Microneedling With Platelet Rich Plasma Versus Microneedling Alone For Gingival Depigmentation: A Randomized Controlled Clinical Trial
Phase: NA
Conditions: Gingival Pigmentation
Sponsor: Cairo University

1 
 
 
 
Microneedling with Platelet Rich Plasma Versus Microneedling Alone 
For Gingival Depigmentation: A Randomized Controlled Clinical 
Trial. 
 
 
Document Date: 
2025-03-15 
 
 
 
 
Protocol Submitted for partial fulfillment of the master requirements in Faculty of Dentistry 
Cairo University 
 
 
By 
Abdelrahman Samir Abdullah 
B.SC. In Oral and Dental Medicine Cairo University 
(2019) 
 
 
 
 
 
 
 
Supervisors’ signature 
Head of department’s signature 
1-Prof.Dr Enji Ahmed Mahmoud 
Prof.Dr Manal Mohamed Hosny 
2-Dr. Manar Taher Elzanaty 
Date 

2 
 
 
Protocol checklist 
Section and topic 
Item no. 
Checked item 
Reported 
on page No. 
Reviewer’s 
check 
 
 
I. Administrative information 
1 
Title 
4 
2 
Protocol registration 
4 
3 
Protocol version 
4 
4 
Funding 
4 
5 
Roles and responsibilities 
4 
 
II. Introduction 
 
 
A) Background and Rationale 
6 a 
Research question 
5 
Statement of the problem 
5 
Rationale for carrying out the trial 
5-6 
Review of literature 
6-7-8 
6 b 
Choice of comparators 
8 
 
B) Objectives 
7 
Aim of the study 
8 
Hypothesis 
8 
Primary and secondary objectives 
8 
C) Trial design 
8 
Trial design 
8 
 
III. Methods 
 
 
A) Participants, interventions 
& outcomes 
9 
Study setting 
9 
10 
Eligibility criteria 
9 
11 
Interventions 
9 
12 
Outcomes 
10-11 
13 
Participant timeline 
12 
14 
Sample size 
13 
15 
Recruitment 
13 
 
 
 
B) Assignment of interventions 
16 
Allocation 
13 
16 a 
Random sequence generation 
(Randomization) 
13 
16 b 
Allocation concealment 
mechanism 
13 
16 c 
Implementation 
13 
17 
Blinding (masking) 
14 
C) Data collection, 
management, and analysis 
18 
Data collection methods 
14 
19 
Data management 
14 
20 
Statistical methods 
14 
 
D) Monitoring 
21 
Data monitoring 
14 
22 
Harms 
15 
23 
Auditing 
15 
 
IV. Ethics and dissemination 
24 
Research ethics approval 
15 

3 
 
 
25 
Protocol amendments 
15 
26 
Informed Consent 
15 
27 
Confidentiality 
15 
28 
Declaration of interests 
15 
29 
Access to data 
15 
30 
Ancillary and post-trial care 
16 
31 
Dissemination policy 
16 
 
V. Appendices 
32 
Informed consent materials 
16 
 
33 
Biological specimens 
16 
 
VI. References 
19-20-21 
 
Evidence based committee (Reviewers) 
Name 
Signature 
Date 
1. 
2. 
 
Research plan committee 
Name 
Signature 
Date 
1. 
2. 

4 
 
I. Administrative information: 
1. Title: 
Microneedling with Platelet Rich Plasma Versus Microneedling Alone for Gingival 
Depigmentation: A Randomized Controlled Clinical Trial. 
2. Protocol Registration: 
Protocol will be registered on ClinicalTrials.gov. 
3. Protocol version: 
First version 
4. Funding: 
Self-funding. 
5. Roles and responsibilities: 
Main supervisor 
Prof. Dr Enji Ahmed Mahmoud 
- 
Professor of Oral Medicine and Periodontology-Cairo University. 
Role: 
- 
Initiating the idea of the clinical trial. 
- 
Randomization of cases. 
- 
Supervising the manuscript. 
- 
Revising & interpretation of collected data. 
 
• Co-supervisor 
Dr. Manar Taher Elzanaty 
- 
Lecturer of Oral Medicine and Periodontology- Cairo University. 
Role: 
- 
Supervising the manuscript. 
- 
Revising & interpretation of collected data. 
 
