Three-Dimensional Evaluation of the Depressor Anguli Oris and Depressor Labii

Inferioris for Botulinum Toxin Injections

You-Jin Choi, PhD; Young-Jun We; Hyung-Jin Lee, PhD; Kang-Woo Lee, PhD; Young-Chun
Gil, PhD; Kyung-Seok Hu, DDS, PhD; Tansatit Tanvaa, MD, PhD; and Hee-Jin Kim, DDS,
PhD

Drs Choi, H.J. Lee, and K.W. Lee are Research Assistant Professors, Department of Oral

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Biology, Division in Anatomy and Developmental Biology, Human Identification Research
Institute, BK21 PLUS Project, Yonsei University College of Dentistry, Seoul, South Korea. Mr

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We is a graduate student, Department of Oral Biology, Division in Anatomy and

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Developmental Biology, Human Identification Research Institute, BK21 PLUS Project, Yonsei
University College of Dentistry, Seoul, South Korea. Dr Gil is a Professor, Department of

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Anatomy, Chungbuk National University School of Medicine, Cheongju, South Korea. Drs Hu
and Kim are Professors, Department of Oral Biology, Division in Anatomy and Developmental

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Biology, Human Identification Research Institute, BK21 PLUS Project, Yonsei University
College of Dentistry, Seoul, South Korea. Dr Tanvaa is a Professor, Chula Soft Cadaver
Surgical Training Center and Department of Anatomy, Faculty of Medicine, Chulalongkorn
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University, Bangkok, Thailand.

Corresponding Author: Dr Hee-Jin Kim, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, South Korea,
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03722.
E-mail: hjk776@yuhs.acww
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Disclosures: The authors declared no potential conflicts of interest with respect to the research,
authorship, and publication of this article.
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Funding: This work was supported by a National Research Foundation of Korea (NRF) grant
funded by the Korean government (MSIP) (NRF-2017R1A2B4003781).
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© 2020 The Aesthetic Society. Reprints and permission: journals.permissions@oup.com. This work
is written by (a) US Government employee(s) and is in the public domain in the US.
Abstract
Background: Botulinum toxin type A (BoNT-A) injection into the inappropriate site and depth
might produce an unwanted change in facial animation because the depressor anguli oris (DAO)
and depressor labii inferioris (DLI) are partially overlapped. Therefore, a simple BoNT-A
injection guideline, based on three-dimensional (3D) facial anatomical references and
landmarks, is essentially required.

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Objectives: This study was performed to establish a novel BoNT-A injection guideline that
includes the soft tissue thickness at the lower perioral region using a 3D scanning system with

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dissections in order to provide accurate injection sites and depths for the DAO and DLI.
Methods: 3D scans of the facial skin, superficial fat, and facial muscle surface were performed

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in 45 embalmed cadavers. The thicknesses of the skin and subcutaneous layer were calculated
automatically from the superimposed images at each of the perioral region’s 5 reference points.

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Results: In every case (100%), P3 and P5 were located in the DLI and DAO areas, respectively
(45/45). Therefore, we defined P3 as the ‘DLI point’ and P5 as the ‘DAO point’. The soft-tissue
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thicknesses at the DLI and DAO points were 6.4±1.7 mm and 6.7±1.8 mm, respectively.
Conclusions: The P3 and P5 described in this study are effective guideline that only target the
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DLI and DAO. Clinicians, specifically, can easily use facial landmarks, such as the cheilion
and pupil, to appoint the DLI and DAO point without any measurement or palpation of the
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modiolus.
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Most people show facial asymmetry, of varying degrees, during animation. Various factors,
such as genetics, aging, smoking, sun damage, injury, stroke, and paralysis of the facial nerve,
can cause facial asymmetry; however, the unbalanced contraction of facial muscles is the main
driving force of asymmetry.1,2 The majority of facial muscles are located adjacent to the
modiolus; therefore, facial asymmetry can be readily observed when the perioral muscles make
contact unequally.

