Assessment and Augmentation of Lip Appearance in Specific Study Populations
Phase: PHASE4
Conditions: Lip Aging, Lip Augmentation, Lip
Sponsor: Dieter Manstein, MD

 
 
1
 
 
 
Assessment and Augmentation of Lip Appearance in Specific Study 
Populations 
 
 
 
 
 
 
 
 
 
 
 
 
NCT04839692  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
December 29, 2021  
 
 
 
 
 

 
 
2
Assessment and Augmentation of Lip Appearance in Specific Study 
Populations 
 
 
 
PRINCIPAL INVESTIGATORS: 
Dieter Manstein, MD, PhD 
Cutaneous Biology Research Center (CBRC) 
Massachusetts General Hospital Research Institute 
149 13th Street, Fl.3, Charlestown MA 02129 
Telephone: (617) 726-4893 
Fax: (617) 726-4453 
e-mail: dmanstein@mgh.harvard.edu 
 
 
CO- PRINCIPAL INVESTIGATOR: 
                                              Neera Nathan, MD, MSHS 
Cutaneous Biology Research Center (CBRC) 
Massachusetts General Hospital Research Institute 
149 13th Street, Fl.3, Charlestown MA 02129 
Telephone: (240) 285-1119 
Fax: (617) 726-4453 
e-mail: nnathan2@mgh.harvard.edu 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
VERSION DATE 
December 29,2021 
 

 
 
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TABLE OF CONTENTS 
 
Page 
I. OBJECTIVE   
 
3 
 
II. BACKGROUND  
3 
 
III. AIMS  
 
 
 
4 
 
IV.  SUBJECT SELECTION 
 
a.  
Inclusion Criteria 
4 
 
b.  
Exclusion Criteria 
4 
 
c.  
Source and recruitment of subjects 
5 
 
V.  SUBJECT ENROLLMENT 
 
a.  
Method of enrollment 
6 
 
b.  
Informed consent form (ICF) 
6 
 
VI. STUDY PROCEDURES 
6 
a. Study visits and procedures 
 
 
 
 
 
 
 
 
 
 
    6 
b. Drugs to be used                                                              
 
 
 
 
 
   9 
c. Devices to be used 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
        9 
d. Procedures/Interventions 
 
 
 
 
 
 
 
 
 
 
 
 
      10 
e. Data to be collected and when data is collected 
 
 
 
      11  
f. Remuneration   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 11 
 
VII. BIOSTATISTICAL ANALYSIS 
11 
 
 
VIII. RISKS AND DISCOMFORTS 
14 
 
 
IX. POTENTIAL BENEFITS 
 
a.   
Potential benefits to subjects 
15 
 
b.   
Potential benefits to society….. 
15 
X. MONITORING AND QUALITY ASSURANCE 
 
a.   
Independent monitoring of source data 
15 
 
b.   
Safety monitoring  
15 
 
c.   
Outcome monitoring 
16 
 
d.   
Adverse Event reporting….. 
16 
XII. Data Management 
      a.       Data collection 
17 
      b.       Record retention 
17 
XIII. IRB Review and Approval 
17 
XIV. References 
18 
 
Appendix A (Study Schema) 
19 

 
 
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I. 
OBJECTIVES 
 
The objectives of this study are: 
• To determine the effects of aging on the lips and perioral area by comparing 
quantitative changes in lip; and 
• To validate the role of optical coherence tomography (OCT) in investigating lip 
microcirculation; and 
• To evaluate any volume, color and texture change of the lips pre and post 
treatment 
• To investigate pain levels from lip filler treatment   
 
To achieve the objectives, the study aims to obtain data from two specific study 
populations, including: 
1) Aged females, otherwise healthy (≥ 60 years); 
2) Young females, otherwise healthy (21-30 years) 
 
 
II. 
BACKGROUND 
 
Skin aging is a complex biological process mainly influenced by two independent 
mechanisms, intrinsic and extrinsic skin aging [1]. Intrinsic skin aging is determined 
primarily by inherent genetic factors, hormonal status, cellular metabolism and function 
[2]. It represents degenerative aging processes seen in other organ systems as a 
consequence of the passage of time. Extrinsic skin aging results from various external 
influences including solar UV radiation, health and lifestyle factors, such as tobacco 
smoking, environmental pollution, and other systemic conditions, such as diabetes and 
renal insufficiency [3-5]. 
In skin with chronic exposure to UV radiation, termed photoaging, alterations in skin 
structure and function depend primarily on the degree of sun exposure and baseline skin 
pigment. UV radiation induces collagen breakdown. Diminution of collagen framework 
that supports vascular plexus within the skin leads to broken vessels [8]. The Langerhans 
cell population representing immune system of the skin is found to be reduced after 
chronic exposure to UV radiation contributing to impaired immune response to antigen 
and skin cancer cells [6,7].  
These age-related changes caused by chronic sun exposure have not been well-
documented in the lips [10]. The majority of literature on the aging lip has focused on 
volume loss. Hyaluronic-acid based fillers have been used primarily for volume 
augmentation, but their effect on lip color and texture has seldom been defined [9,12].  
We propose to study aging of the cutaneous changes of human skin anatomy in the lips 
and perioral area to better understand and quantify the aging process in this, including 
the effect of age on lip redness.  

 
 
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We also aim to test the effects of a common lip augmentation procedure, hyaluronic-acid 
based fillers, on the appearance of aging lips, in addition to the tolerability of this 
procedure. We hypothesize that dermal lip fillers may decrease the redness of the lips.  
Besides, we want to evaluate oxytocin level changes and relate it to subjects’ satisfaction 
with procedure. 
The study will take place at MGH’s Clinical Unit for Research Trials & Outcomes in Skin 
(CURTIS) at 50 Staniford Street, Suite 240 Boston, MA  02114 or Translational Clinical 
Research Center (TCRC) on the 12th floor of the White Building. 
 