• Principle Investigator 
Abdelrahman Samir Abdullah 
Examination of patients. 
- 
Measuring and recording the outcomes of the study 
- 
Data collection and data management. 
- 
Writing the manuscript. 
 
Trial responsibility: 
Department of Oral Medicine and Periodontology, Faculty of Dentistry Cairo University. 
Committees: 
• Research plan committee 
• Periodontology department council 
• Evidence based dentistry committee 
• Faculty Council 

5 
 
• Ethics committee 
 
II.  Introduction: 
6. Background and rationale: 
Research question: 
Will the usage of microneedling with platelet rich plasma as a depigmentation method differ 
in comparison to microneedling alone for pigment intensity reduction? 
 
Statement of the problem: 
The skin and oral mucous membrane normally have a brown pigment called melanin, which is 
not generated from hemoglobin. The melanocytes found in the basal and supra-basal 
epithelium deposit too much melanin, which leads to gingival hyperpigmentation(Javali, 
Tapashetti et al. 2011). Although physiologic gingival hyperpigmentation is not a pathologic 
condition, it is considered one of the main esthetic problems in dentistry. It was found that the 
attached gingiva is the most frequently pigmented intraoral tissues followed by the papillary 
gingiva and the alveolar mucosa(Javali, Tapashetti et al. 2011). Several treatments, such as 
gingivectomy, mucosal removal with a scalpel, abrasion techniques, free gingival grafts, 
chemical approaches involving caustic chemicals, electrosurgery, cryotherapy, and newly 
developed lasers, have been used to achieve pigmentation-free gingiva(Grover, Dadlani et al. 
2014).The scalpel technique, which involves eliminating the gingival epithelium and a layer 
of connective tissue .Subsequently, this will allow the stripped connective tissue to heal by 
secondary intention which is more painful(Gupta, Kumar et al. 2014). Bur abrasion is a 
straightforward, inexpensive, and efficient technique for gingival depigmentation but it causes 
uncomfortable bleeding both during and after the surgery and has the potential to damage 
bone surface when done with rotary tools(Kathariya and Pradeep 2011).Cryosurgery can lead 
to tissue inflammation and damage Moreover, electrosurgery that has some drawbacks that 
include heat accumulation, tissue destruction, and it is a technique sensitive method(Yadav, 
Kumar et al. 2022). 
 
Rationale for conducting the research: 
The term Platelet-Rich Plasma (PRP) is a generic term used to describe a plasma suspension 
obtained from whole blood, prepared so as to contain platelet concentrations higher than those 
normally found in circulating blood. Platelets contain granules consisting of various 
substances, which are released upon activation of the platelets(Smyth, McEver et al. 2009). 
Among the main substances that are released from these granules is growth factors 
(GF)(Mazzucco, Balbo et al. 2009). These factors have a well-known role in the process of 
tissue repair. Thus, the concentration of these substances in injured tissues could be beneficial 
to provide more agility to the regeneration processes(Sommeling, Heyneman et al. 2013). 
Some of the bioactive substances present in the α-granules include platelet-derived growth 
factor (PDGF), transforming growth factor (TGF)-β1, 2, epidermal growth factor (EGF), and 
mitogenic growth factors such as platelet-derived angiogenesis factor and fibrinogen(Eppley, 
Pietrzak et al. 2006). only TGF-β1 and EGF have been investigated about their relation with 
melanogenesis. 

6 
 
Kim et al. investigated the effects of TGF-β1 on melanogenesis by using a spontaneously 
immortalized mouse melanocyte cell line, and asserted that TGF-β1 significantly inhibits 
melanin synthesis in a concentration dependent manner. They declared that TGF-β1 decreases 
melanogenesis via delayed extracellular signal-regulated kinase activation(Kim, Park et al. 
2004).While Yun et al. studied the effect of EGF on melanogenesis by using mouse-derived 
immortalized melanocytes from a laser-treated keratinocyte-conditioned culture media. They 
proposed that treatment with EGF lowers melanin production in melanocytes by inhibiting 
prostaglandin-E2 (PGE2) expression and tyrosinase enzyme activity. They offered that 
treatment with EGF can be used in cosmetics to whiten the skin and for the prevention of post 
inflammatory hyperpigmentation(Yun, Bang et al. 2013). 
PRP is a useful treatment for periorbital hyperpigmentation and further comparative studies of 
other treatment modalities are highly recommended (Salah Hashim Al-Shami 2014). 
 