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The depressor anguli oris muscle (DAO) is a perioral muscle that is located on the
most superficial layer of the perioral region. It arises from the oblique line of the mandible and

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becomes narrower, finally merging with the modiolus. The depressor labii inferioris muscle
(DLI), which is partially covered by the DAO, also arises from the oblique line of the mandible

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and inserts into the lower lip, blending in with the orbicularis oris muscle. These muscles help
to depress the corner of the mouth and the lower lip, respectively. However, the bilateral

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hyperactive status of these muscles can cause a droopy appearance, and asymmetry of the
mouth can be produced as a case of unilateral muscle contraction.3 These cases can be treated
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using an injection of botulinum toxin type A (BoNT-A) to relax the muscles and reduce the
functional imbalance.4
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The most important clinical point is that a BoNT-A injection should be precisely
performed into a target muscle. However, an injection into the inappropriate site and depth
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might produce an unwanted change in facial animation because the DAO and DLI are partially
overlapped. Therefore, it is necessary to identify a detailed anatomical boundary and depth
information when treating perioral asymmetry and a drooping appearance by injecting BoNT-
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A. The best way to only inject into the target muscle is via ultrasound-guided injection, which
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enables clinicians to know where the needle tip is positioned. However, this injection protocol
is rarely performed due to the inconvenience in clinical fields with insufficient knowledge of
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the detailed facial anatomy.

A simple BoNT-A injection guideline based on three-dimensional (3D) facial
anatomical references and landmarks is essentially required. However, previous studies have
shown the thickness of the facial skin with two-dimensional analysis using ultrasound5-7 or
manual dissection.8-11 With these methods, it is possible to identify the relationship between
the anatomical structures; however, there are concerns about interobserver error in the
measurement of the soft tissue thickness.
 Therefore, this study was performed to establish a novel BoNT-A injection guideline
that includes the soft tissue thickness at the lower perioral region using a 3D scanning system
with dissections in order to provide accurate injection sites and depths for the DAO and DLI.

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METHODS
This study was conducted from March 2018 to February 2019 and approved by the institutional
review board of Yonsei University College of Dentistry (No. 2-2017-0023). All cadaveric
specimens in this study were legally donated to the Surgical Anatomy Education Center at
Yonsei University College of Medicine and Soft Cadaver Surgical Training Center at Faculty

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of Medicine, Chulalongkorn University. None of the cadavers had any damage or surgery in
the perioral region.

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The following landmarks (a, b, c, and d) and reference lines (VL1, VL2, VL3, and HL)
were identified before 3D scanning and dissection of the face (Figure 1). The HL line is defined

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as the horizontal line from the midpoint between points a and b to the lobule of auricle. Based
on this reference line, we determined an additional 5 points (P1, P2, P3, P4, and P5) and

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analyzed the positional relationship between each point at the DAO and DLI regions.
The soft tissue thicknesses were measured using a structured-light 3D scanner
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(Morpheus 3D, Morpheus Company, Seongnam, Korea). This system collects distance
information via active triangular technique, which has benefits of precision. A white light-
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emitting diode was source of the light and had 0.1 mm spatial accuracy. The whole scanning
procedure was conducted in about 0.8 seconds. The specimens were scanned from the frontal
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and bilateral oblique aspects and reconstructed. The experiment was divided into the following
three steps: (1) scanning the facial skin surface (Scan 1), (2) scanning the superficial fat surface
after removing only the epidermis and dermis (Scan 2), and scanning the facial muscle surface
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after removing superficial fat tissues (Scan 3) (Figure 2). The obtained 3D scan images were
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reconstructed and combined through geometry analysis using Morpheus Dental Solution (MDS)
software (version 3.0, Morpheus Company, Seongnam, Korea). The dissection was performed
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on only the left half of the face (deviating slightly right of the midline). In other words, the
right half of the facial skin was preserved for later use as a reference in the superimposition
procedure.12,13 For examples, the skin thickness was obtained by superimposing Scan 1 and 2,
while subcutaneous tissue thickness was by Scan 2 and 3. For this reason, 3D scanning cannot
be conducted on both sides of the face.
The thicknesses of the skin and subcutaneous layer were calculated automatically
using MDS 3.0 at each point. Additionally, statistical analyses were performed using standard
software (SPSS version 23.0 for Windows, SPSS, Chicago, IL, USA). A P value of < 0.05 was
considered statistically significant, and differences between males and females were analyzed
using Student’s t-test.

