Biochemical Efficacy of Cymbopogon Citratus Gel as Local Drug Delivery Agent in Reducing Matrix Metalloproteinase-8 Levels Intra-crevicularly in Moderate Periodontitis Patients
Phase: PHASE1
Conditions: Periodontitis
Sponsor: Alexandria University

 
 
Faculty of Dentistry 
Department of Oral Medicine, Periodontology, Oral Diagnosis and Oral 
Radiology   
 
 
 
 
LEVELS OF MATRIX METALLOPROTEINASE-8 IN GINGIVAL 
CREVICULAR FLUID AFTER INTRAPOCKET APPLICATION  
OF CYMBOPOGON CITRATUS GEL ADJUNCTIVE TO  
NON-SURGICAL TREATMENT IN PATIENTS  
WITH MODERATE PERIODONTITIS. 
 
 
Protocol for M. Sc. Degree 
In oral medicine, periodontology, oral diagnosis, and oral radiology
 
 
Academic Year 2019-2020 
 
 
17th October 2019 
 
 
 
 

 
 
 
 
 
 
 
Name of Candidate: Nadein Abd Elnasser El. Sharif 
 
Supervision Committee:
 
1. Prof. Dr. Ahmed M Hommos.  
    Prof. of Oral Medicine, Periodontology, Oral Diagnosis and Oral Radiology. 
    Department of Oral Medicine, Periodontology, Oral Diagnosis and Oral 
    Radiology.        
 Faculty of Dentistry- Alexandria University. 
2. Dr. Souzy Kamal Anwar. 
    Lecturer of Oral Medicine, Periodontology, Oral Diagnosis and Oral   
Radiology. 
    Department of Oral Medicine, Periodontology, Oral Diagnosis and Oral 
    Radiology.  
    Faculty of Dentistry- Alexandria University. 
3. Dr. Riham Mohamed El-Moslemany. 
    Lecturer of Pharmaceutics. 
Department of Pharmaceutics. 
Faculty of Pharmacy- Alexandria University.
 
4. Dr. Neveen Lewis Mikhael. 
    Lecturer of Clinical Pathology. 
    Department of Clinical Pathology. 
    Faculty of Medicine- Alexandria University.  
 
 
 
 
 
 
 
 

 
 
CONTENT 
 
ABSTRACT ........................................................................................................................ 1 
INTRODUCTION ............................................................................................................... 2 
AIM OF THE WORK ......................................................................................................... 5 
PLAN OF THE STUDY ..................................................................................................... 6 
ROLE OF SUPERVISORS ............................................................................................... 11 
ETHICAL CONSIDERATIONS ...................................................................................... 12 
DATA MANAGEMENT ANDSTATISTICAL ANALYSIS .......................................... 13 
DURATION OF THE STUDY ......................................................................................... 14 
BUDGET ........................................................................................................................... 15 
PROBLEMS ANTICIPATED .......................................................................................... 16 
PUBLICATION POLICY ................................................................................................. 17 
REFERENCES .................................................................................................................. 18 
 
 
 

1 
 
ABSTRACT 
 
Background: The gold standard therapy in treating moderate periodontitis is 
mechanical removal of dental biofilm. Using local delivery drugs as adjunctive to scaling and 
root planing is widely used to modulate inflammatory host response and eradicate microbes. 
Nowadays, gingival crevicular fluid (GCF) biological molecular markers located in the exact 
area of contact between bacterial biofilm and host tissues are a suitable way to assess and 
predict periodontal disease progression. Collagenase-2 (MMP-8), which breaks down 
collagen of periodontal ligament, is associated with the onset of inflammation and is 
indicative of the severity of the non-specific inflammatory response. 
 
Study objective: To evaluate gingival crevicular fluid levels of matrix 
metalloproteinase-8 (MMP-8) in the non-surgical management of moderate periodontitis after 
the adjunctive intra-pocket application of Cymbopogon citratus (lemon-grass) gel. 
Materials and Method: This randomized controlled trial will include forty patients 
with moderate periodontitis, divided equally into two groups. Group-I will be managed by 
scaling and root planing with intra-pocket application of 2% lemongrass oil gel. Group- II 
will be managed by scaling and root planing with intra-pocket application of a placebo gel. 
Gingival crevicular fluid will be collected from both groups at baseline before treatment, one 
week, and at twelve weeks after treatment, respectively. Then analysed by Enzyme-linked 
Assay (ELISA) technique. 
 