Microneedling helps to reverse hyperpigmentation and normalize cellular activity and 
signaling between keratinocytes and melanocytes in more than one way including the 
improvement of keratinocyte function so the transfer of melanosomes between keratinocytes 
and melanocytes becomes more regulated(Cardinali, Bolasco et al. 2008). 
the MN technique for gingival depigmentation is a successful procedure and most participants 
were satisfied with the results produced from the MN technique. It successfully reduces 
gingival hyperpigmentation and might be regarded as an alternative minimally invasive 
treatment option. additional investigations may be performed to estimate the effectiveness of 
MN combined with local drug delivery for treating gingival hyperpigmentation(Morsy 2022). 
 
 
Review of literature: 
.Normal appearance of the gingiva is pink to light red, with stippling “orange peel texture.” , 
normal color depends on melanogenesis and distribution of melanin pigment, keratinization 
depth of epithelization, and vascularity of the gingiva(Feller, Masilana et al. 2014). Gingival 
color plays a crucial part in facial aesthetics and contributes to the entire appearance of an 
ideal smile(Altayeb, Hamadah et al. 2021). 
 
Melanin hyperpigmentation has been linked to a number of etiological and pathological 
causes, including smoking, heavy metal toxicity, heredity, endocrine disorders, UV exposure, 
inflammation, benign and malignant lesions, and intentional cultural tattooing. In order to 
determine whether the reason is physiological or pathological, a thorough medical history of 
the patient is essential(Holmstrup, Plemons et al. 2018). 
 
With the introduction of gingival depigmentation as a periodontal surgical procedure, the 
hyperpigmented gingival tissues are removed utilizing a variety of surgical treatment 
methods, such as scalpel excision, bur abrasion, free gingival grafts, ADMA, lasers, 
cryosurgery, electro surgery(Farid, Shinwari et al. 2017). Pigmentation of melanin can also be 
treated by numerous means that contain chemical methods using alcohol, ascorbic acid, 
phenol(Shimada, Tai et al. 2009) 

7 
 
The most popular surgical approaches are associated by anxiety, bleeding, a significant 
postoperative wound, and recurrence (Patil, Joshi et al. 2015). Those methods could also have 
negative consequences include chemical burns, delayed healing, extreme pain and suffering, 
bone loss, and trouble controlling the depth of de-epithelization(Padayatty and Levine 2016). 
 
Needle-based delivery systems are designed to deliver medications by rupturing the skin or 
mucosal barrier, making the medication easily accessible to the targeted tissues (Batra, Dawar 
et al. 2020).The collagen induction therapy method known as microneedling approach 
involves puncturing the skin repeatedly. Microneedling has been widely used in dermatology 
recently since it is a method that is efficient, straightforward, affordable, well-tolerated, and 
advantageous from both a cosmetic and therapeutic standpoint(Singh and Yadav 2016). 
 
Instead of cutting through the cells, the used microneedles create micro-conduits that increase 
the skin's permeability and blood flow into the epidermis. This procedure makes it easier for 
topical drugs to cross the stratum corneum layer. Additionally, growth factors that support the 
regeneration of collagen and elastin are created(Iriarte, Awosika et al. 2017). 
 
The microneedling principle is suggested to be used in the treatment of gingival 
hyperpigmentation, compared to the currently employed techniques such as scalpel surgery, 
laser ablation, bur abrasion, and electrocautery which frequently result in complications, 
microneedling could be a promising minimally invasive, straightforward, painless, and cost- 
effective treatment modality for gingival depigmentation(Batra, Dawar et al. 2020). 
Subsequent randomized controlled clinical trials are required to validate the findings for the 
case report that mentioned the use of Dermapen microneedling with vitamin C topical paste as 
a novel technique resulting in complete gingival depigmentation that lasted for six months 
(Mostafa and Alotaibi 2022). 
 
MN has several advantages, including the minimally invasive procedure for patients who are 
afraid of surgery, minimal bleeding, and painless procedure for patients who did not require 
analgesics after the procedure. Moreover, the gingiva heals faster than other surgical 
procedures because the epithelium remains relatively intact(Morsy 2022). 
 
