The first centrifuge cycle

was done at 2,000 rpm for 15 min. The proceeds was the separation of the whole blood

into the following three layers.

Topmost layer was consisted of straw-coloured plasma (This plasma contains

relatively low concentration of platelets (platelet poor plasma)

The Middle layer was enriched with Platelet rich fibrin clot (This higher

concentration of platelets and WBC in the boundary layer is often called as ‘Buffy coat’).

Bottom layer is Red blood corpuscles


25 | P a g e

2. The upper layer with poor platelet rich plasma was discarded and the middle and bottom

layer again centrifuged at 3,000 rpm for 10 min. After the second centrifuge, the upper half

was discarded and the lower half was used as PRP.

3rd STEP: PRP GEL Preparation

The PRP will be activated with CaCl2 10% to form a PRP gel in a ratio 10:1

ROUTINE POST OPERATIVE CARE:

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

FOLLOW-UP:

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

platelet rich Plasma gel in socket.

• Post-operative follow to be done in case group and control group on post-operative 3rd day

,7th day and 14th day for extraction socket healing.


26 | P a g e

• After 7 days suture removal was done for case group. Succeeding follow-up was done at

4th week, 3rd month postoperatively for the evaluation of radiographic parameters.

PARAMETERS EVALUATED

Evaluation of Soft Tissue Healing by healing index by Landry, Turnbull and howley1

Healing Index 1: Very poor (has 2 or more of the following)

• Tissue colour: C50 % of gingiva red

• Response to palpation: bleeding

• Granulation tissue: present

• Incision margin: not epithelialized, with loss of epithelium beyond incision margin

• Suppuration present

Healing Index 2: Poor

• Tissue colour: C50 % of gingiva red

• Response to palpation: bleeding

• Granulation tissue: present

• Incision margin: not epithelialized, with connective tissue exposed


27 | P a g e

Healing Index 3: Good

• Tissue colour: C25 and 50 % of gingiva red

• Response to palpation: no bleeding

• Granulation tissue: none

• Incision margin: no connective tissue exposed

Healing Index 4: Very good

• Tissue colour: 25 % of gingiva red




Healing Index 5: Excellent

• Tissue colour: all tissues pink





28 | P a g e

RADIOGRAPHIC ANALYSIS

Digital Subtraction Radiography for assessment of bone density. Intra oral radiograph,

RVG will be taken before extraction, immediately after extraction, post- op after 1 month

and 3months. An x-ray unit 70Kvp,80Ma using focal long cone paralleling technique with

focal spot film distance 16inch with an exposure time of 0.08sec and Ren HXCP film

holding device will be used for placing x-ray sensor, and vertical angulation of x-ray beam

is also recorded for each patient every time.(max canines +50, incisors +45, premolars +35,

molars +25. Mandible canines-25, incisors-20, premolars-10, molar-5).and evaluate by

means of DSR, with in subtraction images, a window (experimental region) is defines

covering the visible density changes in the defect area. Background noise measured by

using a similarly sized window (control region) located in an area without calcium chloride

activated platelet rich plasma. By comparing radiographs .Bone density changes was

quantitatively evaluate by calculation of the mean, standard deviation and maximum and

minimum values of the grey level histogram with in these window. Protection measures

was taken for patients to reduce x-ray exposure by using thyroid collor and lead apron.

RVG x-ray exposure is 8.6 times narrower than intra oral periapical radiograph.

BONE DENSITY ANALYSIS

The grey level histogram value was measured in immediate postoperative radiograph 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 postoperative period and the value were compared.


29 | P a g e

STATISTICAL TOOLS EMPLOYED:

The study was analysed by Software(s) to be used for statistical analysis:

Microsoft Excel 2016 and statistical package for social sciences SPSS version 20.0.

Statistical tests used for data analysis are Mean, Standard Deviation, ANOVA, Student‘t’

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

confidence interval.