III. 
Specific AIMS 
 
To: 
• Compare the changes in skin anatomy due to intrinsic aging 
• Correlate clinical assessment with OCT imaging;  
• Compare changes in lip color, texture and volume before and after filler with non-
invasive imaging, including digital photography, OCT and three-dimensional 
imaging 
• To assess the tolerability of lip filler by evaluating pain level from lip filler 
treatment at initial insertion and overall procedural pain   
• To evaluate how oxytocin levels change during and after an aesthetic procedure. 
• To assess lip color change after the procedure. 
• To evaluate patient satisfaction related to the procedure. 
 
 
 
 
IV. 
SUBJECT SELECTION 
 
Subjects will be screened to determine if they meet all the eligibility criteria specified 
below. We will recruit and screen up to 50 subjects with the goal of 40 subjects enroll and 
complete the study ( 20 per study arm).  
 
      a. Inclusion Criteria  
1. Subjects must be able and willing to provide written informed consent and comply 
with the requirements of the study protocol; 
2. In good general health, based on answers provided during the screening visit; 
3. Subjects must be able to read and understand English; 
4. Female subjects; 
5. Aged  21-30 years and Post menopausal women;  
6. Skin Types (Fitzpatrick Skin Phototypes I-VI); 

 
 
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* Smoking history is self-report. 
7. Have very thin to moderately thick lips (Lip Fullness Grading Scale [LFGS] scores 
0–2).  
 
      b. Exclusion Criteria 
1. Participation in another investigational drug or device clinical trial in the past 
30 days;   
2. Are pregnant or lactating; 
3. Intake of hormone replacement therapy (HRT) in the past 6 months; 
4. Have a history of drug or alcohol abuse or have reported habitual alcohol 
intake greater than 2 standard drinks per day [e.g., 2 beers, 2 glasses of 
wine, or 2 mixed drinks]; 
5. Presence of eczema, psoriasis, or any other skin disease on the lips or 
perioral skin; 
6. Have birth marks, tattoos, scars, or any other disfiguration of the skin in the 
skin area of interest; 
7. Use of any anti-aging skin care products containing retinoic acid, retinol, or 
other retinoids (e.g. tazarotene, adapalene) or estrogen on the skin area of 
interest in the past 6 months; 
8. Use of any prescription topical medication, such as corticosteroids or 
hydroquinone on the skin area of interest in the past 6 months; 
9. Have a history of photodynamic therapy treatment or any skin rejuvenation 
procedure on the skin area of interest; 
10. History of blood-clotting abnormality; 
11. History of keloid formation or hypertrophic scarring;  
12. Clinically significant abnormal findings or conditions which might, in the 
opinion of the Investigator, interfere with study evaluations or pose a risk to 
subject safety during the study; 
13. Exhibits any clinical conditions or takes any medication which in the opinion 
of the investigator may interfere with the study or pose a risk to subject 
safety during the study; 
14. Is not able to follow study protocol; 
15. Have permanent lip implant, and lip enhancement or laser therapy 
performed within the preceding 12 months; 
16.  Known history of allergy or sensitivity to glycerol, Tegaderm, lidocaine, 
hyaluronic acid dermal fillers or materials with gram-positive bacterial 
proteins. 
 
 
      c.  Source and Recruitment of Subjects 

 
 
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The study will be posted on the Partner’s clinical research web page (Rally.partners.org) 
to reach an economically and socially diverse population. Potential subjects who 
respond to the Rally posting will be informed of the purpose and procedures involved in 
the study, the location of the study and the remuneration involved for completing study 
visits by study staff as a part of the telephone prescreening.  They will then be asked 
several screening questions regarding whether they are in the age range, sex, skin 
color, and lip assessment required for the study. If the potential subject is not within one 
of the screening criteria described, then study staff will explain that they are not qualified 
for the study and thank them for their time.  If the screening criteria are met and the 
subject is interested in the study, study staff will take contact name, email and phone 
number and schedule the screening visit (phone/video call) with one of the licensed 
physician investigators listed on the protocol to go over the consent process.  
 
V.  SUBJECT ENROLLMENT 
 
a. Method of Enrollment 
All subjects will be subject to a telephone prescreening by study staff before 
scheduling the initial screening visit. All subjects who electronically sign an 
informed consent form (ICF) and are screened will be documented on a screening 
log. All subjects who qualify at the screening visit and who are enrolled in the 
study will be documented on the enrollment log. A note will be made in the source 
documentation verifying that the subject has willingly signed the ICF prior to 
participation in any study procedures. 
 
b. Informed Consent Form (ICF) 
 
One of the licensed physician investigators listed on the protocol will consent and 
inform the potential study subject of all aspects of the study and answer their 
questions. Sub-investigators may assist in the consent process. If the subject 
agrees to be a study subject, they will document consent in electronic form by 
signing the online informed consent form via Adobe eSign. Subjects who need 
more time to decide whether they would like to participate will have access to the 
electronic consent form and reschedule their informed consent call with the 
licensed physician investigator if they are interested in participating in the study. 
 
The investigator is responsible for using a consent form that has been approved by 
the IRB/Mass General Brigham’s HRC and is the most current version. If a new 
version of the consent form is approved by the IRB/MGB’s HRC while a subject is 
still participating in the study and there are major changes to any component of the 
consent form, then the subject will be informed of the changes and, if the subject 
agrees to continue study participation, they should sign the updated form. 
 