RESULTS
Positional Relationship Between the Reference Points and Muscles
Forty-five cadavers (25 males, 20 females; mean age, 76.5 years; range, 51-89 years) from

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Korea (19 males, 13 females; mean age, 76.6 years; range, 51-88 years) and Thailand (6 males,
11 females; mean age, 76.5 years; range, 60-89 years) were used in this study. The locations of

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P1, P2, P3, P4, and P5 were identified on 3D scanning images. In every case, P3 and P5 were
located in the area of DLI and DAO, respectively, in 100% (45/45). In other words, P3 indicates

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only DLI without any overlapping region with DAO, orbicularis oris, and mentalis muscle. In
the same manner, P5 indicates only DAO. P4 was observed in the area of the DLI and at the

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medial border of the DAO, while P1 and P2 were located in the area of the mentalis in most
cases (Figure 3).
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Measurement analysis of P3 and P5
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We measured the minimum distance from the medial border of the DAO to P3 and P5,
respectively. The distance between the medial border of the DAO and P3 was 11.9±3.4 mm
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(male, 11.6±3.3 mm; female, 12.2±3.5 mm; P-value, 0.513) and 10.6±3.3 mm (male, 10.5±3.6
mm; female, 10.6±3.1 mm; P-value, 0.909) between the medial border of the DAO and P5.
There was no significant difference between males and females. The soft-tissue thicknesses (skin
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and subcutaneous tissue) of P3 and P5 are presented in Table 1.

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DISCUSSION
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Unlike skeletal muscles, facial expression muscles have thin and flat structures and are
arranged in multiple layers. These characteristics make it difficult to identify the target muscle
on the skin surface during BoNT-A injection.14 The most common way to identify the DAO is
to use the modiolus. The modiolus is a muscular structure blended with various perioral
muscles, including the DAO. Clinicians can predict the location of the DAO using palpations
of the modiolus on the skin surface. However, this process is inconvenient for both clinicians
and patients because palpation should be performed via an intraoral approach. In addition, the
location of the modiolus varies widely between individuals and races.15 Contrary to the case in
Africans and Caucasians,16 the modiolus is located below the intercheilion line in Asians
(including Korean and Japanese individuals) in 58.4% and 45.1% of cases, respectively. This
means that the modiolus generally lies lower in Asian populations than in Caucasian and black
populations.
This study was designed to overcome these limitations and to suggest simpler
guidelines for clinical fields. In these results, the locations of P3 and P5 belong to the DLI and
DAO, respectively, in every case. First, P5 is the meeting point between the VL3 and HL. This

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means that, if clinicians drew the HL on the skin, the pupil can be used as a guideline for
targeting only the DAO. Therefore, we defined P5 as the ‘DAO point’. In the case of P3, it is

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the three-quarters of the distance between VL1 and VL2. With the same method, if clinicians
designed the HL on the skin, the cheilion and midsagittal line can be used as a guideline for

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targeting only the DLI. Therefore, we defined P3 as the ‘DLI point’. In addition, the medial
border of the DAO was spaced 10.6±3.3 mm and 11.9±3.4 mm apart from the DAO and DLI

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points, respectively. These results would allow clinicians to predict the location of the medial
border of the DAO (Figure 4).
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The depth information is one of the most important factors for successful BoNT-A
injection. The mean skin thicknesses on the DAO and DLI points are 1.9±0.6 mm and 1.8±0.7
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mm, respectively. We believe that the 0.1 mm gap is the tolerance grade for BoNT-A injection.
However, in the case of the subcutaneous layer, the thickness of the DAO point was observed
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to be 0.5 mm thicker than the DLI point. We supposed that the inferior jowl fat either already
existed or migrated until reaching the DAO point, whereas no specific superficial fat
compartment was observed at the DLI point. For this reason, in the case of thickness from the
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skin surface to the muscle, the DAO point was observed to be 0.3 mm thicker than the DLI
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point. The 0.3 mm gap was considered a clinically negligible value because clinicians can
readily determine the length and can administer injections at 0.3 mm intervals, with different
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effects. In addition, a statistically significant difference was observed between males and
females at approximately 1 mm. Therefore, for accurately targeting the muscle, the injection
should differ in depth between males and females. According to our results, we suggest that
the DAO injection be performed at depths of 6.3 mm and 7.3 mm in males and females,
respectively. In addition, DLI injections should be performed at depths of 6.0 mm and 7.0 mm
in males and females, respectively.
The main advantages of 3D scanning include high accuracy, convenient measurement,
and the absence of interobserver error.17,18 The 3D scanner used in this study supports the
spatial accuracy within 0.1 mm. Additionally, the operator can measure the depth by simply
moving the mouse pointer on the analysis software. These advantages allow for more accurate
results by eliminating interobserver error. In addition, the facial landmarks, such as the
midpupil and cheilion, are easy to use in clinical practice. Therefore, the DAO and DLI points
made by these landmarks play an important role in targeting only the DAO and DLI.
Nevertheless, there were some limitations in this study. First, the results of this study
were obtained from only Korean and Thailand cadavers. Therefore, these results can be applied