30 | P a g e

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.

Medical history:


31 | P a g e

Dental history:

Personal history:

Family history

EXAMINATIONS:

Extra oral examination:

Face:

Tmj:

Lymphnodes:


32 | P a g e

Intraoral examination:

Soft tissue examination:

Buccal/labial mucosa:

Tongue:

Alveolar mucosa:

Floor of the mouth:

Hard tissue examination:

Teeth present:


33 | P a g e

PROVISIONAL DIAGNOSIS:

TREATMENT PLAN:

POSTOPERATIVE EVALUATION:

EVALUATION OF SOFT TISSUE HEALING INDEX

Review Case group Control group

3rd post -op day

Date:

7th post -op day

Date:

14th post -op day

Date:


34 | P a g e

RADIOGRAPHIC EVALUATION

Findings

Grey level histogram values

Immediate post-op

Date:

4th week post-op

Date:

3rd month post-op

Date:

Guide/ Co-guide signature

Review

FIGURES

Figures

vii | P a g e

FIG: 1 ARMAMENTARIUM

FIG:2 MEDICO CENTRIFUGE

Figures

viii | P a g e

FIG:3 WITHDRAWAL OF BLOOD

FIG:4 AFTER FIRST CENTRIFUGATION

Figures

ix | P a g e

FIG:5 PRP AFTER SECOND CENTRIFUGATION

FIG:6 PRP ACTIVATED WITH 10 % CALCIUM CHLORIDE

Figures

x | P a g e

FIG:7 CALCIUM CHLORIDE ACTIVATED PLATELET RICH PLASMA GEL

CASE: 1

FIG:8 POST OP CLINICAL EVALUATION OF EXTRACTION SOCKET HEALING

IMMEDIATELY AFTER EXTRACTION

CASE CONTROL

Figures

xi | P a g e

3 DAYS AFTER EXTRACTION

CASE CONTROL


CASE CONTROL

Figures

xii | P a g e


CASE CONTROL

FIG: 9 RADIOGRAPHS OF POSTOPERATIVE PERIODS

IMMEDIATE POSTOPERATIVE DAY

CASE CONTROL

POST OP AFTER 4 WEEKS

CASE CONTROL

Figures

xiii | P a g e

POST OP AFTER THREE MONTHS

CASE CONTROL

CASE:2

FIG: 10 POST OP CLINICAL EVALUATION OF EXTRACTION SOCKET

HEALING


CASE CONTROL

Figures

xiv | P a g e


CASE CONTROL


CASE CONTROL

Figures

xv | P a g e



CASE CONTROL

4 WEEKS POSTOPERATIVE PERIOD

CASE CONTROL

3 MONTH POSTOPERATIVE PERIOD

CASE CONTROL

Figures

xvi | P a g e

CASE:3


HEALING


CASE CONTROL


CASE CONTROL

Figures

xvii | P a g e


CASE CONTROL



CASE CONTROL


CASE CONTROL

Figures

xviii | P a g e


CASE CONTROL

CASE:4


HEALING


CONTROL CASE

Figures

xix | P a g e


CONTROL CASE


CONTROL CASE

Figures

xx | P a g e



CONTROL CASE


CONTROL CASE


CONTROL CASE

RESULT

Results

35 | P a g e

The present study was intended to assess the efficiency of 10 % calcium chloride

activated platelet-rich plasma gel in bone regeneration and soft tissue healing after

extraction. The study was undertaken on 25 patients (age range of 18-32 years) with

bilateral extraction which includes 17 (68 %) females and 8(32 %) males in the ratio

1:2.2(Table: 1, Graph: 1). the age, sex and date of procedure were recorded.

An analysis was done regarding the soft tissue healing on the third day for the two

categories. The case group showed a mean score of 3.46 and for control group

2.46(P<0.05), and after 7 days it was 4.42 and 3.42 for case and control and on 14th day it

was 5 for both case and control groups (P<0.05).

