Annals of Anatomy 246 (2023) 152025

Contents lists available at ScienceDirect

Annals of Anatomy
journal homepage: www.elsevier.com/locate/aanat

Study of the functional relationships between the buccinator muscle and

the connective tissue of the cheek in humans ]]
]]]]]]
]]

⁎
Elena Martínez-Sanz a, Javier Catón a, , Estela Maldonado a, Jorge Murillo-González a,
María Carmen Barrio b, Irene Paradas-Lara b, Moisés García-Serradilla b, Luis Arráez-Aybar a,1,
José Ramón Mérida-Velasco a,1
a
Department of Anatomy and Embryology, Faculty of Medicine, Complutense University of Madrid, Plaza de Ramón y Cajal, s/n, Ciudad Universitaria, 28040
Madrid, Spain
b
Department of Anatomy and Embryology, Faculty of Optics and Optometry, Complutense University of Madrid, Calle de Arcos de Jalón, 118, 28037 Madrid, Spain

a r t i cl e i nfo a bstr ac t

Article history: Background: The buccinator muscle derives from the mesenchyme of the second pharyngeal arch. In adults,
Received 25 July 2022 it has a quadrilateral shape, occupying the deepest part of the cheek region. Its function is complex, being
Received in revised form 17 October 2022 active during swallowing, chewing, and sucking. To our knowledge, there are no studies that have speci­
Accepted 2 November 2022
fically analyzed the relationship of the buccinator muscle fibers and neighboring connective tissue of the
Available online 12 November 2022
cheek in humans, neither during development nor in adults. Such relationships are fundamental to un­
derstand its function. Thus, in this study the relations of the buccinator muscle with associated connective
Keywords:
Buccinator muscle tissue were investigated.
Connective tissue Methods: The buccinator muscle region was investigated bilaterally in 41 human specimens of 8–17 weeks
Functional anatomy of development. Moreover, four complete adult tissue blocks from human cadavers (including mucosa and
Human skin) were obtained from the cheek region (between the anterior border of the masseter muscle and the
Collagens type I/III nasolabial fold). All samples were processed with standard histological techniques. In addition, subsets of
Immunohistochemistry sections were stained with picrosirius red (PSR). Furthermore, immunoreactivity against type I and III
Picrosirius red staining collagen was also studied in adult tissues.
Polarized light microscope
Results: The buccinator muscle showed direct relationships with its connective tissue from 8 to 17 weeks of
development. Collagen fibers were arranged in septa from the submucosa to the skin through the muscle.
These septa were positive for type I collagen and presented elastic fibers. Fibrous septa that were positive
for type III collagen were arranged from the lateral side of the muscle to the skin.
Conclusions: The intimate relationship between buccinator muscle fibers and cheek connective tissue may
explain the complex functions of this muscle.
© 2022 The Author(s). Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction posterior belly of the digastric muscle, and the stylohyoid muscle.
The superficial lamina extends from the external acoustic pathways
The muscles of facial expression are derived from the me­ forming the temporal, occipital, cervical, and mandibular laminae.
senchyme of the second pharyngeal arch and are innervated by the The superficial facial muscles of the face arise from these laminae.
facial nerve. Premuscular facial masses form between the 6th and The buccinator muscle originates from the mandibular lamina
7th week of development. Migrating myoblasts extend from the (Gasser, 1967; Sperber and Sperber, 2018).
region of the second pharyngeal arch in the form of sheets. The deep In human adults, the buccinator muscle is a thin, flattened
mesenchymal layer condenses to become the stapedius, the quadrilateral mass located deep in the cheek. Its origins are the
external surfaces of the alveolar processes of the maxilla and
mandible, along the molars, and the anterior border of the pter­
ygomandibular raphe. In addition, at this origin, the buccinator is
Abbreviations: PSR, Picrosirius red staining; SMAS, Superficial musculoapo­
neurotic system related to the temporalis muscle (Hur, 2017; Rusu et al., 2021). From
⁎
Corresponding author. its origins, the fibers of the buccinator muscle converge toward the
E-mail address: [email protected] (J. Catón). modiolus, near the angle of the mouth, and associate with the fibers
1
These authors have contributed equally to this study.

https://doi.org/10.1016/j.aanat.2022.152025
0940-9602/© 2022 The Author(s). Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
E. Martínez-Sanz, J. Catón, E. Maldonado et al. Annals of Anatomy 246 (2023) 152025