Results: Results will be tabulated and statistically analysed.  
 
Keywords: Enzyme-linked immunosorbent assay (ELISA). Gingival crevicular fluid 
Lemongrass oil, Matrix metalloproteinase -8 (MMP-8)

2 
 
INTRODUCTION 
Periodontal disease is an inflammation of the supporting tissue that surrounds the 
teeth. The major etiological factor for initiating and progressing of this disease is dental 
biofilm, which contains bacteria that have virulence factors that cause reversible or 
irreversible destruction of connective tissue and alveolar bone. (1) Although gingivitis is a 
completely reversible condition and can return to health by removing local factors, it can 
extend into alveolar bone to cause periodontitis, which is irreversible destruction of 
supporting tissues of the teeth, in case causative agents are not removed. (2) 
Caton and his colleague (2018) (3) introduced that periodontitis has two categories. 
According to Tonetti, Greenwell and Kornman (2018), (4) first category consists of four 
stages based on severity and complexity of management. Stage I: initial periodontitis, stage 
II: moderate periodontitis, stage III: severe periodontitis with potential for additional tooth 
loss, and stage IV: severe periodontitis with potential for loss of the dentition. Second 
category consists of three grades based on risk of rapid progression and expected treatment 
response. Grade A: slow rate of progression, grade B: moderate rate of progression, and grade 
C: rapid rate of progression. 
The prevalence of periodontitis remains high globally, especially severe periodontitis 
which is the sixth most prevalent disease of humanity. (5) In case of severe periodontitis, teeth 
may be lost which has an impact on function and aesthetics. Different forms of periodontitis 
could affect any age. (6) 
Appropriate diagnosis can identify the severity, activity and progression of 
periodontitis which is important for reaching the best treatment plan. There are several 
diagnostic procedures such as probing depth, bleeding on probing, mobility assessment, 
plaque index and radiographs to assess alveolar bone level. (7) Goodson (1992) (8) emphasized 
that all previously mentioned methods are easy to use, inexpensive, and relatively non-
invasive. However, they could not assess the current disease status and show only the history 
of disease. 
Nowadays, novel methods of diagnosis can identify periodontal risk and predict its 
progression and thus allow earlier detection of the disease. (9) Those methods include 
microbiological analysis and biomarkers in saliva, (10) and gingival crevicular fluid. (11) 
Biologic markers are substances that are measured as an indicator of normal biologic and 
pathogenic processes, or pharmacologic responses to a therapeutic intervention (12). 
Srivastava, Nayak, and Rana (2017) (13) indicated that prostaglandin E2, tumour necrosis 
factor-α, matrix metalloproteinase (MMP-8, 9 and 13) are biomarkers that not only help in 
diagnosis and predicting the progression of many periodontal diseases, but also help in 
predicting treatment outcomes.  
According to Teles and his co-worker (2006), (14) there are many protocols for 
treatment of periodontitis. Nonsurgical treatment includes mechanical instrumentation alone 
or in conjunction with host modulation or antimicrobial therapy. Removing bacterial biofilm, 