The Comparison of mean soft tissue healing value between the groups at post-

operative day 3rd become significant. (Table: 2)


operative day 7th day become significant. (Table: 3)


operative day 14th day become significant. (Table: 4)

Comparison of mean soft tissue value between the groups at different time periods

become significant. (Table: 5, Graph: 2)

Grey level histogram value recorded from radiograph RVG in immediately after

extraction, 1 month and 3 month postoperatively of all patients of the case and

control groups has been tabulated.

The bone healing overall density values become 47.3342 and 47.3354 of control

and case of immediate postoperative (P= 0.1), During the 4th week the overall bone

Results

36 | P a g e

healing density for case group showed a means score of 57.752 and 55.394 for

control group.(P=0.2)and in 3rd month of postoperative period the values for control

and case group were 73.7181 and 76.4585 respectively(P=0.3).

The Comparison of mean soft tissue value between the groups at 3rd post-operative

day become significant. (Table: 6)

The Comparison of mean soft tissue value between the groups at 7th post-operative


The Comparison of mean soft tissue value between the groups at 14th post-operative


The Comparison of mean soft tissue value between the groups at different time

periods become significant. (Table: 9, Graph: 3)

The multiple comparison of mean soft tissue values within the groups at different

time periods is significant when compared with post-operative value at 3rd day, 7th

day and 14th day. (Graph 4)

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

time periods is significant compared immediate with other time periods, 4th week

with other time periods and 3rd month with other time periods.( Graph 5).

Extracted socket enhanced with 10 % calcium chloride activated PRP gel shown to

heal at 1.5 times faster than of normal healing of extraction socket. The bone density value

and soft tissue healing in extraction socket is higher in case treated with 10% calcium

chloride activated Autologous Platelet Rich Plasma gel compared with control group which

has been not treated with Calcium chloride Activated Autologous Platelet Rich Plasma gel.

TABLES

Tables

37 | P a g e

Table-1: Demographic data of study population

Demographic

data

Age

MEAN

Gender

Male Female

Number Percentage

(%)

Number Percentage

(%)

Groups

2

4

8

32.00

17

68.00

Table-2: Comparison of mean soft tissue values post-operative 3rd day between the

groups

Groups

3rd Day

(MEAN)

p value

Control

2.46

<0.05

Case 3.46

Tables

38 | P a g e

Table-3: Comparison of mean soft tissue values post-operative 7th day between the

groups

Groups

7th DAY

(MEAN)

p value

Control

3.42

<0.05

Case 4.42

Table-4: Comparison of mean soft tissue values post-operative 14th day between the

groups

Groups

14TH

DAY

(MEAN)

p value

Control

5.0

0.00

Case 5.0

Tables

39 | P a g e

Table-5: Mean Soft tissue healing values of different groups at different post-

operative periods

GROUP

Mea

n

Std.

Deviati

on

Std.

Error

Mean

EVALUATION OF SOFT

TISSIUE HEALING - 3rd

Day

CASE 3.46 .508 .100

CONTRO

L

2.46 .508 .100

EVALUATION OF SOFT

TISSIUE HEALING - 7th

Day

CASE 4.42 .504 .099

CONTRO

L

3.42 .504 .099

EVALUATION OF SOFT

TISSIUE HEALING - 14th

Day

CASE 5.00 .000a .000

CONTRO

L

5.00 .000a .000

Table-6: Comparison of mean bone density values immediately after extraction

between the groups

Groups

Immediately

(MEAN)

+/-SD

p value

Control

47.3354+/-

O.58

<0.05

Case 47.3342+/-

0.58

Tables

40 | P a g e

Table-7: Comparison of mean bone density values post-operative 4th week between

the groups

Groups

4th week

(MEAN)

p value

Control

55.39+/-

1.17

<0.05

Case 57.75+/-

1.15

Table-8: Comparison of mean bone density values post-operative 3rd Month between

the groups

Groups

4th week

(MEAN)

p value

Control

73.71+/-

1.66

<0.05

Case 76.45+/-

1.61

Tables

41 | P a g e

Table-9: Mean Bone density values of different groups at different post-operative

periods

Group Mean

Std.