of the orbicularis muscle (Rouvière and Delmas, 2005; Hanawa et al., specimens were the product of ectopic pregnancies or spontaneous
2008; Sun et al., 2018; von Arx et al., 2018; Standring, 2020). Some abortions managed in the Department of Obstetrics and Gynecology
studies have described a fourth bundle of fibers reaching the men­ at UCM. No clinical information or genetic analysis was available to
talis muscle (D’Andrea, Barbaix, 2006; Hur et al., 2011). explain the abortions. However, all specimens were thoroughly
The buccinator muscle has been reported to be active during studied morphologically and histologically and no material sus­
swallowing, chewing, blowing, and sucking (Perkins et al., 1977; pected of malformation was included in this study. The specimens
Schieppati et al., 1989; Standring, 2020; Rathee and Jain, 2022). The were classified by postconceptional developmental age as follows:
buccinator muscle also helps close the lips by pulling the corners of four 8-week-old, seven 9-week-old, six 10-week-old, four 11-week-
the mouth (Shiratori et al., 2021). In addition, the buccinator muscle old, five 12-week-old, four 13-week-old, four 14-week-old, three 15-
may act as a sphincter and play a role in regulating the parotid duct week-old, two 16-week-old, and two 17-week-old specimens. The
(Kang et al., 2006; Amano et al., 2010, 2013). This muscle is also parameters used to determine the postconceptional age were the
present in other species and it has been reported that it may have greatest length and the external and internal criteria (O’Rahilly,
similar functions in cats (Tomo et al., 2002) and minipigs (Dutra Müller, 2000, 2010). All specimens were fixed in 10 % formalin and
et al., 2010). embedded in paraffin following standardized procedures. Specimens
Collagen fibers are important structural elements of the extra­ greater than 30 mm greatest length, from the ninth week of devel­
cellular matrix. Collagens have structural functions and contribute to opment onwards, were previously decalcified with 5 % tri­
the organization and shape of tissues and provide mechanical chloroacetic acid. Each specimen was cut completely in a single
properties (Ricard-Blum, 2011). Skeletal muscle is surrounded by plane. Sections were cut 7–10 µm thick, depending on the size of the
connective tissue that forms the epimysium, and within the muscle specimen.
there are two distinct extracellular matrix structures: perimysium
and endomysium. Together, these three structures form the in­ 2.3. Human adult samples
tramuscular connective tissue. The mechanical activity of a muscle
depends on both its muscle fibers and its intramuscular connective Four hemi-heads of formalin-fixed cadaveric specimens (2 males
tissue (Purslow, 2020; Turrina et al., 2013; Zhang et al., 2021). There and 2 females aged between 65 and 85 years at the time of death)
are large variations in the amount and composition of intramuscular were used for this study. In all cases, resection of the cheek region
connective tissue between muscles with different functions. In­ was performed. The four tissue blocks with the adult cheek samples
tramuscular connective tissue acts as a scaffold for muscle fibers and included a full thickness, from mucosa to skin, of the region between
serves as a carrier for blood vessels and nerves to muscle cells. The the anterior border of the masseter muscle and the nasolabial fold.
variability of the intramuscular connective tissue between different After fixation, the samples were processed and embedded in paraffin
muscles conditions the variations in their active and passive me­ following standardized procedures. They were then serially sec­
chanical properties (Purslow, 2020). tioned (at a thickness of 7 µm) along the transverse plane using a
Numerous studies have analyzed and provided a solid basis for standard microtome. The specimens came from the Center for Body
the expression of different types of collagens in the intramuscular Donation and Dissection Rooms at UCM.
connective tissue of skeletal muscles (Light and Champion, 1984;
Turrina et al., 2013; Zhang et al., 2021). Nevertheless, to our 2.4. Histological staining procedures
knowledge there are no studies on the presence of type I and III
collagen in the buccinator muscle. This information is crucial for Sections for histological analysis were routinely stained with H&
understanding the function of this muscle. Thus, the aim of the E, Azan stain, Verhoeff’s stain for elastic fibers, or Mallory’s triple
present study was to examine the relationships between the buc­ stain. The stained sections were examined with a Nikon Eclipse
cinator muscle and the connective tissue of the cheek. For this E400. Digital microphotographs of the samples were taken with a
purpose, serial sections of human specimens from 8 to 17 weeks of Nikon DXM 1200 camera (Nikon Corporation, Tokyo, Japan), and
development were studied. In addition, the relationships between transferred to and edited on a Pentium IV PC, using Act One software
type I and III collagen and the buccinator muscle in adult human and Adobe Photoshop CS6. In addition, a subset of sections—two 13-
tissue were examined by conventional histology and im­ week-old specimens, one 17-week-old specimen and four cheek
munohistochemical techniques. This study finally suggests the pos­ adult samples—were stained with picrosirius red (PSR) stain (PSR
sible role of the buccinator muscle within the cheek through the Stain Kit; Abcam, Cambridge, UK). In PSR-stained sections, under
surrounding connective tissue. polarized light, collagen fibers appear red-orange and green-yellow;
the colors indicate the different organization and/or heterogeneity in
2. Materials and methods the orientation of collagen fibers in connective tissues (Lattouf et al.,
2014). The PSR-stained sections were analyzed with a Nikon Eclipse
2.1. Ethical statement Ti microscope equipped with a polarizing filter and photographed
with a Nikon DS-Fi1 digital camera (Nikon Corp.). No less than three
The authors declare that all local and international ethical different sections per sample were evaluated. Two independent in­
guidelines and laws regarding the use of human cadaveric donors in vestigators examined the serial sections in all cases.
anatomical research were followed. Prior to death, all individuals
gave written informed consent for the use of their donation for 2.5. Immunohistochemistry
scientific purposes. In addition, descriptive studies with embry­
ological and fetal material from the collections of the Universidad In adult cheek tissue samples, immunohistochemistry was per­
Complutense de Madrid (UCM) were approved by the Clinical formed on paraffin sections following the manufactures protocol for
Research Ethics Committee of the Hospital Clínico San Carlos. ready to use (R.T.U.) Vectastain® Universal Elite® ABC Kit (PK-7200,
Vector Laboratories LTD, Peterborough, UK). Briefly, paraffin blocks
2.2. Prenatal human specimens were sectioned at 7 µm. The sections were rehydrated with xylol
followed by a series of ethanol solutions ending with distilled H2O.
The buccinator muscle region was investigated bilaterally in 41 Heat-induced epitope retrieval was performed on the samples using
developing human specimens belonging to the embryo library of the Tris-EDTA-T buffer (10 mM Tris-base, 1 nM EDTA solution, 0.05 %
Department of Anatomy and Embryology of the UCM. All the Tween 20, pH 9.0) using a pressure cooker. Endogenous peroxidases