3 
 
calculus, and toxins from periodontally involved root surfaces is the main goal of periodontal 
therapy to stop progression of bacterial infection. It also reduces the levels of inflammation, 
probing pocket depth and helps in clinical attachment gain. Although removing dental biofilm 
mechanically is still the gold standard therapy, there are many factors that reduce its 
effectiveness such as severity of periodontal disease, rate of progression and response to 
therapy. Therefore, systemic host modulation and antimicrobial therapy are used as adjunctive 
therapy to nonsurgical treatment to overcome these limitations. (15) Despite  controlling or 
eradicating many pathogens, systemic antibiotics cause several side effects to patients, 
including the development of resistant species. (16)  
Localized intra-pocket drug delivery systems were developed to reduce these side 
effects. Local drug delivery systems have controlled release ability for extended periods of 
time. Moreover, they provide high concentrations of active ingredients at the targeted site. 
However, the use of local delivery antibiotics did not overcome antimicrobial resistance. (17,18) 
Thereafter, phytotherapics were advanced as an alternative option to antibiotics to reduce side 
effects. Phytochemicals are derived from plants that have therapeutic anti-inflammatory, anti-
collagenase, antimicrobial, antioxidant, and antiseptic properties. Nowadays, periodontists use 
local herbal drugs as adjuncts to conventional periodontal therapy. (19) 
Phytochemicals (herbal) drugs such as Aloe vera gel, (20) Emblica officinalis gel, (21) a 
bark of Mimusops elengi (Bakul) and a bark of Vachellia nilotica (Acacia arabica), (22) and 
Cymbopogon citratus (lemongrass) essential oil gel were investigated in several  
studies. (23,24,25,26)  
Cymbopogon citratus (lemongrass) is native plant to Asia, Africa, and the Americas, 
but it is widely planted in moderate and equatorial regions of the world. (24) Generally, 
lemongrass is recognized as safe (GRAS) in the US. According to Federal Regulations 
Section in 1982, lemon grass plant extract/essential oil is safe for human consumption. 
Pharmacological study on lemon grass, two months daily administered orally in male and 
female rat, in dose up to 20 times larger than the estimated corresponding human dosage. 
Results showed no inducing to any effect which could be taken as evidence of toxicity. (25) 
Another study on the assessment of eventual toxic, hypnotic and anxiolytic effects of lemon 
grass herbal tea on human. It had taken for two weeks of daily oral administration. The tea has 
created no changes in urea, serum glucose, creatinine, alkaline phosphates, cholesterol, 
indirect bilirubin, lipid total bilirubin, total protein and albumin, but there was slight raise of 
amylase and direct bilirubin in some of the volunteers but without any clinical  
manifestation. (26) It has been used in ancient India as a therapy to inflammation, food 
flavouring agent, for pharmaceutical application, and as ornamental plant. (27,28) It is used as a 
popular medicine for throat infections, catarrh, coughs, bronchitis, asthma and sinusitis 
associated with respiratory system. (29,30) In addition, it can be used for headache, leprosy, and 
malaria. (31,32,33,34) Moreover, it used for arthritis and inflammatory disorders. (35,36,37) 
Cymbopogon citratusessential oil can be added to ointments, creams and in massages 
to treat topical inflammation because of the citral, main component of Cymbopogoncitratus, 

4 
 
has shown a high inhibitory effect to inflammatory mediators. (38) Accordingly, it was used as 
an adjunctive mouth wash in nonsurgical treatment of periodontitis. (39) 
 
Many researchers stated that lemongrass oil at concentration 2% or less than 2% 
when applied subgingivally with scaling and root planning shows more probing depth 
reduction and inhibits the growth of several kinds of microorganisms including periodontal 
pathogens, especially the reference strains Actinomyces naeslundii and Porphyromonas 
gingivalis. (18,23,40,41) Semisolid formulations consisting of mucoadhesive polymers such as 
hydroxypropyl cellulose, polyvinylpyrrolidone, polycarbophil, and carbopol have been added 
to increase the contact time of agent to the tissue, in the periodontal pocket. (41,42) In a 
longitudinal study in 2018, the effectiveness of lemongrass oil gel was more than 
chlorhexidine gel. (18) 
To assess the response to periodontal therapy, there are several molecular biomarkers 
in plaque, saliva, and gingival crevicular fluid (GCF). (7,43) Gingival crevicular fluid is located 
between the bacterial biofilm and host tissues. This location is the most suitable place to 
monitor inflammatory changes. (44) Matrix metalloproteinases (MMPs), are a group of 
enzymes responsible for the degeneration of most extracellular matrix proteins and basement 
membrane (BM) components. They are assessed by collecting gingival tissue extracts, 
gingival crevicular fluid and saliva. (45,46) 
There are twenty four genetically separate types of matrix metalloproteinases which 
are divided into subgroups, such as collagenases (MMP-1, -8, and -13), gelatinases (MMP-2 
and -9), membrane-type MMPs (MMP-14,-15,-16, -17,-24, and -25), and others. Collagenases 
show cleavage of type I collagen. The major collagenases in periodontitis are human 
collagenase-2 (MMP-8) accompanied by gelatinase B (MMP-9). (7) 
Matrix metalloproteinase-8 (MMP-8) is considered the main collagenolytic matrix 
metalloproteinase detected in gingival tissue and oral fluids and is used as a periodontal 
biomarker. The major cellular source for this marker is neutrophils (PMN), but it can be 
released 
from 
gingival 
sulcular 
epithelial 
cells, 
fibroblasts, 
endothelial 
cells, 
monocyte/macrophages and plasma cells. (7) Matrix metalloproteinase-8 (MMP-8) has been 
related to the diagnosis, severity of inflammation, progression, and treatment follow-up of 
periodontitis. (47) 
 