Deviati

on

Std.

Error

Mean

Evaluation of bone

density - immediately

after extraction

Case 47.3342 .58579 .11488

Control 47.3354 .58512 .11475

Evaluation of bone

density - 1 month

Case 57.752 1.3563 .2660

Control 55.397 1.1798 .2314

Evaluation of bone

density - 3rd month

Case 76.4588 1.6179

4

.31730

Control 73.7181 1.6673

7

.32700

GRAPHS

Graphs

xxi | P a g e

GRAPH:1 Demographic Data of Study population

GRAPH:2 Mean soft tissue healing values of different groups at different post-

operative periods

3.46

4.425

2.46

3.42

5

0

1

2

3

4

5

6

3rd Day 7th Day 14th Day

ME

AN

±SD

CASE CONTROL

Male32%

Female68%

Graphs

xxii | P a g e

GRAPH:3 Mean bone density values of different groups at different post-operative

periods

GRAPH 4:Multiple Comparison of mean soft tissue healing values within the group

at different time periods

47.3342

57.752

76.4588

47.335455.397

73.7181

0

20

40

60

80

100

immediately after extraction 1 month - 3rd month

ME

AN

±SD

Case Control

0

1

2

3

4

5

6

3rd Day 7th Day 14th Day

Chart Title

Control Case

Graphs

xxiii | P a g e

GRAPH 5:Multiple Comparison of Mean bone density values within the group at

different time periods

0

10

20

30

40

50

60

70

80

90

Immediate 4 Weeks 3rd Month

Control Case

DISCUSSION

Discussion

42 | P a g e

Immediately following tooth removal, a healing process commences that affects the

final alveolar bone volume and architecture of the alveolar ridge. Satisfactory and timely

healing is crucial to obtain ideal functional reconstruction. Traumatic removal of a tooth,

or inadequate healing response, may lead to excessive bone loss delaying tooth

replacement11. Bone tissue repair is a convoluted process helpful for cellular functions and

mineralization of defects to remodelling the surgical defect to regain the original structure.

In the future, protecting the wound and regain the bone will become standard care for all

extractions. Platelet-rich plasma is a new step in the platelet therapeutic concept helpful for

artificial biochemical modification. PRP progress via centrifugation has significantly been

explained so that it can be done in the office environment as well as in the operating room9.

Nevertheless, the centrifugation process must be sterile and precisely suited to

platelet separation from red blood cells and their sequestration in high concentrations

without dropping the platelets or damaging them so that they no longer can actively secrete

their growth factors11.

The platelet rich plasma is first generation platelet concentrate factor obtained from

freshly drawn venous blood, centrifuged twice without anticoagulants which contain

diverse growth factors such as platelet derived growth factor, vascular endothelial growth

factor and transforming growth. Fibrin, fibronectin and vitronectin are the three proteins

present which is helpful for epithelial migration and osteo-conduction1.

During the last decade, there have been numerous in vivo animal studies, which

have adopted biological mediators such as polypeptide growth factors to expedite soft

tissue and bony healing. It is, therefore, a reasonable hypothesis that raising the

concentration of platelets in bone defects may lead to improved, faster healing and

Discussion

43 | P a g e

stimulate new bone formation. May lead to advanced, faster healing and spur new bone

formation 13. Surgical sites improved with PRP have been bestowed to heal at two to three

times that of regular surgical sites. PRP can be a great supplement to many surgical

procedures14, and PRP accelerates wound maturity and epithelialization, hence decreased

scar formation. PDGF and epidermal growth factor (EGF) are the primary growth factors

associated with fibroblast migration, proliferation, and collagen synthesis. Raised

congregations of these growth factors is a likely cause for the accelerated soft tissue wound

healing1.