 
 
Electronic informed consent will be obtained by trained study staff prior to 
performance of any protocol-specific procedures. 
 
 

 
 
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VI.  STUDY PROCEDURES 
 
a. Study visits and procedures 
 
Screening visit (phone/video call) 
 
During the screening visit, the investigator will discuss with each subject the nature of the 
study, its requirements and its restrictions.  
The following will be performed to determine eligibility: 
• Review of inclusion/exclusion criteria 
• Review of medical history, medications and demographics  
• Lip examination  
 
Subjects will be consented remotely by a licensed physician investigator, either over 
video conference or telephone, depending on subject’s preference. Adobe eSign will be 
used to perform the consent signing, and a copy of the e-signed consent form will be 
kept in an encrypted file within the lab documents for this study. Subjects who qualify for 
the study will be scheduled for visit 1. Subjects who fulfill all inclusion and exclusion 
criteria may enroll and begin the Visit 1 procedures that same day. 
 
 
Visit 1 (1.5 hours) 
 
The following assessments will be performed during Visit 1: 
• Review of inclusion/exclusion criteria 
• Review of medical history, medications and demographics  
 
The following procedures and evaluations will be performed: 
• OCT imaging for microvasculature 
• Three-dimensional imaging using the Cherry Imaging system (before and after 
the procedure)  
• Digital photography of the lips and perioral area 
Filler treatment of the lips with Restylane Kysse (if the subject has completely inverted 
lips the lip filler procedure will be optional and only imaging will be collected in visit 1)  
• Lip volume assessment with Lip Fullness Grading Scale 
• Subjects will be instructed how to report a pain score for first needle insertion and 
overall procedural pain.  A visual 10 point pain scale will be used. 

 
 
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• Intravenous access will be placed to facilitate four blood draws to analyze serum 
oxytocin levels, all of which will occur on Visit 1 as follow: after photography and 
OCT, and before the filler procedure starts, immediately after the filler procedure, 
after the “mirror reveal” (when we show the lips to the subject using a mirror”) 
minutes and 30- 35  minutes  after the filler procedure has been concluded. 
• The questionnaires: “FACE-QTM – satisfaction with outcome” and other questions 
will be applied right after the procedure to evaluate  subject’s satisfaction and 
about lip color and shape changes. 
• Documentation of Adverse Events 
• Video recording (15-30 seconds-video) from subjects’ face and lips after the lip 
filler procedure. The video recording is optional.  
• Upon completion of all treatments, the subject will be provided with aftercare 
instructions, remuneration and parking voucher if needed 
 
 
 
 
Follow-up Call ( 3-7 days after Visit 1, 30 minutes) 
Study staff will call the subject 3-7 days after Visit 1 to check-in and inquire about any 
post-procedure pain or any side effects. If there are any negative side effects from 
treatment, they will be evaluated and treated in accordance with standard medical 
procedures.  
 
 
Visit 2 (2 weeks after Visit 1, 1 hour) 
 
Subjects will be asked to return 2 weeks after Visit 1. Visit 2 is expected to last up to 1 
hour. The following procedures and evaluations will be performed: 
• Three-dimensional imaging using the Cherry Imaging system 
• OCT imaging for microvasculature 
• Digital photography of the lips and perioral area 
• Lip volume assessment with Lip Fullness Grading Scale (LFGS) 
• Documentation of adverse events 

 
 
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• The questionnaires: “FACE-QTM – satisfaction with outcome” and other questions 
will be applied right after the procedure to evaluate  subject’s satisfaction and 
about lip color and shape changes. 
 
 
b. Drugs to be used 
 
Restylane® Kysse (Galderma Laboratories, L.P, Fort Worth, TX) 
 
Restylane® Kysse injectable gel is a clear, colorless gel. It is a hyaluronic acid based 
dermal filler with local anesthetic lidocaine, which is FDA approved for lip augmentation 
and wrinkles around lips. The content of the syringe is sterile and each syringe comes 
with 1mL of injectable gel. In the event that a syringe contains material that is not clear 
or is cloudy, that syringe will not be used. Refrigeration of the product is not required, 
store at 25ºC/77ºF [9].  
 
c. Devices to be used 
 
Optical coherence tomography (OCT) Device (Thorlabs, Inc., Newton, NJ) 
 
OCT imaging is performed using the commercially available, non-modified spectral-
domain OCT scanner Telesto II (Thorlabs Inc.). It operates with a similar light exposure 
as FDA-approved systems such as the Vivosight Scanner (Michelson Diagnostics, Kent, 
UK) and Skintell (Afga HealthCare, Greenville, SC). The Telesto II consists of a 
broadband SLD light-source Class 1M (Class I FDA) at a center wavelength of 1300 nm 
and a maximum power of <50 mW. While the system achieves A-Scan rates of up to 76 
kHz, note that the irradiation of the SLD is not pulsed.  
 
Using a lens with an optical lateral resolution of 13 μm (LSM03, Thorlabs Inc. – spot size 
> 20 μm), we acquire images with an oversampling factor of 2× leading to voxel sizes of 
6.5 × 6.5 × 3.5 μm3 and a field-of-view (FOV) of 6 × 6 × 3.5 mm3 (length × width × depth).  
 
Although the system has not yet been approved by the FDA and is available in the United 
States for investigational use, its classification aligns with ANSI 136.1 class 1M and is 
thought to be incapable of causing harm.  
 