The null hypothesis of this research is that there will be no difference in GCF matrix 
metalloproteinase-8 (MMP-8) levels in moderate periodontitis patients treated by the non-
surgical treatment and Cymbopogon citratus (lemongrass) gel in comparison to those with 
non-surgical treatment only. 
 
 
 

5 
 
AIM OF THE WORK 
The present work aims to evaluate gingival crevicular fluid levels of matrix 
metalloproteinase-8 after intrapocket application of Cymbopogon citratus gel adjunctive to 
non-surgical treatment in patients with moderate periodontitis. 
 
 
 

6 
 
Statistical analysis of results 
(Figure-1) 
PLAN OF THE STUDY 
I. Materials 
i. 
Study design: 
 
This study will be a randomized controlled design following CONSORT  
guidelines. (48) Assess and compare the effect of adjunctive intra-pocket application of 
Cymbopogon citratus (lemon-grass) gel to placebo gel on the matrix metalloproteinas-8 
(MMP-8) in the gingival crevicular fluid in the non-surgical management of moderate 
periodontitis. (Figure-1) 
 
 
 
 
Randomized controlled clinical trial sample selection n=40 
Baseline Examination probing depth (PD), clinical attachment loss (CAL) and collect 
gingival crevicular fluid from gingival sulcus by paper point to assess MMP-8 
Group II (control group)  
n=20 
Scaling and root planing +intra-pocket 
application of placebo gel 
Group I (study group) 
n=20 
Scaling and root planing + intra-pocket 
application of 2% Cymbopogon citratus 
(lemon-grass) gel 
Collecting gingival 
crevicular fluid for 
MMP-8 assessment by 
ELISA 
Probing depth 
Week after non-surgical 
management 
Twelve weeks after non-surgical 
management 
Clinical assessment 
Attachment loss 

7 
 
ii. 
Study sample and setting 
A total number of forty moderate periodontitis patients will be included in the study. They 
will be recruited from the outpatient clinic of the Department of Oral Medicine, 
Periodontology, Diagnosis and Oral Radiology Faculty of Dentistry, Alexandria University. 
Sample size calculation 
A sample size of twenty patients per group (number of groups = 2) (total sample size = 
40 patients) is the sufficient required sample as statistically significant with 80% power. (41,49) 
The sample size was calculated using G Power version 3.1.9.2. (50) 
Inclusion criteria  
(1)  Patients of both sexes having moderate periodontitis (stage II), CAL 3-4mm. (4) 
(2) Patients’ age between 25 and 45 years old. 
(3) Systemically healthy patients. 
(4) No history of periodontal therapy (surgical and non-surgical) or taken any antibiotic 
therapy for the past six months. 
Exclusion criteria  
(1) History of smoking.  
(2) Patients having previous adverse reaction to the products (or similar products) used in 
this study.  
(3) Grade C category that has rapid rate of progression. (4) 
(4) Pregnant and lactating women.  
 
      iii.     Materials 
a. Cymbopogon citratus (lemongrass) gel 
Preparation of gel will be at the Department of Pharmacology; Alexandria University. 2% 
Lemongrass essential oil gel will be prepared by a method like other anti-inflammatory gel 
formulations. For this preparation, certain quantity of carbopol * 934 will be soaked in water 
for a period of two hours. Carbopol will be then neutralized with triethanolamine (TEA)† by 
stirring. In a pre-weighted amount of propylene glycol and ethanol, 2% lemongrass essential 
oil will be dissolved. The solvent blend will be transferred to carbopol container and will be 
agitated for an additional twenty minutes. Then, the dispersion will be allowed to hydrate and 
swell for sixty minutes. pH will be adjusted with 98% triethanolamine (TEA) until it will 
reach (6.8-7). During pH adjustment, the mixture will be stirred gently with a spatula until a 
homogeneous gel will be formed. (18) 
 
 
__________________________________________________________________________         
*Kind gift from Alexandria company for pharmaceuticals and chemicals industry.  
†Kind gift from Alexandria company for pharmaceuticals and chemicals industry. 