Wound healing involves a sequence of physiological events that restore and

replace damaged tissue functions. After extraction has been done, the healing is revealed

in the coagulative phase, proliferative phase, and osteogenic remodelling phase. The

coagulative phase starts immediately after the extraction and lasts until 3days, which

involves filling the socket with clot and inflammatory process initiation. In this phase,

platelets, endothelium, and fibroblast start to release numerous growth factors. After this,

the proliferative phase starts and lasts until 20 days to 2 months following the postoperative

period. After this, the blood clot becomes dissolved, and the 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 most extended phase is an osteogenic-remodeling

phase. The healing of the extracted socket lasts from 8weeks to months. This phase

involves mineralization of the matrix, secretion of osteoid, and bone remodeling.

The platelets are helpful for blood clotting by hemostatic plug formation. By using

platelet concentrate, it is helpful for natural clot formation, which induces wound healing

and bone regenerative process21. The PRP creates a gel-like substance that contains

Discussion

44 | P a g e

functional, activated, intact platelets present in the plasma matrix, which immediately starts

to release growth factors. It is mainly used as a protective layer for the Schneiderman

membrane in the sinus lift procedure to fill the material6.

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 formation76.

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. The active

secretion of these growth factors is initiated by the clotting process of blood and begins

within 10 minutes after clotting. More than 95% of the presynthesized growth factors are

secreted within 1 hour2 . Therefore, PRP must be developed in an anticoagulated state and

should be used on the graft, flap, or wound, within 10 minutes of clot initiation. Studies

that have not used anticoagulated whole blood, which is then clotted to activate the PRP14.

The discharged growth factors instantly bind to the outer surface of cell membranes of cells

in the graft, flap, or wound through transmembrane receptors. After the beginning blast of

Discussion

45 | P a g e

PRP-related growth factors, the platelets synthesize and secrete additional growth factors

for the prevailing seven days of their life span. Once the platelet is drained and dies off the

macrophage, it has reached in the region through the vascular in-growth stimulated by the

platelets, considers the function of wound healing management by secreting some of the

same growth factors as well as others. Therefore, the number of platelets in the blood clot

within the graft, wound, or adherent to a flap fastens the pace of wound healing. PRP

slightly rises this number. Researches have revealed that adult mesenchymal stem cells,

osteoblasts, fibroblasts, endothelial cells, and epidermal cells signify the cell membrane

receptors to growth factors in PRP. These transmembrane receptors, in turn, persuade

activation of an endogenous internal signal protein, which causes the expression of

(unlocks) a typical gene sequence of the cell, such as cellular proliferation, matrix

formation, osteoid production, collagen synthesis, etc. The significance of this knowledge

is that the PRP growth factors never access the cell or its nucleus, they are not mutagenic,

and they act through the stimulation of natural healing, just much faster. Therefore, PRP

cannot persuade tumor formation and has never done so9.

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 is helpful for faster maturation of collagen

in wounds. Platelet derived growth factor (PDGF) is helpful to increase the wound healing

cells which helpful for increase wound healing properties21.

Moreover, the increase in soft tissue healing was found to occur at earlier time

points than non-PRP treated control sites. Of note was the immediate increase in healing

index readings which indicates enhanced early re epithelisation.. Accelerate tissue healing

Discussion

46 | P a g e

is in contrast to the drop in wound healing seen at the control site before wound healing

began to take place. It took approximately 1weeks for the control sites to reach the same

bone density that the PRP-treated site had reached by 3 days Further examination of the

soft tissue healing changes shows that the finding of parallel increases in bone density in

later weeks most likely represents normal healing taking place at both sites, subsequent

changes in soft tissue healing with the PRP-treated and control sites are parallel, with no

significant differences, except for the 3rd day and 7th day time points.