2
E. Martínez-Sanz, J. Catón, E. Maldonado et al. Annals of Anatomy 246 (2023) 152025

Fig. 1. Photomicrographs of coronal sections of the cheek region of early developing human specimens stained with hematoxylin–eosin. (A and B) Sections of a 27 mm greatest
length human embryo at 8 weeks of development. (B) Higher magnification from (A), of the yellow dashed line box area. (C–H) Sections of a 65 mm greatest length human fetus at
11 weeks of development. The segments marked with the numbers 1, 2, and 3 in (C), (G), and (H) indicate the upper maxillary, middle, and lower mandibular portions of the
buccinator muscle, respectively. (C) Posterior region of the buccinator muscle, dorsal to the opening of the parotid duct. (D) Higher magnification from the upper maxillary portion
of the buccinator muscle. (E) Higher magnification from the middle portion of the buccinator muscle. (F) Higher magnification from the lower mandibular portion of the
buccinator muscle. (G) Middle region of the buccinator muscle, at the level of the opening of the parotid duct. (H) Anterior region of the buccinator muscle, anterior to the opening
of the parotid duct. The arrows in (B), (D), (E), and (F) indicate fibers of collagen that extend from the outline of the submucosa and penetrate the buccinator muscle. Abbreviations
in this figure: AM inferior alveolar nerve, B buccinator muscle, FA facial artery, M mucosa, MA mandible, MC Meckel’s cartilage, MM masseter muscle, P platysma muscle, PD parotid
duct, S submucosa, T tongue, TG tooth germ, Z zygomatic muscle. Scale bars: 200 µm in (A), (C), (G), and (H); 50 µm in (D), (E), and (F); and 20 µm in (B).

3
E. Martínez-Sanz, J. Catón, E. Maldonado et al. Annals of Anatomy 246 (2023) 152025

Fig. 2. Photomicrographs of coronal sections of the cheek region of human fetuses at the 13th and 15th weeks of development. (A and B) Picrosirius red staining from a 90 mm
greatest length human fetus at 13 weeks of development; sections as viewed using brightfield (A) and polarized light microscopy (B). (C and D) Hematoxylin–eosin staining from a
116 mm greatest length human fetus at 15 weeks of development. The arrows in (A–D) indicate tracts of collagen fibers extending from the outline of the submucosa, penetrating
the buccinator muscle (A and B) to the loose connective tissue superficial to the buccinator muscle (C and D). Asterisks in (A) indicate collagen fibers condensed between the
submucosa and buccinator muscle. Abbreviations in this figure: B buccinator muscle, M mucosa, S submucosa. Scale bars: 200 µm in (C), and 100 µm in (A), (B), and (D).

were quenched with 3 % H2O2 in water at room temperature for 3. Results

30 min. Specimens were then blocked with the kit’s normal horse
serum and thereafter incubated with primary antibodies overnight 3.1. Human development
at 4 °C. The following primary antibodies were used: mouse anti-
collagen type 1 (1:200; sc‐59772; Santa Cruz Biotechnology, Dallas, During the 8th week of development, the buccinator muscle is
TX, USA) and rabbit polyclonal antibody anti-collagen type III located deep in the cheek. Superficially, it is related to a loose me­
(1:200; bs-0549R; Bioss Antibodies, Woburn, MA, USA). For negative senchyme in which the facial artery runs. The buccinator muscle is
controls, the primary antibody was omitted. The sections were then formed by myoblasts, among which collagen fibers can be seen.
incubated with the kit’s biotinylated universal secondary antibodies, These fibers extend through the outline of the submucosa
followed by incubation with the R.T.U. reagent for 30 min. The sec­ (Fig. 1A, B).
tions were developed with 3,3'-diaminobenzidine (DAB) peroxidase During the 9th–12th week of development, there are three ori­
substrate solution (SK-4100; Vector Laboratories LTD) until staining ginal insertion portions of the buccinator muscle: the upper max­
was revealed; the sections were then counter-stained with hema­ illary, the lower mandibular, and the middle (Fig. 1C–H). In the upper
toxylin, washed, dehydrated, and mounted in Permount™ mounting and lower portions, the muscle fibers appear more condensed, se­
media (Fisher Scientific, Pittsburgh, PA, USA). The stained sections parated by collagen fibers (Fig. 1D, F). The fibers of the upper part are
were examined with a Nikon Eclipse E400 microscope and photo­ arranged toward the submucosa in an oblique craniocaudomedial
graphed with a Nikon DXM 1200 digital camera. direction (Fig. 1D). The fibers of the lower portion also run obliquely
Immunohistochemical analysis of the developing tissues was not toward the submucosa, but in a caudocraniomedial direction
possible, as the samples were very old had been decalcified with 5 % (Fig. 1F). The muscle and collagen fibers in the middle portion appear
trichloroacetic acid, which had a negative effect on the im­ less dense; they are arranged perpendicular and continue with the
munoreactivity for the study antigens. fibers of the submucosa (Fig. 1E). The excretory duct of the parotid