8 
 
              b.   Placebo gel: (Carbopol®-Based pH-Sensitive Gels) 
 
Preparation of gel will be in the Department of Pharmacology; Alexandria University. 
For this preparation, certain quantity of carbopol 934 will be soaked in water for a period of 
two hours. Carbopol will be then neutralized with triethanolamine (TEA) by stirring. Then, 
the dispersion will be allowed to hydrate and swell for sixty minutes. pH will be adjusted with 
98% triethanolamine (TEA) until it will reach (6.8-7). During pH adjustment, the mixture will 
be stirred gently with a spatula until a homogeneous gel will be formed). (51) 
 
c.    Human Matrix metalloproteinase 8/Neutrophil collagenase (MMP-8) 
ELISA Kit * 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
________________________________________________ 
*Bioneovan Co., Ltd., Beijing, China. 

9 
 
II. Methods 
i. 
Randomized Controlled Clinical Study  
Patients diagnosed as having moderate periodontitis requiring non-surgical 
management after clinical and radiographic examination will be included in this study. The 
purpose and nature of the study will be explained, and an informed consent will be obtained 
from patients who agree to participate in this study prior to any procedure.  
Grouping and Randomization  
The selected forty patients will be randomly assigned to the following groups:  
Group I: twenty patients will be treated with scaling and root planning and intra-pocket 
application of 2% lemongrass oil gel.  
Group II: twenty patients will be treated with scaling and root planning and intra-pocket 
application of placebo gel. 
Allocation concealment 
Simple randomization of subjects into two groups will be carried out using a computer-
generated list of random numbers of the patients, and the two treatment modalities used in the 
study and will be performed by an expert statistician. The list will be generated using random 
allocation software. Each allocation will be represented by a code and sealed in sequentially 
numbered opaque envelopes that will be only opened at the time of intervention. (52,53) 
ii. 
Clinical examination includes: 
1. Bleeding on probing. (54) 
2. Plaque index, and probing depth (PPD) and clinical attachment loss (CAL). (55,56) 
3. Collection of gingival crevicular fluid by paper point to analyses MMP-8. (60) 
iii. 
Preliminary Phase 
           All the patients who have emergencies such as dental or periapical lesion will be 
treated, and nonrestorable teeth will be extracted. (57) 
iv. 
Non-surgical Phase  
1. Oral hygiene instructions will be given to the patients which include tooth 
brushing using proper technique twice daily. 
2. Scaling and root planing will be performed.  
3. Coronoplasty will be done when required. (58) 
4. The gels (lemongrass oil gel and placebo) will be administered by means of a 
syringe with a bent, blunt-end needle. The needle will be carefully inserted into 
the periodontal pocket and the gel will be applied in the test sites in a gentle 
probing manner, attempting to fill the full extent of the pocket. The gel will be 
applied up to the gingival margin and the excess gel will be removed with sterile 
gauze.  

10 
 
5. Periodontal dressing will be applied after delivery of the drug, and they will be 
also asked not to eat for thirty minutes. (59) 
6. After placement of the gel in situ, patients will be instructed to follow strict oral 
hygiene protocol and not chew hard or sticky foods at the gel placement sites for 
rest of the week.  
Sample collection and analysis  
1. The sites to be sampled will be isolated with cotton rolls and gently air-dried. 
2. Gingival crevicular fluid will be collected by paper point which will be gently 
placed for 30 seconds into the pocket until a minimum of resistance was felt. (60)  
3. The paper points will be inserted into tubes containing 500 μl phosphate-buffered 
saline and will be eluted at 4°C overnight, then will be centrifuged at 400 g for 4 
minutes. (44,60) 
4. The samples will be frozen immediately to the temperature of –20°C and stored 
until the analysis. (60) 
 
v. 
Follow up 
Clinical evaluation 
a. Patients’ oral hygiene status will be reassessed after first week, four weeks, and 
twelve weeks post-non-surgical follow up. (18, 23) 
b. Collection of gingival crevicular fluid by paper point to analyses MMP-8 after first 
week and twelve weeks. (61) 
c. Probing depth (PBD) and clinical attachment loss (CAL) after twelve weeks. (18, 23)  
vi. 
Outcomes 
 