Intra-oral digital radiographs taken of the individual surgical sites revealed that the

effects of PRP were significantly beneficial (P<.05) for increasing bone density following

extraction. The increase in bone density suggests a greater volume of new bone formation

with PRP treatment. Moreover, the increase in bone density (presume as increased volume

of new bone formation) was found to occur at earlier time points than non-PRP treated

control sites. Of note was the immediate increase in grayscale readings which indicates

enhanced early bone formation. This corresponds with results from the Lucarelli molecular

study that demonstrated PRP treatment of human mesenchymal stem cells induced early

proliferation of these cells and possibly differentiation into osteoblasts. Accelerate bone

formation is in contrast to the drop in bone density (representing bone loss) seen at the

control site before bone formation began to take place. It took approximately 6 weeks for

the Control sites to reach the same bone density that the PRP-treated site had reached by 4

week. The PRP-induced acceleration in bone formation may be due to the presence of bone

morphogenetic proteins (BMPs)-2 and -6 in PRP that stimulates mesenchymal stem cells

to begin osteoblast differentiation and subsequent calcification. Healing at control sites

would be dependent upon initial osteoclast activity to break down existing bone thereby

Discussion

47 | P a g e

releasing BMPs9. The findings of this study correspond with the known, accepted bone

repair timeline. The immediate start of bone formation seen with PRP treatment is of

clinical relevance because it is the initial 2 weeks following bone manipulation oral surgery

that are crucial in countering infection, loss of the blood clot and/or AO (dry socket)

formation. with greater bone density for the PRP-treated sites due to earlier, more rapid

bone formation. The significant differences seen at 4th week and 3rd month may be due to

bone remodeling or inherent variations in the radiographic evaluation.In this study we used

calcium chloride activated platelet rich plasma to regenerate the bony defects in the

extraction sockets and a faster soft tissue healing.

The use of PRP is a recently proposed technique for the regeneration of periodontal

defects. Limited studies have assessed the effect of activated and non-activated PRP

(Graziani et al. 2006; Creeper and Ivanovski 2012; Slapnicka et al. 2008) and their different

concentrations (Graziani et al. 2006; Slapnicka et al. 2008; Ogino et al. 2006; Choi et al.

2005; Kanno et al. 2005) on proliferation of HGF and MG-63 cell lines.

Graziani et al. reported the highest proliferation rate of fibroblasts in the low

concentration PRP group at 24 h. In their study, in contrast to ours, the 10% activate PRP

group caused the highest proliferation rate of HGFs at 72 h after culture; however, the FBS-

rich DMEM group showed the highest proliferation rate among other groups. The 10%

activated PRP had the most significant effect on the proliferation of HGFs among PRP

groups at 24, 48, and 72 h10. PRP enhances osteoprogenitor cells in the host bone. It has

found clinical applications in fully autogenous bone grafts and composites of autogenous

bone grafts with a variety of bone substitutes with as little as 20% autogenous bone. PRP

has shown improved results in continuity defects, sinus lift augmentation grafting,

Discussion

48 | P a g e

horizontal and vertical ridge augmentations ridge preservation grafting, and

periodontal/peri-implant defects and had also recognized PRP allows first implant filling

and improved Osseointegration when used in compromised bone such as osteoporotic bone

after radiotherapy. Because PRP also improves soft tissue mucosal and skin healing, it is

utilized in connective tissue grafts, palatal grafts, gingival grafts, mucosal flaps13.

PRP gel is formed from PRP for the degranulation of a-granules which are present

in the platelets and are responsible for releasing the growth factors.

Platelet activation is a crucial step that might influence the availability of bioactive

molecules and, therefore, tissue healing. The most commonly used activation methods in

the current clinical practice were directly compared. CaCl2, autologous thrombin, their

combination, and collagen type I were used to mimicking the clinical situations where their

proximity in the administered connective tissues should provoke an “in situ” platelet

activation. The latter is currently taken for several PRP applications since it was considered

as a more convenient and more robust strategy to deliver platelet bioactive molecules.