4
E. Martínez-Sanz, J. Catón, E. Maldonado et al. Annals of Anatomy 246 (2023) 152025

Fig. 3. Photomicrographs of Azan-stained sections of the cheek region of human fetuses at the 16th and 17th weeks of development. (A and B) Coronal sections from a 137 mm
greatest length human fetus at 16 weeks of development. The arrows in (B) indicate fibrous tracts connecting the perimysium of the buccinator muscle and extending to reach the
connective tissue surrounding the neurovascular (VN) structures and parotid duct (PD). (C and D) Transverse sections from a 150 mm greatest length human fetus at 17 weeks of
development. The arrows in (D) indicate tracts of collagen fibers extending from the perimysium of the buccinator muscle to reach the newly formed fibroadipose tissue (lateral to
the buccinator muscle). The asterisk in (D) indicates a thin sheet of epimysium over the buccinator muscle. Abbreviations in this figure: B buccinator muscle, BF buccal fat pad, F
collagen fiber, FA facial artery, M mucosa, P platysma muscle, PD parotid duct, S submucosa, VN vasculonervous bundle. Scale bars: 1 mm in (A) and (C), and 200 µm in (B) and (D).

gland passes through the buccinator muscle between the muscle buccinator muscle, the deep fibroadipose tissue, the superficial
fibers of the upper maxillary portion (Fig. 1G). Anterior to the musculoaponeurotic system (SMAS), the superficial fibroadipose
opening of the parotid duct, most of the muscle belongs to the tissue, and the skin. In the deep fibroadipose tissue, fibrous tracts
middle portion, with the maxillary and mandibular portions being join the buccinator muscle to the SMAS. In the superficial fi­
denser (Fig. 1H). In addition, the muscle fibers and connective tissue broadipose tissue, thin tracts run parallel in an oblique direction
are denser in this region along the medial surface of the muscle, from the SMAS to the skin, forming the retinaculum cutis super­
relative to the submucosa (Fig. 1H). ficialis (Fig. 4).
Between the 13th and 15th week of development, there are some The different conventional histological stains used, as well as PSR
changes in the relationships between the muscle and collagen fibers. staining, showed that collagen fibers are very abundant in the sub­
The partitions of collagen fibers between the fascicles of muscle fi­ mucosa (Fig. 5A–C). Some fibers run from the submucosa to the
bers are evident and constitute the perimysium of the muscle. buccinator muscle and connect with the perimysium. There are
Collagen fibers are condensed between the submucosa and the some elastic fibers in the submucosa and in the perimysium of the
muscle (Fig. 2A, B). The perimysium has connections with the fibers buccinator muscle (Fig. 5C and D). Immunohistochemical analyses
of the submucosa and extends to the loose connective tissue su­ showed that there are many type I collagen fibers in the submucosa
perficial to the buccinator muscle (Fig. 2C, D). and perimysium of the buccinator muscle (Fig. 5E). These fibers did
Between the 16th and 17th week of development, two aspects not show immunoreactivity for type III collagen (Fig. 5F). A re­
are very evident. The fibrous tracts connected with the perimysium presentative image of a negative control during im­
of the muscle reach the connective tissue that surrounds the neu­ munohistochemistry, omitting the primary antibody, is shown in
rovascular structures and the parotid duct (Fig. 3A, B). In addition, by Fig. 5G.
the 17th week, the fibroadipose tissue that is related to the lateral Thicker tracts of collagenous fibers, which are connected to the
aspect of the buccinator muscle has been formed. The perimysium of perimysium of the buccinator muscle, traverse the deep fibroadipose
the muscle is connected to the newly formed fibroadipose tissue tissue and reach the SMAS (Fig. 6A–D). These tracts are attached to
(Fig. 3C, D), and a thin sheet of epimysium over the buccinator the connective tissue surrounding the neurovascular bundles
muscle is visible (Fig. 3D). (Fig. 6C, D). Fibers emerging from the buccinator muscle into deep
fibroadipose tissue, reaching the SMAS, showed positive im­
3.2. Human adults munoreactivity for type I collagen (Fig. 6C) and type III collagen
(Fig. 6D).
In human adults, the cheek consists of seven layers arranged Between the SMAS and the dermis is the superficial fibroadipose
from depth to surface as follows: the mucosa, the submucosa, the tissue and numerous fine tracts of collagen fibers with positive

