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Journal of Oral Biosciences 55 (2013) 208–216

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Journal of Oral Biosciences


journal homepage: www.elsevier.com/locate/job

Original Article

Origin of mandibular condylar cartilage in mice, rats, and humans:


Periosteum or separate blastema?
Shunichi Shibata a,n, Rei Sato a, Gen Murakami b, Hiroki Fukuoka c,
José Francisco Rodríguez-Vázquez d
a
Maxillofacial Anatomy, Department of Maxillofacial Biology, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan
b
Division of Internal Medicine, Iwamizawa Koujin-kai Hospital, Iwamizawa, Hokkaido, Japan
c
Maxillofacial Orthognathics, Department of Maxillofacial Reconstruction and Function, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan
d
Departamento de Anatomía y Embriología Humana II, Facultad de Medicina, Universidad Complutense de Madrid, Spain

art ic l e i nf o a b s t r a c t

Article history: Objective: To investigate if mandibular condylar cartilage is derived from the periosteum of the ossifying
Received 23 May 2013 mandible or from a separate, programmed blastema.
Received in revised form Materials and methods: Fetal mice at E14.0–16.0, fetal rats at E16.0–18.0, and human embryos at 9 and
25 July 2013
10 wks of gestation were used. The initial formation of rat condylar cartilage was investigated by using
Accepted 2 August 2013
Available online 10 September 2013
serial sections and enzyme-histochemistry to detect alkaline phosphatase activity. Histological observa-
tions of serial sections of human fetuses as well as 3D-reconstruction models were also analyzed. The
Keywords: expression of collagen type mRNA in developing rat condylar cartilage was directly compared with that
Mandibular condylar cartilage in mice by performing in situ hybridization.
Origin
Results: An anlage of the rat condylar process (condylar anlage) was clearly identified in the posterior
Alkaline phosphatase
position of the ossifying mandible and was continuous with it at E16.0. Newly formed rat condylar
Rat
Human cartilage was observed at E16.5 and was continuous with the ossifying mandible. Mesenchymal cells in
the condylar anlage at E16.0 showed alkaline phosphatase activity and chondrocytes in the newly formed
condylar cartilage also showed enzymatic activity. Thus, rat mandibular condylar cartilage that derives
from alkaline phosphatase-positive periosteum-like cells is continuous with the ossifying mandible, as
previously demonstrated in mice, but rapid differentiation into hypertrophic chondrocytes in rats is not
remarkable compared to that in mice. The condylar anlage and the newly formed cartilage were also
continuous with the ossifying mandible in human embryos.
Conclusions: Mammalian mandibular condylar cartilage derives from the periosteum of the ossifying
mandible in mice, rats, and humans.
& 2013 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.

1. Introduction separate, programmed blastema (termed the “blastema theory”),


however, has long been debated.
Mandibular condylar cartilage is classified as secondary carti- The blastema theory is supported by previous studies in humans
lage in embryology, and it differs somewhat from primary carti- [7,8], rats [9], and the Syrian hamster [10]. The periosteum theory is
lage, for example, the time of its appearance, its cell alignment, the supported by studies in humans [11], rats [12,13], and mice [14–17].
invasion pattern of capillaries [1,2], and the expression pattern of We previously reported that mouse mandibular condylar cartilage
different types of collagen [3,4]. Secondary cartilage has several derives from alkaline phosphatase (ALP)-positive, periosteum-like
definitions, one of the widest being that it appears later in cells that are continuous with the preliminarily formed ossifying
embryonic development, untouched by the primary cartilaginous mandible, and asserted that the periosteum theory applies to mouse
skeleton. A narrower definition is that it arises from the periostea condylar cartilage [15–17]. The origin of this cartilage, however, may
of membrane bone after (secondary to) bone formation [2,5,6]. differ between species, and this topic has not yet completely been
Whether or not the initial chondrogenesis of this cartilage starts resolved. A study by Duterloo and Jansen [9] has significantly
from the periosteum (termed the “periosteum theory”) or from a advanced the blastema theory. They reported that rat condylar
cartilage is initially formed within the blastema, which is separate
from the ossifying mandible at embryonic day (E) 17.0, and then
n
fuses with it at E18.0. The proposed schematic diagram in their paper
Correspondence to. Maxillofacial Anatomy, Department of Maxillofacial Biology,
Graduate School, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkuo-ku,
was adopted in a recently published textbook [6] despite the fact it
Tokyo, 113-8549, Japan Tel.: þ81 3 5803 5435; fax: þ 81 3 5803 0185. has not yet been confirmed. The blastemal origin theory was also
E-mail address: sshibata.mfa@tmd.ac.jp (S. Shibata). adopted in a popular orthodontic textbook [18]. To confirm whether