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to Asians, but it is limited to other ethnic populations. Second, this study 3D scanned the face
of fixed cadavers aged 50-80y. We expect the soft tissue thickness of the face to change with

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age. Therefore, there will be a limitation when applying this result to young people.

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CONCLUSION
The P3 and P5 described in this study is an effective guideline that can target only DLI and

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DAO. Therefore, we defined P3 and P5 as DLI and DAO point, respectively. Clinicians,
especially, can easily use facial landmarks such as cheiliion and pupil to appoint the DLI and
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DAO point without any measurement or palpation of the modilolus.
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REFERENCES
1. Liu MT, Iglesias RA, Sekhon SS, et al. Factors contributing to facial asymmetry in
ed

identical twins. Plast Reconstr Surg. 2014;134(4):638-646.

2. Lee HJ, Kim JS, Youn KH, Lee J, Kim HJ. Ultrasound-Guided Botulinum Neurotoxin
Type A Injection for Correcting Asymmetrical Smiles. Aesthet Surg J.
pt

2018;38(9):NP130-NP134.
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3. Choi YJ, Kim JS, Gil YC, et al. Anatomical considerations regarding the location and
boundary of the depressor anguli oris muscle with reference to botulinum toxin
Ac

injection. Plast Reconstr Surg. 2014;134(5):917-921.

4. Hur MS, Hu KS, Cho JY, et al. Topography and location of the depressor anguli oris
muscle with a reference to the mental foramen. Surg Radiol Anat. 2008;30(5):403-407.
5. Pellacani G, Seidenari S. Variations in facial skin thickness and echogenicity with site
and age. Acta Derm Venereol. 1999;79(5):366-369.
6. Mlosek RK, Malinowska S. Ultrasound image of the skin, apparatus and imaging basics.
J Ultrason. 2013;13(53):212-221.
7. Vyas S, Meyerle J, Burlina P. Non-invasive estimation of skin thickness from
hyperspectral imaging and validation using echography. Comput Biol Med.
 2015;57:173-181.
8. Lee Y, Hwang K. Skin thickness of Korean adults. Surg Radiol Anat. 2002;24(3-4):183-
189.
9. Ha RY, Nojima K, Adams WP, Jr., Brown SA. Analysis of facial skin thickness:
defining the relative thickness index. Plast Reconstr Surg. 2005;115(6):1769-1773.
10. Hwang K, Kim DJ, Hwang SH. Thickness of Korean upper eyelid skin at different

Downloaded from https://academic.oup.com/asj/advance-article-abstract/doi/10.1093/asj/sjaa083/5814121 by guest on 12 April 2020

levels. J Craniofac Surg. 2006;17(1):54-56.
11. Chopra K, Calva D, Sosin M, et al. A comprehensive examination of topographic

t
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thickness of skin in the human face. Aesthet Surg J. 2015;35(8):1007-1013.
12. Kim YS, Lee KW, Kim JS, et al. Regional thickness of facial skin and superficial fat:

cr
Application to the minimally invasive procedures. Clin Anat. 2019;32(8):1008-1018.
13. Lee KW, Kim SH, Gil YC, Hu KS, Kim HJ. Validity and reliability of a structured-light

us
3D scanner and an ultrasound imaging system for measurements of facial skin thickness.
Clin Anat. 2017;30(7):878-886.
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14. Choi YJ, Lee KW, Gil YC, Hu KS, Kim HJ. Ultrasonographic Analyses of the Forehead
Region for Injectable Treatments. Ultrasound Med Biol. 2019;45(10):2641-2648.
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15. Yu SK, Lee MH, Kim HS, Park JT, Kim HJ, Kim HJ. Histomorphologic approach for
the modiolus with reference to reconstructive and aesthetic surgery. J Craniofac Surg.
ed