This system was preferred over other commercial devices because of its detailed control 
of imaging parameters and the accessibility of the raw spectral data. This enables 
reproducible imaging as well as to obtain the imaging data needed to evaluate skin 
vascular characteristics.   
 
Cherry Imaging Photography (Cherry Imaging Ltd, Yokneam, Israel) 
The Cherry Imaging system is a 3-dimensional photographic imaging system designed 
to accurately measure aesthetic treatments to deliver objective patient data before and 
after aesthetic treatments. The handheld camera captures thousands of three-
dimensional images of the face and/or body from multiple field views and angles that 

 
 
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are analyzed to provide 100-micron accuracy level data of the body and/or face for real-
time evaluation of treatment results and traceability over time. The Cherry Imaging 
device will be used to take images of the face before the filler treatment takes place and 
during the last visit. No special lighting is needed for the imaging device, so the images 
will be taken and processed directly in the room where the filler procedure will take 
place. The Cherry imaging software has multiple capabilities, including the ability to 
calculate topographic changes, volume changes, and color changes in the skin.  
 
 
d. Procedures/Interventions 
 
Optical coherence tomography (OCT) imaging 
 
The OCT imaging system will be cleansed with 70% isopropyl alcohol wipe in patient’s 
presence prior to perform any imaging procedure. The subject’s lips and surrounding skin 
at the intended OCT imaging sites will be cleansed with 70% isopropyl alcohol prep and 
allowed air dry thoroughly. First, OCT imaging will be performed in non-contact setting 
focused on the skin surface to image its profile. It takes about 1-2 minutes to properly 
position the patient and align OCT device per site and about 30 seconds per scan. 
Secondly, a z-spacer (OCT-Imm03, ThorLabs, Inc., Newton, NJ) will be attached to the 
OCT probe and glycerol will be utilized for optical coupling (immersion) between the OCT 
spacer and the skin. In this setting, a sequence of three OCT angiographic images will be 
acquired. For imaging of the lips, a waterproof film polymer (i.e., Tegaderm) dressing will 
be used to minimize direct contact of the OCT spacer with the skin. Glycerol will be 
applied to the lips with a Q-tip. The middle portion of the bottom part of the lip will be 
imaged on each patient. One image will be taken. Then the OCT imaging device will be 
pressed onto the skin until the skin is blanched.  When the skin is blanched, a second 
OCT image will be acquired.  After the second image is acquired, the pressure will be 
held with the skin blanched for a further 60 seconds and then released.  A third image will 
be taken about 10 seconds after pressure is released. The pressure required for 
blanching the skin will be measured and recorded.  A brief test may be done near the 
imaging site prior to imaging in order to determine the blanching threshold pressure.  The 
acquisition and saving time per image will be less than 1 minute. Taking these three 
images will allow assessment of the vascular density and vessel thickness as well as the 
vessel refilling characteristics after occlusion.  If study staff operating the OCT imaging 
device decide the images are of too low quality or contain artifacts that would affect 
analysis, this procedure may be repeated on an area of the skin within close proximity to 
the original imaging location but at least 2-3 cm away from the original location. After 
finishing the OCT imaging procedures, the z-spacer will be detached and cleansed with 
70% isopropyl alcohol wipe then allowed to air dry thoroughly prior to next use. 
 
Cherry Imaging Photography 
The Cherry Imaging system will be used to take images of the lips. The Cherry Imaging 
photography can take place at the MGH’s Clinical Unit for Research Trials & Outcomes 
in Skin (CURTIS) or Translational Clinical Research Center (TCRC) since there is no 
special background or lighting condition needed to use the Cherry Imaging system. 

 
 
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Digital Photography 
A DSLR camera will be used to take images of the lips and perioral area before the 
dermal filler procedure takes place during visit 1 and then at the second study visit. 
Subjects will be asked to stand with limited facial expression so that consistent, 
photographic images can be obtained. 
 
Video Recording  
Study’s encrypted Iphone 12 (no chip inside, used only for the study) will be used by 
study staff (investigator or research coordinators) to take a video recording from the 
subjects’ face/ reaction during the “mirror reveal”. From 15 to 30 seconds of video 
recording will be taken from subject’s face after a mirror is used to show them their “new 
lips”. This will happen after the dermal filler procedure takes place during visit 1. This 
video will be optional and subjects will express if they allow this video recording or not  
in the consent form. Subject’s reaction after the “ mirror reveal” will be correlated to 
Oxytocin levels.  
 
Restylane Kysse Lip Filler Treatment 
After the OCT imaging, Cherry Imaging and digital photography, Restylane Kysse lip filler 
will be injected into the submucosal layer of the lips and subcutaneous layer of the perioral 
area. This hyaluronic acid filler formulation contains local anesthetic lidocaine so no 
topical anesthetic will be applied. The subject will be positioned in a seated position and 
the treatment area will be cleaned prior to injection. One syringe with 1mL will be injected 
to lips and perioral area in accordance with standard aesthetic lip filler practices. We aim 
to inject the full amount of one syringe, but upon the investigators discretion we may inject 
+/- 0.25mL as needed. The area will then be gently massaged with Vaseline to ensure 
the dermal filler gel is distributed evenly [9]. The subject will experience sharp pain and 
mild pressure from initial injection, which will subside with subsequent insertions as the 
lidocaine numbing takes effect. Standard of care for aesthetic lip filler treatment will be 
followed. 
 