The combination of microneedling with PRP therapy emerges as a promising and effective 
solution for pigmentation reduction. Overall, microneedling with PRP holds great promise as 
a non-invasive and effective approach for revitalizing the skin and reducing pigmentation, 
offering individuals an opportunity to achieve a more even and rejuvenated 
complexion(Shahraki, Khazaei et al.). This therapy presents a valuable option for individuals 
seeking to address pigmentation issues while simultaneously improving overall skin health. 
Its ability to promote collagen production, minimize downtime, and offer long-lasting results 
makes it a compelling choice in the field of aesthetic dermatology .Further research and 
clinical trials are still needed to explore the optimal treatment protocols, long-term efficacy, 
and potential combination therapies for enhancing the outcomes of microneedling with PRP 
in pigmentation reduction (Mohamed, Attwa et al. 2023) (Wu, Zhang et al. 2022). 

8 
 
In order to treat Periorbital Hyperpigmentation, patients receive intradermal injection of PRP 
on their face for three sessions at a 3-week interval. To prepare PRP, 10mL of the patient's 
venous blood sample was taken manually with a sterile 10mL syringe and then centrifuged 
with 1800G/6min, buffy coat and plasma were then extracted and re-centrifuged with 
2500G/15min. The injection of 1mL of PRP was immediately initiated intradermally into the 
infraorbital area on one side of the face with a 1 cc insulin syringe 30G, (0.1mL in each point 
of injection) (Iranmanesh, Rastaghi et al.). 
 
Explanation for choice of comparators: 
Microneedling has been widely used in dermatology due to its ability to create micro-conduits 
that increase the skin's permeability and blood flow into the epidermis (Iriarte, Awosika et al. 
2017). 
MN technique for gingival depigmentation is a successful procedure and most participants 
were satisfied with the results produced from the MN technique. additional investigations may 
be performed to estimate the effectiveness of MN combined with local drug delivery for 
treating gingival hyperpigmentation(Morsy 2022). 
 
 
 
7. Objectives: 
The aim of this study is to evaluate efficacy of the microneedling technique with Platelet 
Rich Plasma application as a depigmentation method in comparison to microneedling Alone 
for pigment intensity reduction. 
 
Hypothesis: 
Null hypothesis: there is no significant difference between both groups 
 
Primary objective: 
Oral Pigmentation intensity score (DOPI) which will be measured at baseline, 3 months and 
6 months after gingival depigmentation procedures. 
 
Secondary objectives 
Pain 
Gingival thickness 
Wound healing 
Patient Satisfaction 
 
8. Trial design: 
The trial will be a parallel-group, two-arm, superiority, randomized controlled clinical trial 
with allocation ratio of 1:1. 
 
 
III.  Methods 

9 
 
A) Participants, interventions & outcomes 
9. Study settings: 
Study is to be conducted in the Oral Medicine and Periodontology department, Faculty of 
Dentistry – Cairo University, Egypt. 
Patients are to be selected from the outpatient clinic of the department of Oral Medicine 
and Periodontology-Cairo University. 
 
10. Eligibility criteria: 
Inclusion criteria: (Mostafa and Alotaibi 2022) 
• Patients exhibiting melanin hyperpigmentation in the anterior region of the upper or 
lower gingiva. 
• Patients should be free from any systemic diseases according to modified Cornell 
Medical index (Abramson 1966). 
• Non- smokers. 
 
Exclusion criteria:(Mostafa and Alotaibi 2022) 
• Fully edentulous patients. 
• Patients with endocrine disorders causing hyperpigmentation or drug induced gingival 
pigmentation. (Sreeja, Ramakrishnan et al. 2015) 
• Pregnant or lactating females. 
 
 
11. Interventions 
Pre-operative preparation phase includes ultrasonic scaling and oral hygiene instructions for 
all patients. Eligible patients will be randomly allocated into two groups. 
Clinical photographs: Clinical photographs will be taken at baseline, 1 and 6 months 
postoperatively. 
 
• Intervention Group (Group A): Microneedling will be done with application of 
Platelet Rich Plasma for depigmentation. 
• Control Group (Group B): microneedling will be done alone for depigmentation. 
 