5
E. Martínez-Sanz, J. Catón, E. Maldonado et al. Annals of Anatomy 246 (2023) 152025

4.1. Morphological and immunohistochemical aspects

Skeletal or striated muscles are made of muscle fibers and con­

nective tissue. Numerous studies have analyzed the morphological,
structural, and functional aspects of the intramuscular connective
tissue. These reports suggest that connective tissue has very dif­
ferent structural features than described previously. The en­
domysium forms a three-dimensional network that connects
adjacent muscle fibers rather than separating them. The perimysium
is also a three-dimensional network that runs the length and
breadth of the muscle, joining muscle fascicles. The epimysium
surrounds the entire muscle and defines its volume. The en­
domysium, perimysium, and epimysium constitute the in­
tramuscular connective tissue (Purslow, 2020).
The epimysium, perimysium, and endomysium of skeletal muscle
contain type I collagen as a major component and type III collagen as a
minor component (Light and Champion, 1984; Zhang et al., 2021). In
the facial region there are some studies on collagen in relation to the
functionality of adipose tissue (Kruglikov et al., 2016). However, to our
knowledge, there have been no immunohistochemical studies to de­
termine the type of collagen that forms these fibrous tracts in the
cheek. Macchi et al. (2010) showed by conventional histological
techniques that these fibrous septa contained elastic fibers and col­
lagen. Nevertheless, this study is the first to show the relationships of
the buccinator muscle to the connective tissue of the cheek during
development. Furthermore, in adults, colocalization of collagens I and
III was demonstrated in the lateral connective tracts from the muscle
to the skin. However, only type I collagen and elastic fibers were ob­
served in the submucosa and intramuscular convective tissue.
Numerous studies have reviewed and characterized superficial
and deep cheek fat in relation to facial aging in adults (Cotofana
et al., 2016; Kruglikov et al., 2016; Stuzin et al., 2019). Other research
has focused on the development, anatomy, and histology of the
SMAS of the cheek (Ghassemi et al., 2003; de la Cuadra-Blanco et al.,
2013; Hwang and Choi, 2018; Sandulescu et al., 2018, 2019). In stu­
dies on fibroadipose tissue of the cheek region, researchers have
reported that the skin is attached to the superficial part of the SMAS
Fig. 4. Reconstruction of the full thickness of an adult human cheek from photo­ by undulating fibrous tracts that separate the superficial adipose
micrographs of Mallory-stained sections. Notice the seven layers of the cheek from layer and form the retinaculum cutis superficialis. Other longer fi­
depth to surface: the mucosa (M), the submucosa (S), the buccinator muscle (B), the brous tracts join the SMAS to the buccinator muscle and compart­
deep fibroadipose tissue (DFA), the superficial musculoaponeurotic system (SMAS),
the superficial fibroadipose tissue (SFA), and the skin (SK). The arrows indicate tracts
mentalize the deep adipose tissue layer (Macchi et al., 2010;
of collagen fibers that extend from the buccinator muscle and DFA to the SMAS. The Sandulescu et al., 2019). Our results confirm this arrangement, but in
arrowheads indicate tracts of collagen fibers that extend from the SMAS to the SFA cases where there is no SMAS we have verified that there are thick
and skin. Scale bar: 2 mm. fibrous tracts that extend from the submucosa to the skin, crossing
the buccinator muscle. It has been described that these septa con­
immunoreactivity for type I and III collagen, as well as abundant nect the mimic musculature to the skin and are part of the type I
elastic fibers that constitute the retinaculum cutis superficialis SMAS (Ghassemi et al., 2003; Sandulescu et al., 2019). In specimens
(Fig. 6E–G). of the 17th week of development, in the upper maxillary portion of
the cheek these tracts reach the superficial fibroadipose tissue, while
the SMAS does not appear in this area. According to some authors,
4. Discussion these collagen fiber tracts are related to the connective tissue that
surrounds the nerves and vessels of the cheek region and to the
Facial expression muscles derive from the mesenchyme of the excretory duct of the parotid gland (Macchi et al., 2010; Cotofana
2nd pharyngeal arch and are innervated by the facial nerve (Sperber et al., 2016; Sandulescu et al., 2019).
and Sperber, 2018). The extracellular matrix is essential for muscle
formation during myogenic differentiation. The extracellular matrix
provides a stable microenvironment for the migration, adhesion, 4.2. Functional aspects
proliferation, and differentiation of myogenic stem cells and is es­
sential for physiological functions of muscle cells, such as the Some authors have considered it to be a neuromuscular forma­
transmission of mechanical force (Zhang et al., 2021). The organi­ tion “orbiculo-buccinato-linguo-velo-pharyngo-oesophageale” (Plas
zation of these muscles differs from that of skeletal muscles in other et al., 2004). It has been reported to be active during swallowing,
regions. They lack muscle spindles and tendon organs of the Golgi chewing, blowing, and sucking (Rathee and Jain, 2022) and, together
apparatus. Ruffini corpuscles and sensory mechanoreceptor forma­ with the orbicularis oris muscle, acts in the articulation of words
tions have been immunohistochemically described in some facial (Plas et al., 2004). Fascial connections between the temporalis and
muscles (Cobo et al., 2017). In addition, their terminal insertions are buccinator muscles have been described, suggesting the involve­
made in the skin or mucosa. ment of this muscle during chewing (Hur, 2017; Rusu et al., 2021).

6
E. Martínez-Sanz, J. Catón, E. Maldonado et al. Annals of Anatomy 246 (2023) 152025

Fig. 5. Photomicrographs of adult human cheek sections showing the submucosa and buccinator muscle. (A) Picrosirius red staining as viewed using polarized light microscopy.
(B) Mallory staining (C and D) Verhoeff's staining for elastic fibers. (D) High magnification showing a tract of elastic fibers in the perimysium pervading the buccinator muscle
(arrow). (E) Immunoexpression of type I collagen revealing positive immunostaining for collagen fibers in the submucosa and perimysium of the buccinator muscle (arrows). (F)
Immunoexpression of type III collagen revealing negative immunostaining throughout the section. (G) Negative control for immunohistochemistry. The arrows in (A–C) and (E)
indicate tracts of collagen fibers that extend from the submucosa and penetrate the buccinator muscle, constituting its perimysium. Abbreviations in this figure: B buccinator
muscle, M mucosa, S submucosa, V vessel. Scale bars: 1 mm in (A), (B), (E), (F) and (G); 200 µm in (C); and 50 µm in (D).