Blood Draw  
 
 
 Four blood draws to analyze serum oxytocin levels, all of which will occur on Visit 1 as 
follow: 
- 
After photography and OCT, and before the filler procedure starts, an 
intravenous access will be placed and approximately 3 cc of blood will be drawn 
into EDTA tubes containing Aprotinin supplied by the Brigham Research Core 
lab. 
- 
Immediately after the filler procedure, approximately 3 cc of blood will be drawn 
into EDTA tubes containing Aprotinin supplied by the Brigham Research Core 
lab. 
- 
after the after the “mirror reveal” (when we show the lips to the subject using a 
mirror approximately 3 cc of blood will be drawn into EDTA tubes containing 
Aprotinin supplied by the Brigham Research Core lab. 

 
 
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- 
30-35  minutes after the filler procedure has been  concluded, a final approximate 
of 3 cc of blood will be drawn into EDTA tubes containing Aprotinin supplied by 
the Brigham Research Core lab. 
Each sample will be allowed to clot for 30-60 minutes at room temperature to prepare 
serum, then centrifuged at 1600 x g for 15 minutes at 4°C by study staff, and the serum 
will be transferred to plastic tubes.  
The samples will be placed on dry ice and hand-delivered by the research staff to the 
Brigham Research Core lab or stored at -80 degrees freezer located at the MGH Navy 
Yard in the Manstein lab when immediate delivery is not possible.  
The Brigham Research Core lab will process the ELISA assay to evaluate oxytocin 
levels. 
 
 
e. Data to be collected and when data is collected 
 
Study data will be collected during each study visit. Study data to be collected includes 
OCT imaging (various images as described above, visit 1 and 2), digital photography 
and Cherry Imaging of the lips and perioral area (visit 1 and 2), visual assessment of 
treated area (visit 2), video recording (optional) from subject’s reaction after the mirror 
reveal, Lip volume assessment with Lip Fullness Grading Scale (screening visit, visit 1 
and 2), pain assessment of initial injection and overall procedure (visit 1), blood samples 
( visit 1)  and satisfaction questionnaire answers (visit 1 and 2).  Medical history and 
medications will be documented at visit 1. There are no other biological data or health 
information to be collected in this study. 
 
Information about side effects in the treatment area or any adverse events will be 
collected during the follow-up call and visits. 
 
f. Remuneration 
 
Subjects will be paid $50 for completing all imaging activities and filler treatment during 
visit 1, and $100 for completing visit 2. The total remuneration amount is $150 upon 
completing the study. We will provide a parking voucher at MGH main campus for each 
visit upon request.  
 
We will be using an approved, outside vendor (Forte Research) to make these 
payments via a reloadable credit card-based system, called Forte Payments. This 
secure system is similar to a gift card or credit card. 
  
If subjects are paid by this system, they will be given a Forte Payments Via card (which 
is just like a debit card) when subjects enroll in the study. Once the card is activated, the 
study team will add a payment after each paid visit completed by the subjects. The 
payment should be available within one (1) business day. Research staff will not know 
where subjects spend the money. You may use the card anywhere Visa cards are 
accepted, such as at a grocery store. 
  

 
 
14
We will need to collects subjects’ Social Security number in order to make these 
payments, and it will be shared securely with the company that runs the card-based 
system. Payments like this are considered taxable income. If subjects receive more than 
$600, the payment will be reported to the IRS as income by the hospital. 
 
VII. BIOSTATISTICAL ANALYSIS 
 
a. Study Variables  
 
Aesthetic Procedure Improvement Analysis  
The Global Aesthetic Improvement Scale (GAIS) is a five-point scale used to assess 
effectiveness of aesthetic procedures comparing pre-treatment and post treatment. This 
scale ranges from 1= exceptional improvement, 2 = very improved patient, 3 = improved 
patient, 4 = unaltered patient, 5 = worsened patient [11, 13]. 
 
 
 
 
 
 
 
Lip Volume Analysis  
Lip Fullness Grading Scale (LFGS) will be used to assess the lip volume change from 
pretreatment compared to post treatment. This photonumeric rating scale ranges from 0 
= very thin; 1 = thin; 2 = moderately thick; 3 = thick; and 4 = full. Improvement of lip 
volume is defined as ≥1 increase at post-treatment compared to pretreatment [11, 14].  
 

 
 
15
 
 
Pain Scoring Analysis 
The Wong-Baker FACES Pain Rating Scale will be used to score subject pain from the 
first insertion of dermal filler and the overall pain from the procedure.   
 
 
b. Study Analyses  
 
OCT analysis 
 
OCT imaging is performed using the commercially available spectral-domain OCT 
scanner TELESTO II (Thorlabs Inc., Newton, New Jersey). It operates at a central 

 
 
16
wavelength of 1,300 nm with a maximum output power of less than 10 mW. Using a lens 
with an optical lateral resolution of 13 μm (LSM03, Thorlabs Inc.), we acquire images with 
an oversampling factor of 2× leading to voxel sizes of 6.5 × 6.5 × 3.5 μm3 and a field-of-
view (FOV) of 6 × 6 × 3.5 mm3 (length × width × depth), acquired at an A-Scan rate of 76 
kHz. 
 
The imaging data is further processed and analyzed by custom made software tools 
implemented in MATLAB 2016b (MathWorks Inc). All OCT image data files will be labeled 
with the study subject number assigned to each subject upon consent. No identifying 
information will be used in labeling the image data files and the files will be stored on 
Partners OneDrive. 
 
With angiographic image post processing, the capillary layer, typically in between 70 − 
400 μm depth in skin, is displayed and converted to 2-dimensional images as maximum 
intensity projections. These 2-dimensional images are further segmented by an 
automatic, customized software and quantitative metrics such as vessel area, vessel 
length, vascular network complexity (fractal dimension) and vessel curvature are 
assessed. These characteristics of the capillary network are further used for inter- and 
intra-cohort investigations. 
 