In both groups: 
Local anesthesia (Articaine with 1:100,000 Epinephrine) will be administered by infiltration 
technique. 
• A Dermapen device will be used to microneedle the gingival tissue (model M8) with 
24 microneedles arranged in rows, which is adjusted according to the gingival 
thickness at the 6th mode speed of 700 cycles/min(Mostafa and Alotaibi 2022). 
• It will be laid perpendicular to gingival surface and MN will be carried out in 
horizontal, vertical and diagonal directions about four to five times for the whole 
hyperpigmented gingiva until mild microbleeding and mild erythema was clearly 

10 
 
visible. 
• Once bleeding points are observed on all areas of the pigmented gingiva, it will be 
irrigated using saline solution and dried using sterile gauze. 
 
Intervention group: 
• 
Patients receive injection of PRP in their Gingiva. To prepare PRP, 10mL of the 
patient's venous blood sample was taken manually with a sterile 10mL syringe and 
then centrifuged with 1800G/6min, buffy coat and plasma were then extracted and re- 
centrifuged with 2500G/15min. The injection of 1mL of PRP was immediately 
initiated with a 1 cc insulin syringe 30G, (0.1mL in each point of injection) 
(Iranmanesh, Rastaghi et al.). 
 
 
Postoperative Care: 
Patients were instructed to refrain from mechanical oral hygiene practices in regard to 
the target region for the day of the operation following each visit to minimize mechanical 
damage to the treated areas. If discomfort or itching was reported on the first day, analgesics 
were prescribed (Brufen 400 mg tablets). Restricted spicy, acidic, and coloring food. (Yussif, 
Abdel Rahman et al. 2019, Morsy 2022). 
 
Assessment: 
Photos will be taken preoperative, during, postoperative and at follow-up 
appointments until 6 months. Clinical assessment will be done at baseline, after 3 month and 
6 months including the following parameters: 
 
1- Dummett-Gupta Oral Pigmentation Index (DOPI): the degree of gingival pigmentation 
is scored as 0, pink tissue [no clinical pigmentation]; 1 = mild light brown tissue [mild clinical 
pigmentation]; 2, medium brown or mixed brown and pink tissue [moderate clinical 
pigmentation]; or 3, deep brown/blue–black tissue [heavy clinical pigmentation] (Dummett 
and Gupta 1964). 
 
2- Gingival thickness: the gingival tissue thickness was measured starting at 1.5 mm apical 
to the marginal gingiva using an endodontic spreader number 25 with a rubber stopper that 
was inserted perpendicularly into the soft tissues until a hard surface was felt and the rubber 
stopper rest in close contact with the gingival surface. The gingival biotype will be considered 
thin if the measurement is ≤1.0 mm and thick if it measured >1.0 mm (Couso-Queiruga, 
Tattan et al. 2021). 
 
3- Pain: using Visual analogue score questionnaire. (Becker, Heeschen et al. 2018). The rate 
of analgesic consumption will be recorded 7 days after the intervention. (Palheiros, Cunha et 
al. 2021) 

11 
 
4- Patient Satisfaction: questionnaire to score the degree of patient satisfaction with the 
cosmetic results of the procedure. It will be documented depending on the patient’s opinion, 
measured on a five-point scale 0: Very dissatisfied 1: Dissatisfied 2: Neither satisfied nor 
dissatisfied 3: Satisfied 4: Very satisfied (Becker, Heeschen et al. 2018). 
4- Wound healing: Healing Index by Landry et al.: is used to describe the extent of healing 
after periodontal surgery (Landry RG, Turnbull RS, Howley T. 1988), Score 1: Very Poor, 
Score 2: Poor, Score 3: Good, Score 4: Very Good, Score 5: Excellent. 
12. Outcomes: 
Recordings 
Measuring Device 
Measuring Unit 
Primary 
Outcome 
Pigmentation Score 
(Dummett and Gupta, 
1964) 
Visual 
It will be measured using 
Dummett Oral 
Pigmentation Index (DOPI 
1964) : 
Score 0: No clinical 
pigmentation (pink 
gingival) 
Score 1: Mild clinical 
pigmentation (mild light 
brown color) 
Score 2: Moderate clinical 
pigmentation (medium 
brown or mixed pink and 
brown) 
Score 3: Heavy clinical 
pigmentation (deep brown 
or bluish black). 
Secondary 
Outcomes 
Pain (Becker et al., 
2018) 
Questionnaire 
Visual Analogue Scale 
(VAS) 
Gingival thickness 
(Couso-Queiruga, 
Tattan et al. 2021) 
Standard no. 25 
endodontic finger 
spreader with a 
rubber stop 
The gingival biotype will 
be considered thin if the 
measurement is ≤1.0 mm 
and thick if it measured 
>1.0 mm. 
Wound healing 
Photographic 
clinical pictures 
Landry healing index 