Carola Cavallo et al. 2016 study shows CaCl2, thrombin, CaCL2/thrombin, and collagen

type I induced different platelet aggregation. In particular, PRP activated with CaCl2,

thrombin, and CaCL2/thrombin formed clots recognized in the 15-minute evaluation and

continuing up to 24 hours (thrombin and CaCL2/thrombin already macroscopically stable

at 15 minutes, CaCl2 starting at 15 and visually stabilized at 30 minutes).

In contrast, in collagen-type-I-activated samples, no clot formation was noticed for

any of the time points evaluated. At 15 and 30 minutes, thrombin and CaCl2/thrombin

produced a significantly higher amount of PDGF concerning that of CaCl2The release

pattern of PRP activated with CaCl2 was similar for all the GFs evaluated, with a

Discussion

49 | P a g e

significant and progressive release of GFs starting from 15 minutes and increasing up to

24 hours. CaCl2/thrombin induced a significantly higher VEGF release. At 15 minutes,

thrombin and CaCl2/thrombin showed a more significant amount of TGF-𝛽 concerning

that of CaCl2 and collagen type I (𝑝 < 0.05), whereas no significant difference was noted

between CaCl2 and collagen type I14.

In our technique CaCl2 alone was mixed with PRP to form an autologous platelet

gel. This platelet gel was free of eliciting any antigen–antibody reaction as it was prepared

from patients’ own blood.

The present study to evaluate soft tissue healing and bone regeneration in

the extraction socket by using calcium chloride activated platelet rich plasma. Bilateral

extraction of same tooth in a patients is chosen 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 were treated with 10% calcium chloride

activated Platelet rich plasma gel shows1.5 times more bone regeneration and faster

extraction socket healing without any complications than the patients not treated with

calcium chloride activated PRP gel. And the rate of soft tissue healing was much faster and

case group attained a healing of socket in 3 days which the control group attained on 7th

day. And on the 7th day the socket was almost closed by secondary intension.

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 AND CONCLUSION

Summary And Conclusion

50 | P a g e

The procedure for the preparation of 10% calcium chloride activated platelet-rich

plasma is a simple chairside procedure, patients own blood, no allergic reactions, little cost,

time-consuming is very less, no side effects, and shows good results. In this study, soft

tissue healing and bone generation in the extraction socket is analyzed in different time

intervals. There are significant changes seen in the extracted socket treated with 10%

Calcium chloride activated platelet-rich plasma when compared to the socket alloyed to

heal naturally.

Study Concluded that the 10%calcium chloride activated platelet-rich plasma

shows a significant role in soft tissue healing and bone regeneration process in the extracted

socket when compared to the non-treated socket.

This advancement in wound healing, reduction in pain, and improvement in the

bone density imply and highlights the use of PRP, certainly as a proven method in

producing and accelerating soft and hard tissue regeneration.

It was also helpful for osseous regeneration in other post-surgical defects, implant

placement, mandibular reconstruction, ridge augmentation, graft for bone substance, etc.

An appended advantage of PRP noted in the present study is its capacity to develop

a biologic gel that provided clot stability and functions as an adhesive and enhances wound

healing much faster.

The limitations of the study were bone width, and bone height was not assessed

Platelets quality, quantity, and its growth factor was not assessed. The present study was

Summary And Conclusion

51 | P a g e

done with a follow up of 3 months. Further clinical trials with longer duration follow up

with a larger sample size should be done to get a more affirmative and conclusive result.