However, morphological studies are lacking to clarify some of these by increasing their volume, would prevent food from remaining in
functions. the upper and lower bottoms, emptying the vestibular space. The
The buccinator muscle is considered to participate in mastication middle portion in which the connective tissue is arranged perpen­
by bringing food to the occlusal surface of the teeth and pulling on dicular to the mucosa would condition its traction to avoid it being
the mucosa to avoid being pinched. We have verified that, in the pinched by the dental occlusal surface. This hypothesis is consistent
posterior part of the muscle, three portions can be defined by the with the findings of Reid et al. (2003) of the vestibule morphology
arrangement of muscular fibers and connective tissue. The con­ during buccinator muscle movements. In adults, the predominant
traction of the upper (maxillary) and lower (mandibular) portions, collagen in the submucosa is type I, and some tracts are also

7
E. Martínez-Sanz, J. Catón, E. Maldonado et al. Annals of Anatomy 246 (2023) 152025

Fig. 6. Photomicrographs of adult human cheek sections showing the skin and connective tissues lateral to the buccinator muscle. (A–D) Sections from the deep fibroadipose
tissue (DFA) of the cheek. (A and B) Picrosirius red staining sections as viewed using brightfield (A) and polarized light microscopy (B). (C) Immunoexpression of type I collagen
revealing positive immunostaining for tracts of collagen fibers. (D) Immunoexpression of type III collagen revealing positive immunostaining for tracts of collagen fibers. The
arrows in (A–D) indicate tracts of collagen fibers through the DFA, lateral to the buccinator muscle. (E–G) Sections from the superficial fibroadipose tissue (SFA) and skin of the
cheek. (E and F) Picrosirius red staining sections as viewed using brightfield (E) and polarized light microscopy (F). (G) Verhoeff’s staining for elastic fibers. The arrows in (E–G)
indicate tracts of collagen fibers through the superficial fibroadipose tissue (SFA) that reach the skin and constitute the retinaculum cutis superficialis. Abbreviations in this figure:
SK skin, VN vasculonervous bundle. Scale bars: 1 mm in (A–F), and 200 µm in (G). (For interpretation of the references to color in this figure legend, the reader is referred to the
web version of this article.)