2013;24(4):1414-1417.
16. Greyling LM, Meiring JH. Morphological study on the convergence of the facial
muscles at the angle of the mouth. Acta Anat (Basel). 1992;143(2):127-129.
pt

17. Hammond P, Hutton TJ, Allanson JE, et al. 3D analysis of facial morphology. Am J
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Med Genet A. 2004;126A(4):339-348.

18. Kim SC, Kim HB, Jeong WS, et al. Comparison of Facial Proportions Between Beauty
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Pageant Contestants and Ordinary Young Women of Korean Ethnicity: A Three-

Dimensional Photogrammetric Analysis. Aesthetic Plast Surg. 2018;42(3):748-758.
Figure Legend
Figure 1. Reference points and lines based on the facial landmarks. a, gnathion; b, midpoint of
the vermillion border of the lower lip; c, cheilion; d, pupil; VL1, midsagittal line; VL2, vertical
line passing through the cheilion; VL3, midpupillary line; HL, horizontal line from the
midpoint between a and b to lobule of auricle; P1, one-quarter of the distance between VL1
and VL2; P2, one-half of the distance between VL1 and VL2; P3, three-quarters of the distance

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between VL1 and VL2; P4, intersection point between VL2 and HL; P5, intersection point
between VL3 and HL.

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Figure 2. The three steps for acquiring 3D scanning images. (A) Scanned image of the facial

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skin surface (scan 1), (B) scanned image of the superficial fat surface (scan 2), and (C) scanned
image of the facial muscle surface (scan 3). Each blue line represents the VL1, VL2, and VL3

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according to the curvature of the face. VL1, mid-sagittal line; VL2, vertical line passing
through the cheilion; VL3, mid-pupillary line.
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Figure 3. The overlapping image between the reference points, lines and dissected face. VL1,
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mid-sagittal line; VL2, vertical line passing through the cheilion; VL3, mid-pupillary line; P1,
one-quarter of the distance between VL1 and VL2; P2, one-half of the distance between VL1
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and VL2; P3, three-quarters of the distance between VL1 and VL2; P4, intersection point
between VL2 and HL; P5, intersection point between VL3 and HL; DLI, depressor labii
inferioris; DAO, depressor anguli oris.
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Figure 4. The DLI and DAO points and its relationship with the medial border of the DAO.
The P3 (yellow point) and P5 (red point) represent the DLI and DAO point, respectively. The
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star symbol, the horizontal distance between P3 and medial border of DAO; The asterisk
symbol, the horizontal distance between P5 and medial border of DAO; VL1, mid-sagittal line;
VL2, vertical line passing thtough the cheilion; VL3, mid-pupillary line; a, gnathion; b,
midpoint of the vermillion border of the lower lip; c, cheilion; P1, one-quarter of the distance
between VL1 and VL2; P2, one-half of the distance between VL1 and VL2; P3, three-quarters
of the distance between VL1 and VL2; P4, intersection point between VL2 and HL; P5,
intersection point between VL3 and HL; DLI, depressor labii inferioris; DAO, depressor
amguli oris.
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Figure 1
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Figure 2A
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Figure 2B
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Figure 2C
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Figure 3
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Figure 4
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Table 1. The Soft-Tissue Thickness at P3 and P5
Skin (A) Subcutaneous tissue (B) Skin
All Male Female p-value All Male Female p-value All
P3 1.9±0.6 1.9±0.5 1.8±0.7 0.437 4.5±1.7 4.1±1.7 5.1±1.5 *0.031 6.4±1.7 6
P5 1.8±0.7 1.8±0.8 1.8±0.6 0.944 5.0±1.8 4.5±1.8 5.5±1.6 *0.049 6.7±1.8 6
The results are expressed as the mean ± SD values in millimeters; *, p value < 0.05.

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