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ANNEXURE

CONSENT FORM

PART 1 OF 2 INFORMATION FOR PARTICIPANTS OF THE STUDY

1. Name of the Principal Investigator: Dr.Aneesha.S

. Post Graduate student

Dept. of Oral and Maxillofacial

surgery

SMIDS, Kulasekharam, pin 629161

2. Name of the Guide: Dr. Mathew Jose

Professor & H.O.D


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 have any doubts or if you want more information, you are free to ask the study

personnel or the contact person mentioned below before you give your consent and also at any

time during the entire course of the project.

3. Name of the Co-Guide: Dr Sajesh

Professor


surgery


4. Institue: Sree Mookambika Institute of Dental

Sciences

V.P.M Hospital complex,

Padanilam,

Kulasekharam, pin: 629161

TamilNadu

5. Title of the study:

“COMPARISON OF POST OPERATIVE HEALING OF EXTRACTION

SOCKET WITH AND WITHOUT CALCIUM CHLORIDE ACTIVATED

PLATELET RICH PLASMA”

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. PRP preparation is very simple. For this 5 ml blood

will be taken from patient and immediately centrifuged at 2,000 rpm for 15 minutes.

the separated PPP and buffy coat layer including 1mm below is collected in sterile

tube and centrifuge at 3000 rpm for 10 min. separated PRP will be activated with

10% calcium chloride to form a PRP gel.

It accelerates wound maturity and epithelialization, hence decreased scar

formation. PDGF and epidermal growth factor (EGF) are the main growth factors

responsible for this.

Activated PRP gel causing degranulation of αgranules present in the

platelets and releasing the growth factor which is 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.

7. Aim and objectives:

To compare the healing and bone formation of molar extraction socket

with and without autologous PRP gel

8. Scientific justification of the study:

Platelets are very important in the wound healing process. They arrive

quickly at the wound site and begin coagulation. They release multiple wound

healing growth factors and cytokines, including platelet derived growth factor

(PDGF), transforming growth factors (TGF)/b1 and b2, vascular endothelial growth

factor (VEGF), platelet derived endothelial cell growth factor (PDEGF),

interleukin- 1 (IL-1), basic fibroblast growth factor (bFGF), and platelet Activating

factor-4 (PAF-4). These growth factors are consider to be contributing to bone

regeneration and increased vascularity, vital features of a healing bone graft [1]

[Journal of oral and maxilla facial surgery (july-sept2015) 14 (3) : 808 – 815

9. Procedure for the study:

• Under aseptic condition, local anaesthesia will be given.

• Atraumatic extraction will be done with appropriate forceps

• Prepared calcium chloride activated PRP gel will be place in extraction socket and

sutured Prp is prepared from patients own blood for that,

• 5ml venous blood drawn from patient

• PRP will be prepare by double centrifugation of patient’s blood

• Separated PRP mixed with 10% calcium chloride and formed into gel form.

• Post-op record will be maintain to measure the bone regeneration and tissue

healing.

10. Expected risks for the participants:

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

thrombophlebitis at Injecting site.

11. Expected benefits of research for the participants:

The placement of autologous calcium chloride activated PRP gel in the

extraction site will improve the healing property of tissue and help for bone

formation, which will 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 publish 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

14. How many people will be in the study? 25 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

provide 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 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: 3 month

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.ANEESHA.S

Post Graduate student.

Department of Oral & Maxillofacial Surgery,

SreeMookambika Institute of Dental Sciences,

Kulasekharam, Kanyakumari District-629161.

Mobile No: 8547148230

[email protected]

Place: signature of principal investigator

Date:

Signature of the participant

mailto:[email protected]

CONSENT FORM

PART 2 OF 2

PARTICIPANTS CONSENT FORM

The details of the study have been explain 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 title.

“COMPARISON OF POST OPERATIVE HEALING OF EXTRACTION

SOCKET WITH AND WITHOUT CALCIUM CHLORIDE ACTIVATED

PLATELET RICH PLASMA”

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.

Date:

Place: Signature of principal

Investigator

COMPARISON OF POST OPERATIVE HEALING OF EXTRACTION …

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