               The primary endpoint with respect to efficacy in moderate periodontitis 
will be achieving CAL gain 3.20 ± 0.75 mm and PBD reduction 2.40 ± 0.73 mm 
baseline to 12 weeks as measured by Michigan 'o' probe after twelve weeks. (18, 23) 
The GCF concentration of MMP-8 will be decreased after therapy but reduced 
even more dramatically (approximately 50%) following a 3-month period of 
maintenance. (61) 
 
 
 
 
 
 

11 
 
ROLE OF SUPERVISORS 
 
 
1. Prof. Dr. Ahmed M Hommos. 
He will supervise managing patients and collecting samples. 
He will also help in interpreting results and revising the thesis. 
 
2. Dr. Souzy Kamal Anwar. 
She will supervise managing patients and collecting samples. 
She will also help in interpreting results and revising the thesis. 
 
3. Dr. Riham Mohamed El-Moslemany. 
She will supervise preparing the medicine drugs (2% Cymbopogon citratus oil gel and 
placebo gel). 
She will also help in interpreting results and revising the thesis. 
 
4. Dr. Neveen Lewis Mikhael. 
She will supervise analyzing the matrix metalloproteinases-8 gingival crevicular fluid 
levels. 
She will also help in interpreting results and revising the thesis. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

12 
 
ETHICAL CONSIDERATIONS 
 
The purpose and nature of the study will be explained to the patients and an informed 
consent will be obtained from patients who agree to participate in this study prior to any 
procedure of the study. (Appendix -1) Treatment will be in accordance with the principles of 
the modified Helsinki code for human clinical studies (2013). (62) 
 
The clinical study will be conducted following the ethical guidelines for conduct of 
research on human subjects, by the Faculty of Dentistry, Alexandria University (IRB 
NO:00010556 - IORG 0008839). 
 
 
 
 
 
 
 

13 
 
DATA MANAGEMENT AND STATISTICAL ANALYSIS 
 
The data will be processed and analysed using Statistical Package for Social Sciences 
program SPSS (20.0) software*. The study will include descriptive and analytical data. A P-
value of less than 0.05 will be considered statistically significant. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
___________________ 
Armonk, NY: IBM Corp, USA.  

14 
 
DURATION OF THE STUDY 
 
Estimated time: Nine Months. 
Study activities/ 
Months 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
Materials collection 
and preparation 
✓ 
 
 
 
 
 
 
 
 
 
 
 
Patient selection  
 
✓ 
 
 
 
 
 
 
 
 
 
 
Clinical study 
 
 
✓ 
✓ 
✓ 
 
 
 
 
 
 
 
Data management 
and statistical 
analysis 
 
 
 
 
 
✓ 
 
 
 
 
 
 
Writing thesis 
 
 
 
 
 
 
✓ 
✓ 
 
 
 
 
Thesis submission 
 
 
 
 
 
 
 
 
✓ 
 
 
 

15 
 
BUDGET 
 
Item 
Cost in LE 
Lemongrass essential oil and other ingredients 
1000 
Miscellaneous instruments and materials 
6,000 
Human Matrix metalloproteinase 8/ Neutrophil 
collagenase (MMP-8)ELISA Kit 
7,500 
Statistical Analysis  
700 
Thesis printing and Binding  
5000 
Publication  
2000 
Total 
22,200 EL 
 
 
 
 
 
 
 
 
 
 
 

16 
 
PROBLEMS ANTICIPATED 
Availability of indicated patients who have moderate periodontitis with satisfactory 
oral hygiene and are not medically compromised. Cooperation of the patients participating in 
the study. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

17 
 
PUBLICATION POLICY 
 
This study will be sent for either national or international Journal for publication, 
in the following order: 
 
- 
Nadein Abd Elnasser El. Sharif. 
- 
Prof. Dr. Ahmed M Hommos. 
- 
Dr. Souzy Kamal Anwar. 
- 
Dr. Riham Mohamed El-Moslemany. 
- 
Dr. Neveen Lewis Mikhael. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

18 
 
 
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APPENDIX 2