Cherry Imaging Photography Analysis  
The Cherry Imaging analysis software contains distinct algorithms that process the 
generated three-dimensional images to calculate volume in cubic centimeters, precise 
color using saturation and hue, roughness detecting local variations in height/depth, and 
a ruler function that measures the distance between two distinct points. These will all be 
employed to analyze photos of lips,  lip redness, roughness and volume.  
 
 
DSLR Camera Photography Analysis 
Standard photographs will be blindly reviewed by a physician and assessed using the Lip 
Fullness Grading Scale and Global Aesthetic Improvement Scale.  
 
Video Recording  
Videos will be blindly reviewed by a physician and assessed using the following grading 
scale (this grading scale is based on the model of answers that are given to subjects in 
the questionnaire):  
 
Very 
satisfied  
Somewhat 
satisfied 
Somewhat  
dissatisfied 
Very 
dissatisfied  
  
All video recording data files will be labeled with the study subject number assigned to 
each subject upon consent. No identifying information will be used in labeling the video 
data files and the files will be stored on Partners Dropbox. The videos will be stored at 
Partners Dropbox until 7 years after the end of this study when all the copies will be 
permanently deleted from this drive.  
 

 
 
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Only study staff will have access to the videos (for storage purposes- like research 
coordinators or for assessment - investigators) unless the subject authorizes, by 
signalizing it in the consent form, the use of their video recordings for the following 
purposes:  
 
• medical or pharmaceutical publications, or presentations in conferences   
• educating health care professionals (such as physicians and nurses) through 
electronic materials (iPads), brochures and the healthcare professional 
website   
• patient-focused websites and patient-handouts / industry ads    
If the subject agrees (in the consent form) with the use of her video recording for the 
purposes cited above the video will be shared with pharmaceutical companies ( ex. 
Galderma- Lip filler producer) or will be shared with other doctors during lectures in 
medical conferences.  
 
Oxytoxin  
The Brigham Research Core lab will process the ELISA assay to evaluate oxytocin levels 
per the Oxytocin EIA kit which is a colorimetric competitive enzyme immunoassay kit, with 
very low reactivity with vasopressin. 
 
 
c. Statistical Methods 
The study is intended to gather empirical data about the volume, color, texture and 
microcirculation change in the lips and perioral area; and pain assessment of treatment. 
Descriptive statistics will be collected to present quantitative descriptions of changes in 
lips and perioral area from pretreatment compared to post-treatment.   
 
 
 
VIII. RISKS AND DISCOMFORTS 
 
a. Complications of surgical and non-surgical procedures 
 
Subjects might encounter an experience of discomfort during OCT imaging procedure 
due to the application of pressure to occlude blood vessels in the skin.  There will be no 
harm caused by vessel occlusion and study staff will explain the procedure to subjects 
prior to taking the OCT images. There are no known adverse effects of the three-
dimensional imaging system (Cherry) other than psychological distress from seeing a 
high-resolution photo that may highlight imperfections. This is no different than routine 
professional photography.  
 
The IV access can cause pain during its placement and any infection, thrombophlebitis 
or other adverse outcome will be treated according to standard medical care and 
recorded in the Adverse Event Log. 
 
 

 
 
18
b. Drug side effects and toxicities 
 
Possible side effects of Restylane Kysse dermal filler include redness (very common)*, 
pain/tenderness (very common), swelling (very common), lumps/bumps (very common), 
bruising (very common), itching (very common), or discoloration (very common). 
Potential risks included allergic reaction (rare but serious), infection (rare but serious), 
scarring (rare but serious), skin ulceration (rare but serious), ischemia (rare but serious), 
infarction (rare but serious) or change in skin tone (very common) [9].  
 
Dermal fillers in the lips and perioral area are considered to be low risk but rare 
instances of localized skin necrosis are possible. In this case, if there are symptoms of 
occlusions, intense pain, or purple patches upon injection, we will have hyaluronidase 
(FDA approved for vascular occlusion necrosis ) available in clinic.  
 
A subject could have an allergic reaction to glycerol, Tegaderm or dermal filler (rare but 
serious). In this case, the allergic reaction would be recorded in the Adverse Events Log 
and would be monitored and treated by study staff in accordance with standard medical 
procedures.  
 
*Please refer to the National Cancer Institute frequency categories listed below  
for reporting of possible side effects:  
a. Very Common (more than 1 out of 10 people) 
b. Common (between 1 and 10 out of 100 people) 
c. Uncommon (between 1 and 10 out of 1,000 people) 
d. Rare but Serious (less than 1 out of 1,000 people) 
 
c. Device complications/malfunctions 
 
Telesto II OCT imaging device (Thorlabs, Newton, NJ) 
 
The Telesto II system is controlled and triggered by an external PC workstation. In case 
the OCT acquisition software (ThorImage, Thorlabs Inc.) or the operating system 
malfunctions, the operator will reboot the system. 
 
Cherry Imaging, Version 19.08.3322 (Yokneam, Israel) 
 
The Cherry Imaging system is a photographic system that captures patient images using 
standard lighting conditions with no special positioning required. The images are analyzed 
using Cherry Imaging algorithms to create volumetric images of the photographed area. 
We expect no device complications or malfunctions due to the Cherry Imaging system. 
 
d. Psychosocial (non-medical) risks 
 
There is a potential risk of loss of privacy.  We will protect privacy by labeling samples, 
information, and data files only with a study subject number code, and keeping the key to 
the code in a password protected database.  