12 
 
Pain (Palheiros, Cunha 
et al. 2021) 
Questionnaire 
Rate of analgesic 
consumption 
Patient 
Satisfaction(Taher 
Agha and Polenik 
2020) 
Questionnaire 
It will be documented 
depending on the patient’s 
opinion, measured on a 
five-point scale 
0: Very dissatisfied 
1: Dissatisfied 
2: Neither satisfied nor 
dissatisfied 
3: Satisfied 
4: Very satisfied 
13. Participant timeline 
After accepting to be enrolled in this study, all patients will receive proper oral hygiene 
instructions and will be motivated to stick to these instructions. 
After receiving the treatment, patients will be followed-up for 6 months. 
Time point 
T0 
T1 
T2 2 weeks 
(post 
intervention) 
T3 4 weeks 
(post 
intervention) 
T4 3 months  
(post 
intervention) 
T5 
6 months 
(post 
intervention) 
Enrollment 
X 
Eligibility 
screen 
X 
Informed 
consent 
X 
Initial 
phase 
(Oral hygiene 
measures) 
X 
Allocation 
X 
Intervention 
X 
X 
X 
 
Pigmentation 
Score 
X 
X 
X 
X 
Wound 
healing 
X 
X 
X 
Gingival 
thickness 
X 
 
X 
Pain 
X 
X 
Patient 
satisfaction 
X 
X 

13 
 
 
14. Sample size: 
We are planning a study of a continuous response variable from independent control and 
experimental subjects with (1) control per experimental subject. In a previous study the 
response within each subject group was normally distributed with standard deviation 0.444. if 
the true difference in the experimental and control means is 0.5, we will need to study 13 
experimental subjects and 13 control subjects to be able to reject the null hypothesis that the 
population means of the experimental and control groups are equal with probability (power) 
0.8. The type I error probability associated with this test of this null hypothesis is 0.05. To 
compensate for drop outs, 3 subjects will be added to each group to become 16 subjects, with 
a total of 32. Calculations were conducted using PS Power and Sample Size Calculations 
version 3.1.2. (Vanderbilt University, Nashville, Tennessee, USA). 
 
 
15. Recruitment: 
Patients will be selected from the outpatient clinic of the Oral Medicine and Periodontology 
Department, Faculty of Dentistry-Cairo University. 
Screening of patients will continue until the target sample is achieved. 
Identifying and recruiting potential subjects will be achieved through patient database 
 
 
B) Assignment of interventions 
16. Allocation: 
16a-Random sequence generation: 
Methods of generating randomization: 
Patients will be randomly assigned to either test or control group using computer generated 
randomization (www.randomizer.org) which will be performed by the supervisor. The 
patients will be allocated to either test or control group 
. 
16b. Allocation concealment mechanism: 
The two groups will be equally prepared for both procedures. Then the decision of which 
group will receive (microneedling with application of platelet rich plasma) and which will 
receive (microneedling alone) will be taken according to the randomized numbers placed in 
opaque sealed envelopes. The number will be picked by the supervisor. 
 
16c. Implementation 
- All patients who provide an informed consent for participation as well as fulfill the inclusion 
criteria will be randomized. 
- The supervisor will generate the allocation sequence as well as assign the participants to the 
test or control groups. The principle investigator will enroll the participants. 
 
17. Blinding: 

14 
 
Single blinded: 
- 
Blinding of the participants is not applicable. 
- 
Blinding of the operator is not applicable. 
- 
Outcome assessor (primary and secondary outcomes) & biostatistician will be blinded. 
 
 
C) Data collection, management, and analysis: 
18. Data collection methods 
All the date of the patients included in the study will be recorded from them or extracted from 
the patient’s file. 
 
- Plans to promote participant retention and complete follow- up 
 
Telephone numbers of all patients included in the study will be recorded as a part of the 
written consent. 
All patients will receive a phone call at the time of the pre-determined follow up dates. 
 
19. Data management: 
All data will be entered and saved electronically. 
Patient files are to be stored in numerical order and stored in secured file. Data will be 
encrypted using a password. 
All data will be maintained after completion of the study. 
 