8
E. Martínez-Sanz, J. Catón, E. Maldonado et al. Annals of Anatomy 246 (2023) 152025

continuous with the perimysium of the buccinator muscle. Type I individuals gave written informed consent for the use of their do­
collagen provides solidity and tensile strength in tissues and bio­ nation for scientific purposes. This study was approved by the UCM
mechanically defines properties related to load and traction (Gelse Ethics Committee.
et al., 2003).
Type I collagen is the most common form of collagen, accounting Data availability
for 90 % of total collagen in mammals. It is usually organized in thick
bundles, which confer strength. These bundles are found in liga­ Any additional data supporting this study are available from the
ments, tendons, and fasciae. Type III collagen forms shorter and corresponding author (J.C.) upon reasonable request.
thinner fibers. It is generally found associated with type I collagen in
variable proportions and is more abundant in tissues with some Declaration of Competing Interest
degree of elasticity, including skin, muscle, and fascia (Calvi et al.,
2014). There are tracts containing colocalized collagen I and III in the The authors declare that they have no known competing fi­
superficial part of the buccinator muscle which are connected to the nancial interests or personal relationships that could have appeared
connective tissue surrounding the vasculonervous bundles of the to influence the work reported in this paper.
cheek. This arrangement in the adult was also observed during de­
velopment and could be explained by the greater thickness and Acknowledgements
expandability of the cheek in this superficial region of the muscle
with respect to the deep region of the submucosa. Furthermore, the The authors wish to sincerely thank those who donated their
tracts would support the movement of the neurovascular bundles bodies to science so that anatomical research could be performed.
that run through the cheek to which they are related. The results from such research can potentially improve patient care
Recently, it has been reported that connection between the su­ and increase mankind’s overall knowledge. Therefore, these donors
perior constrictor pharynx and buccinator muscles occurs during the and their families deserve our highest gratitude. The authors would
13th week of development (Jin et al., 2022). During that time, the also like to acknowledge Proof-Reading-Service.com (UK) for the
morphology of the temporomandibular joint allows movements English revision of the manuscript. Finally, thanks also to the funding
(Mérida-Velasco et al., 1999); the pharynx and esophagus can also sources for this study: a Banco Santander/Universidad Complutense
present ascending and descending movements during swallowing de Madrid research project [PR108/20-18] and the Complutense
(López-Fernández et al., 2019). Our study showed the arrangement Research Group 920202 [GRFN14/22].
and progression of connective tissue tracts from the submucosa to
the skin during development, which is necessary to ensure bucci­ References
nator and cheek muscle movements.
Amano, K., Moriyama, H., Shimada, K., Matsumura, G., 2010. Morphological study of
the fetal parotid duct and buccinator muscle and the relationship to salivary se­
5. Conclusion cretion. Clin. Anat. 23 (6), 642–648.
Amano, K., Moriyama, H., Shimada, K., Matsumura, G., 2013. Study of human adult
In summary, this work evidenced the organization and progres­ parotid duct in the area of penetration through buccinator muscle and their
functional relationship as a sphincter. Ital. J. Anat. Embryol. 118 (1), 6–18.
sion during development of the connective tissue of the cheek, and Calvi, E.N.C., Nahas, F.X., Barbosa, M.V., Calil, J.A., Ihara, S.S.M., Juliano, Y., Ferreira, L.M.,
its relationships with the buccinator muscle. In adults, the ar­ 2014. Collagen fibers in the rectus abdominis muscle of cadavers of different age.
rangement and distribution of type I and III collagen fibers, as well as Hernia 18 (4), 527–533. https://doi.org/10.1007/s10029-014-1213-0
Cobo, J.L., Abbate, F., de Vicente, J.C., Cobo, J., Vega, J.A., 2017. Searching for proprio­
elastic fibers of the cheek, could suggest the complex function of the
ceptors in human facial muscles. Neurosci. Lett. 640, 1–5.
buccinator muscle. Cotofana, S., Fratila, A.A., Schenck, T.L., Redka-Swoboda, W., Zilinsky, I., Pavicic, T.,
2016. The anatomy of the aging face: a review. Facial Plast. Surg. 32 (3), 253–260.
D’Andrea, E., Barbaix, E., 2006. Anatomic research on the perioral muscles, functional
Author contributions
matrix of the maxillary and mandibular bones. Surg. Radiol. Anat. 28 (3), 261–266.
de la Cuadra-Blanco, D., Peces-Peña, M., Carvallo-de Moraes, L., Herrera-Lara, M.,
J.R.M.-V. and L.A.-A. conceived and designed the study. E.M.-S., Mérida-Velasco, J., 2013. Development of the platysma muscle and the superficial
J.C., E.M., J.M.-G., and J.R.M.-V. processed, studied, photographed, musculoaponeurotic system (human specimens at 8–17 weeks of development).
Sci. World J. 2013, 716962. https://doi.org/10.1155/2013/716962
and analyzed all the samples. M.C.B., I.P.-L., and M.G.-S. contributed Dutra, E.H., Caria, P.H., Rafferty, K.L., Herring, S.W., 2010. The buccinator during
to interpret the histological findings. J.R.M.-V., E.M.-S., J.C., and J.M.- mastication: a functional and anatomical evaluation in minipigs. Arch. Oral. Biol.
G. drafted the manuscript. All authors critically revised and com­ 55 (9), 627–638.
Gasser, R.F., 1967. The development of the facial muscles in man. Am. J. Anat. 120 (2),
mented on previous versions of the manuscript. All authors re­ 357–375.
viewed and approved the final manuscript. Gelse, K., Pöschl, E., Aigner, T., 2003. Collagens—structure, function, and biosynthesis.
Adv. Drug Deliv. Rev. 55 (12), 1531–1546. https://doi.org/10.1016/j.addr.2003.08.
002
Funding Ghassemi, A., Prescher, A., Riediger, D., Axer, H., 2003. Anatomy of the SMAS revisited.
Aesthetic Plast. Surg. 27 (4), 258–264.
This work has been supported by a Banco Santander/Universidad Hanawa, S., Tsuboi, A., Watanabe, M., Sasaki, K., 2008. EMG study for perioral facial
muscles function during mastication. J. Oral Rehabil. 35 (3), 159–170.
Complutense de Madrid research project [PR108/20-18] and funding
Hur, M.-S., 2017. Anatomical connections between the buccinator and the tendons of
from the Complutense Research Group 920202 [GRFN14/22]. the temporalis. Ann. Anat. 214, 63–66.
Hur, M.-S., Hu, K.-S., Kwak, H.-H., Lee, K.-S., Kim, H.-J., 2011. Inferior bundle (fourth
band) of the buccinator and the incisivus labii inferioris muscle. J. Craniofac. Surg.
Ethical statement
22 (1), 289–292.
Hwang, K., Choi, J.H., 2018. Superficial fascia in the cheek and the superficial mus­
All the specimens were the product of ectopic pregnancies or culoaponeurotic system. J. Craniofac. Surg. 29 (5), 1378–1382.
spontaneous abortions managed in the Department of Obstetrics Jin, Z.-W., Kim, J.H., Yamamoto, M., Katori, Y., Abe, H., Murakami, G., Abe, S.-I., 2022.
Growth in fetuses of the constrictor pharyngis superior with special reference to
and Gynecology at UCM, and none of the material indicated a pos­ its meeting with the buccinator: an embryological basis of adult variations in
sible malformation. The adult specimens came from the Center for palatopharyngeal anatomy. Surg. Radiol. Anat. 44 (4), 559–571. https://doi.org/10.
Body Donation and Dissection Rooms at UCM. The study was con­ 1007/s00276-022-02907-w
Kang, H., Kwak, H., Hu, K., Youn, K., Jin, G., Fontaine, C., Kim, H., 2006. An anatomical
ducted in accordance with the 1964 Declaration of Helsinki and its study of the buccinator muscle fibres that extend to the terminal portion of the
subsequent amendments. All cadavers were obtained following the parotid duct, and their functional roles in salivary secretion. J. Anat. 208 (5),
legal procedures governing body donation. Prior to death, all 601–607.