 
 
19
 
 
 
 
IX.  POTENTIAL BENEFITS 
  
a.  Potential Benefits to Subjects 
Subjects who participate in this study will receive Restylane Kysse dermal filler treatment 
of the lips, which may improve the appearance of their lips. 
 
b.  Potential Benefits to Society 
Information gathered from this study may improve the understanding of the pathological 
alteration of intrinsic aging to lip microvasculature and changes in color, roughness and/or 
appearance of lips before and after a lip filler procedure. This study may also aid in 
understanding pain tolerance with the lip filler procedure to improve pain management 
techniques in the future as needed. 
 
X.  MONITORING AND QUALITY ASSURANCE 
 
a.  Independent monitoring of source data 
Experienced study personnel (study monitor) who are not assigned to complete 
procedures of this study will conduct monitoring after the first subject is enrolled and 
periodically thereafter. The monitor will be responsible for confirming the completion and 
correctness of the study procedures as well as record collection and keeping. 
    
b.  Safety monitoring 
Prior to enrollment, subjects will be screened for eligibility; at which time a complete 
medical history, including a baseline assessment of the subject’s lips will be done. 
Evaluations will be ongoing throughout the study to detect adverse events and changes 
in existing medical conditions. 
At any time after enrollment, a subject may be discontinued. Reasons for discontinuation 
of a subject from the study will include, but may not be limited to, the following: 
1. Subject is found to be intolerant to a required study procedure at any time point. 
2. Subject is noncompliant with protocol restrictions and requirements. 
3. Subject develops an intercurrent illness that would, in the judgment of the 
investigator, affect assessments of clinical status to a significant degree.  
4. Subject becomes pregnant while participating in the study. 
5. Subject enrolls in another investigational study. 
6. Subject requests to withdraw from the study. 
7. The study staff decides to suspend or terminate the study. 
 
 
If possible, a final set of assessments will be performed on all subjects who end their 
participation prior to study completion. 

 
 
20
 
c. Outcomes monitoring 
 
The study will be conducted in accordance with applicable regulations and Good Clinical 
Practice Guidelines. Keeping files locked with access limited to study staff will ensure 
confidentiality and data integrity. 
 
d. Adverse Event Reporting 
 
Definition 
Adverse Event (AE) is any untoward medical occurrence in a subject that does not 
necessarily have a causal relationship with this treatment. An AE can therefore be any 
unfavorable and unintended sign, symptom, or disease temporally associated with the 
use of an investigational product, whether or not related to the investigational product. 
 
 
Serious Adverse Event (SAE) is any untoward medical occurrence that: 
• Results in death 
• Is life-threatening 
• Requires inpatient hospitalization or prolongation of existing 
hospitalization 
• Results in persistent or significant disability or incapacity 
• Is a congenital anomaly/birth defect 
• Is another medically important condition 
 
Reporting and Documenting Adverse Events 
All untoward medical occurrences that occur after the subject signs a consent form will 
be documented as an AE. The Investigator will ensure that all events that occur during 
the study period are recorded. All AEs will be followed until resolution or until, in the 
Investigator’s judgment, they are chronic and stable. If an emergency situation should 
occur, appropriate medical measures should be taken to stabilize the subject. 
 
Documentation of AEs includes: date and time of onset and resolution of AE, intensity, 
frequency, seriousness, related interventions and outcome. The Investigator will also 
evaluate the probability of a causal relationship of the AE to the study treatment as being: 
“definite, probable, possible, unlikely, or unrelated.” Intensity of adverse events will be 
graded as mild, moderate, or severe according to the following criteria: 
 
• Mild: symptoms that are easily tolerated and transient in nature with 
minimal or no impairment of normal activity 
• Moderate: symptoms that are poorly tolerated, are sustained, and 
interfere with normal activity 
• Severe: symptoms that are incapacitating and render the subject 
unable to work or participate in many or all usual activities 

 
 
21
 
All SAEs will be reported to the IRB according to the IRB’s requirements.  
Adverse events will be reported to the PHRC as described in the PHRC policy on 
Unanticipated Problems Involving Risks to Subjects or Others Including Adverse 
Events, which can be found on the Partner’s Research Navigator website. 
 
XII.  Data Management 
a. 
Data collection 
All study data will be collected during both study visits.  Study data to be collected includes 
clinical photography, Cherry images, OCT images and qualitative patient responses to 
treatment and laboratory results. Pain scores will be recorded on the case report form for 
visit 1, LFGS for screening visit, visit 1 and 2. Subjects’ answers for satisfaction evaluation 
questions will be also recorded.  All physical documentation and IRB correspondence will 
be stored in study binders maintained in restricted lab space only accessible by study 
staff and members of the Manstein Lab. Digital data including photographs will be 
deidentified and stored on MGB computers. Any identifiable information in photographs 
such as eyes and tattoos will be blacked out and deidentified. All study documents 
containing PHI will be password encrypted, including the enrollment log and identification 
key. An encrypted external hard drive will be used to store the data as a backup. 
 
b. 
Record retention 
The Investigator or designees will retain all study records in accordance with the test 
facility's SOP's.  
 
XIII.  IRB Review and Approval 
The study will not begin prior to the receipt of written confirmation of approval by the IRB 
and any relevant regulatory authority.  It is the responsibility of the Investigator to obtain 
the IRB approval (per the U.S. Code of Federal Regulations, Title 21, Part 56 and 
applicable ICH guidelines) for the protocol, amendments, informed consent, subject 
information sheet, questionnaires, and advertising materials used to recruit study 
subjects, if appropriate.   
 