20. Statistical methods: 
Nominal data will be reported as numbers and frequency and will be analyzed using chi- 
square test or Fisher's exact test. Numerical data will be explored for normality using 
Kolmogorov-Smirnov test and Shapiro-Wilk test. In the case of normally distributed 
numerical variables, a comparison between both groups will be done using independent t-test 
while for non-normally distributed variables, Mann Whiney U test will be utilized. For 
intragroup analysis in normally distributed data, Paired t-test will be utilized while Wilcoxon 
signed rank test will be used for non-normally distributed data. All test will be two-tailed and 
P-values less than 0.05 will be considered statistically significant. 
 
D) Data monitoring: 
21. Monitoring 
No formal monitoring committee is currently available. 
 
22. Harms 
Any temporary or permanent adverse effect will be recorded and documented. 
 
23. Audit 

15 
 
No formal auditing committee is currently available. 
 
 
IV.  Ethics and dissemination 
24. Research ethics approval 
This protocol and the template informed consent form will be reviewed by the Ethics 
Committee of Scientific Research -Faculty of Oral and Dental Medicine- Cairo University. 
 
25. Protocol amendments 
Any modifications to the protocol which may have an impact on the conduct of the study, 
potential benefit of the patient or may affect patient safety, including changes of study 
objectives, study design, sample sizes, study procedures, or significant administrative aspects 
will require a formal amendment to the protocol. Such amendment will be agreed upon by the 
Council of Oral Medicine and Periodontology Department and the Faculty of Oral and Dental 
Medicine, Cairo University. 
 
26. Informed consent 
Researcher will introduce the trial to patients and provide full explanation of its aim and 
benefits in layman language. Patients will then be able to have an informed discussion with 
the researcher. Researcher will obtain written consent from patients willing to participate in 
the trial. All consent forms will be translated into Arabic. 
 
27. Confidentiality 
All study-related information will be stored securely. All participant information will be 
stored in locked file cabinets in areas with limited access. All laboratory specimens, reports, 
data collection, process, and administrative forms will be identified by a coded ID 
[identification] number only to maintain participant confidentiality. All records that contain 
names or other personal identifiers will be stored separately from study records identified by 
code number. All local databases will be secured with password-protected access systems. 
 
28. Declaration of interest 
If there will be any conflict of interest it will be declared. 
 
29. Access to data 
The principal investigator and the supervisors will have access to the data sets. All data sets 
will be password protected. To ensure confidentiality, data dispersed to project team members 
will be blinded of any identifying participant information. 
 
30. Post-trial care 
All patients will be followed up for nine months after the clinical trial is over. 
 
31. Dissemination policy 

16 
 
- Study results will be published as partial fulfillment of the requirements for master degree in 
Periodontology. 
- Topics suggested for presentation or publication will be circulated by the authors. 
 
33. Biological specimens 
Blood sample collected from intervention group and centrifuged then injected. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
V. References 
 
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(2021). "Gingival depigmentation with diode and Er,Cr:YSGG laser: 
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• Batra, P., A. Dawar and S. Miglani (2020). "Microneedles and 
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• Becker, V., V. Heeschen, K. Schuh, H. Schieb and T. Ziemssen (2018). 
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1756285617748845. 

17 
 
• Cardinali, G., G. Bolasco, N. Aspite, G. Lucania, L. V. Lotti, M. R. Torrisi and 
M. Picardo (2008). "Melanosome transfer promoted by keratinocyte 
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Dermatol 128(3): 558-567. 
• Couso-Queiruga, E., M. Tattan, U. Ahmad, C. Barwacz, O. Gonzalez-Martin 
and G. Avila-Ortiz (2021). "Assessment of gingival thickness using digital 
file superimposition versus direct clinical measurements." Clin Oral 
Investig 25(4): 2353-2361. 
• Dummett, C. O. and O. P. Gupta (1964). "ESTIMATING THE 
EPIDEMIOLOGY OF ORAL PIGMENTATION." J Natl Med Assoc 56(5): 419- 
420. 
• Eppley, B. L., W. S. Pietrzak and M. Blanton (2006). "Platelet-rich plasma: 
a review of biology and applications in plastic surgery." Plast Reconstr 
Surg 118(6): 147e-159e. 
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