9
E. Martínez-Sanz, J. Catón, E. Maldonado et al. Annals of Anatomy 246 (2023) 152025

Kruglikov, I., Trujillo, O., Kristen, Q., Isac, K., Zorko, J., Fam, M., Okonkwo, K., Mian, A., Rouvière, H., Delmas, A., 2005. Anatomía humana: Vol. Tomo 1. Cabeza y cuello,
Thanh, H., Koban, K., 2016. The facial adipose tissue: a revision. Facial Plast. Surg. eleventha ed. Masson, Barcelona, Spain.
32 (06), 671–682. Rusu, M., Hostiuc, S., Jianu, A., 2021. A glimpse of past, the temporo-buccinator band
Lattouf, R., Younes, R., Lutomski, D., Naaman, N., Godeau, G., Senni, K., Changotade, S., of Hovelaque, or the buccotemporal fascia of Zenker. Morphologie 105 (350),
2014. Picrosirius red staining: a useful tool to appraise collagen networks in 189–195.
normal and pathological tissues. J. Histochem. Cytochem. 62 (10), 751–758. Sandulescu, T., Buechner, H., Rauscher, D., Naumova, E.A., Arnold, W.H., 2019.
https://doi.org/10.1369/0022155414545787 Histological, SEM and three-dimensional analysis of the midfacial SMAS – New
Light, N., Champion, A.E., 1984. Characterization of muscle epimysium, perimysium morphological insights. Ann. Anat. 222, 70–78. https://doi.org/10.1016/j.aanat.
and endomysium collagens. Biochem. J. 219 (3), 1017–1026. 2018.11.004
López-Fernández, P., Murillo-González, J., Arráez-Aybar, L.A., de la Cuadra-Blanco, C., Sandulescu, T., Spilker, L., Rauscher, D., Naumova, E., Arnold, W., 2018. Morphological
Moreno-Borreguero, A., Mérida-Velasco, J.R., 2019. Early stages of development of analysis and three-dimensional reconstruction of the SMAS surrounding the na­
the alar fascia (human specimens at 6-12 weeks of development). J. Anat. 235 (6), solabial fold. Ann. Anat. 217, 111–117.
1098–1104. https://doi.org/10.1111/joa.13074 Schieppati, M., Di Francesco, G., Nardone, A., 1989. Patterns of activity of perioral facial
Macchi, V., Tiengo, C., Porzionato, A., Stecco, C., Vigato, E., Parenti, A., Azzena, B., muscles during mastication in man. Exp. Brain Res. 77 (1), 103–112. https://doi.
Weiglein, A., Mazzoleni, F., De Caro, R., 2010. Histotopographic study of the fi­ org/10.1007/BF00250572
broadipose connective cheek system. Cells Tissues Organs 191 (1), 47–56. Shiratori, T., Ofusa, W., Tada, M., Yamamoto, M., Sato, A., Asakura, S., Yamada, Y., 2021.
Mérida-Velasco, J.R., Rodríguez-Vázquez, J.F., Mérida-Velasco, J.A., Sánchez- New method of recording the functional activity pattern of the buccinator from
Montesinos, I., Espín-Ferra, J., Jiménez-Collado, J., 1999. Development of the the mucosal surface. Physiol. Behav. 237, 113455. https://doi.org/10.1016/j.
human temporomandibular joint. Anat. Rec. 255 (1), 20–33. https://doi.org/10. physbeh.2021.113455
1002/(SICI)1097-0185(19990501)255:1<20::AID-AR4>3.0.CO;2-N Sperber, G.H., Sperber, S.M., 2018. Craniofacial Embryogenetics and Development,
O’Rahilly, R., Müller, F., 2000. Prenatal ages and stages-measures and errors. (thirda ed.), PMPH-USA Ltd., Raleigh, NC, USA.
Teratology 61 (5), 382–384. https://doi.org/10.1002/(SICI)1096-9926(200005) Standring, S., 2020. Gray’s anatomy: The anatomical basis of clinical practice, forty
61:5<382::AID-TERA10>3.0.CO;2-5 seconda ed. Elsevier, Amsterdam, The Netherlands.
O’Rahilly, R., Müller, F., 2010. Developmental stages in human embryos: revised and Stuzin, J.M., Rohrich, R.J., Dayan, E., 2019. The facial fat compartments revisited:
new measurements. Cells Tissues Organs 192 (2), 73–84. clinical relevance to subcutaneous dissection and facial deflation in face lifting.
Perkins, R.E., Blanton, P.L., Biggs, N.L., 1977. Electromyographic analysis of the “bac­ Plast. Reconstr. Surg. 144 (5), 1070–1078.
cinator mechanism” in human beings. J. Dent. Res. 56 (7), 783–794. https://doi. Sun, M., Chen, G.C., Wang, Y.Q., Song, T., Li, H.D., Wu, D., Yin, N.B., 2018. Anatomical
org/10.1177/00220345770560071301 characterization and three-dimensional modeling of the muscles at the corner of
Plas, E., Deliac, P., Garuet Lempirou, A., Caix, P., Bioulac, B., 2004. The buccinator the mouth: an iodine staining technique based on micro–computed tomography.
muscle: an original morphogenetical study. Morphologie 88 (280), 27–30. https:// Plast. Reconstr. Surg. 142 (3), 782–785.
doi.org/10.1016/s1286-0115(04)97995-7 Tomo, S., Tomo, I., Nakajima, K., Townsend, G.C., Hirata, K., 2002. Comparative
Purslow, P.P., 2020. The structure and role of intramuscular connective tissue in anatomy of the buccinator muscle in cat (Felis domestica). Anat. Rec. 267 (1),
muscle function. Front. Physiol. 11, 495. 78–86.
Rathee, M., Jain, P., 2022. Anatomy, Head and Neck, Buccinator Muscle. StatPearls Turrina, A., Martínez-González, M.A., Stecco, C., 2013. The muscular force transmission
Publishing, Treasure Island, FL. 〈http://www.ncbi.nlm.nih.gov/books/ system: role of the intramuscular connective tissue. J. Bodyw. Mov. Ther. 17 (1),
NBK546678/〉. 95–102.
Reid, S.E., Broad, M.T., Dickson, M., McDonald, S.W., 2003. The anatomy of von Arx, T., Nakashima, M., Lozanoff, S., 2018. The face–a musculoskeletal perspective.
buccinator—Insights from functional casts of the oral vestibule. Arch. Oral. Biol. 48 A literature review. Swiss Dent. J. 128 (9), 678–688.
(11), 787–795. https://doi.org/10.1016/s0003-9969(03)00136-5 Zhang, W., Liu, Y., Zhang, H., 2021. Extracellular matrix: an important regulator of cell
Ricard-Blum, S., 2011. The collagen family. Cold Spring Harb. Perspect. Biol. 3 (1), functions and skeletal muscle development. Cell Biosci. 11 (1), 1–13.
a004978. https://doi.org/10.1101/cshperspect.a004978

10