 
XIV. REFERENCES  
 
1. Uitto J. Understanding premature skin aging. N Engl J Med. 1997 Nov 
13;337(20):1463-5.  
2. Zouboulis ChC. Intrinsic skin aging. A critical appraisal of the role of hormones. 
Hautarzt. 2003 Sep;54(9):825-32.  

 
 
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3. Leung WC, Harvey I. Is skin ageing in the elderly caused by sun exposure or 
smoking? Br J Dermatol. 2002 Dec;147(6):1187-91.  
4. Argyropoulos AJ, Robichaud P, Balimunkwe RM, Fisher GJ, Hammerberg C, 
Yan Y, Quan T. Alterations of Dermal Connective Tissue Collagen in Diabetes: 
Molecular Basis of Aged-Appearing Skin. PLoS One. 2016 Apr 22;11(4).  
5. Van Waateringe RP, Mook-Kanamori MJ, Slagter SN, van der Klauw MM, van 
Vliet-Ostaptchouk JV, Graaff R, Lutgers HL, Suhre K, El-Din Selim MM, Mook-
Kanamori DO, Wolffenbuttel BHR. The association between various smoking 
behaviors, cotinine biomarkers and skin autofluorescence, a marker for 
advanced glycation end product accumulation. PLoS One. 2017 Jun 20;12(6) 
6. Gilchrest BA, Soter NA, Hawk JL, Barr RM, Black AK, Hensby CN, Mallet AI, 
Greaves MW, Parrish JA. Histologic changes associated with ultraviolet A--
induced erythema in normal human skin. J Am Acad Dermatol. 1983 
Aug;9(2):213-9. 
7. Rogers GS1, Gilchrest BA. The senile epidermis: environmental influences on 
skin ageing and cutaneous carcinogenesis. Br J Dermatol. 1990 Apr;122 Suppl 
35:55-60. 
8. Bentov I, Reed MJ. The effect of aging on the cutaneous microvasculature. 
Microvasc Res. 2015 Jul;100:25-31. 
9. Restylane Kysse Patient Brochure. Accessed December 28, 2020. 
https://www.galderma.com/us/sites/g/files/jcdfhc341/files/2020-
04/Restylane_Kysse-
Patient_Brochure.pdf?_ga=2.59052845.1698235642.1604334264-
853585669.1604334264&_gac=1.157524424.1604378565.CjwKCAiA-
f78BRBbEiwATKRRBPyH8_64L0BLjGEHNISljih9I6tz-
fSQohCtDMdwKAe7hz57dYQAihoCieQQAvD_BwE 
10. Tamura E, Ishikawa J, Sugata K, Tsukahara K, Yasumori H, Yamamoto T. Age-
related differences in the functional properties of lips compared with skin. Skin 
Research and Technology. 2018;24(3):472-478. 
doi:https://doi.org/10.1111/srt.12456 
11. Hilton S, Sattler G, Berg A-K, Samuelson U, Wong C. Randomized, Evaluator-
Blinded Study Comparing Safety and Effect of Two Hyaluronic Acid Gels for Lips 
Enhancement. Dermatol Surg. 2018;44(2):261-269. 
doi:10.1097/DSS.0000000000001282 
12. Stojanovič L, Majdič N. Effectiveness and safety of hyaluronic acid fillers used to 
enhance overall lip fullness: A systematic review of clinical studies. Journal of 
Cosmetic Dermatology. 2019;18(2):436-443. 
doi:https://doi.org/10.1111/jocd.12861 
13. Savoia A, Accardo C, Vannini F, Di Pasquale B, Baldi A. Outcomes in Thread Lift 
for Facial Rejuvenation: a Study Performed with Happy LiftTM Revitalizing. 
Dermatol Ther (Heidelb). 2014;4(1):103-114. doi:10.1007/s13555-014-0041-6 

 
 
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14. Carruthers A, Carruthers J, Hardas B, et al. A Validated Lip Fullness Grading 
Scale. Dermatologic Surgery. Published online November 2008. 
15. Safety/Efficacy Study of Restylane® in Lip Augmentation - Study Results. Study 
Results - ClinicalTrials.gov. 
https://clinicaltrials.gov/ct2/show/results/NCT00935272?view=results. Published 
January 30, 2012. Accessed April 19, 2021.  
16. King M, Walker L, Convery C, Davies E. Management of a Vascular Occlusion 
Associated with Cosmetic Injections: JCAD: The Journal of Clinical and 
Aesthetic Dermatology. Journal of Clinical and Aesthetic Dermatology (JCAD). 
https://jcadonline.com/management-vascular-
occlusion/#:~:text=The%20incidence%20of%20necrosis%20related,of%201%20
in%20100%2C000%20cases. Published January 1, 2020. Accessed April 19, 
2021.  
 
 
Appendix A: Study Schema 
 
 
 
 
Screening 
Visit 1 
Follow-up Call 
Visit 2 
Study Visit 
<30 days 
Day 0 
 
3-7  days after Visit 
1 
2 weeks after Visit 1 
Informed Consent 
X 
 
 
 
Inclusion/Exclusion 
X 
X 
 
 
History/Demographics 
X 
 
 
 
Review side effects/check-in   
 
X 
 
Investigator Assessments 
 
X 
X 
X 
Digital Photography  
 
X 
 
X 
3D Cherry Imaging 
 
X  
 
X 
OCT Imaging 
 
X 
 
X 
Filler Treatment 
 
X 
 
 
Questionnaires 
 
X 
 
X 
LFGS 
X 
X 
 
X 
Con Meds/Adverse Events 
 
X 
X 
X 
Blood Draw 
 
X