1349-0079/$ - see front matter & 2013 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.job.2013.08.001
S. Shibata et al. / Journal of Oral Biosciences 55 (2013) 208–216 209

the periosteum theory or the blastema theory applies to rat man- polymorphic cell zone, a flattened cell zone, and a hypertrophic
dibular condylar cartilage, we investigated the initial chondrogenesis cell zone. In previous papers, we used the term “anlage” to mean
of rat mandibular condylar cartilage by using coronally cut serial “blastema” [14–17]; therefore, in the present paper we continue to
sections and enzyme-histochemistry for ALP. use the term anlage.
In humans, chondrogenesis of mandibular condylar cartilage starts
at 9 wks of gestation [19], and has been well demonstrated to be 2.3. In situ hybridization
followed by morphological and histological changes during weeks 10–
15 of gestation [20]. However, it remains unclear which theory applies We used the same RNA probes for mouse collagen types I, II
to developing human mandibular condylar cartilage. Therefore, we and X as described in our previous reports [16,24]. For in situ
investigated serial sections and 3D reconstruction models of available hybridization of rat collagen type X, total RNA was extracted from
human samples at 8–9 wks of gestation to address this issue. the rib cartilage of rat fetuses at E18.0 and cDNA for rat collagen
Furthermore, we demonstrated that progenitor cells of this type Xα1 (col10a1); (NCBI: XM_002725875.2) was synthesized by
type of cartilage in mice rapidly differentiate into hypertrophic using the reverse transcription-polymerase chain reaction. The
chondrocytes that simultaneously express collagen types I, II, and following primers were used: forward, 5′(1394)-CTGGTCCAA-
X, and that this is another nature of secondary cartilage that is GAGGTCTCT-3′(1413); and reverse, 5′(1962)-TCATATGGGAGCCAC-
different from that of primary cartilage [15,16]. To confirm TAGGA-3′(1943). The PCR product was subcloned into a pCRII
whether this is also a characteristic of rat condylar cartilage, we vector (Strategene, La Jolla, CA). Antisense and sense probes were
directly compared the expression of collagen types I, II, and X labeled with 35S-UTP by using a Riboprobe in vitro Transcription
mRNA in rats with that in mice by performing in situ hybridization System (Promega, Madison, WI).
by using 35S-UTP labeled RNA probes. In situ hybridization by using 35S-UTP labeled probes was
performed as previously described [24,25]. Sense probes were
used as negative controls.
2. Materials and methods
We examined three different samples for each embryonic day
and the same results were obtained each time.
2.1. Samples collected

In total, 10 pregnant Wister-Albino rats at E16.0–18.0 and 10 2.4. Three dimensional reconstruction
pregnant ICR mice at E14.0–E16.0 were used in this study. The
animals were housed in facilities that were approved by Tokyo In human specimens, mesenchymal condensation (condylar
Medical and Dental University. Our animal-use protocol and anlage), ossifying matrix, condylar and Meckel's cartilages were
experimental system were reviewed and approved by the Screen- reconstructed in three dimensions (3D) as previously described
ing Committee for Animal Research of Tokyo Medical and Dental [26,27]. Briefly, serial sections were photographed every 50 μm
University (No. 0130081A). and the outlines of each structure were traced (see Fig. 5b and d,
In addition, 2 human specimens at 8 wks of gestation (Br-4, 28 mm and Fig. 6b). Then, the traced outlines were reconstructed by using
Crown rump length (CRL); Ca-2, 29 mm CRL) and those of 9 wks of a Tri-surf system (Ratoc system engineering, Tokyo, Japan).
gestation (Pe, 35 mm CRL; OY-2, 38 mm CRL) from the collection of
the Embryology Institute of the Universidad Complutense de Madrid
were used. The parameters that were used to determine the ages of 3. Results
gestation were CRL, weight, and cranial perimetry [21]. Approval of
our study was granted by the Ethics Committee of the Faculty of 3.1. General histology of developing rat mandibular condylar
Medicine of the University Complutense of Madrid. Samples from this cartilage in coronally-cut resin sections
collection have been well analyzed previously [19,20,22].
At E16.0, the anlage of the future condylar process (termed the
2.2. Tissue preparation and ALP staining condylar anlage), which appeared as mesenchymal cell condensa-
tion, was clearly identified in the posterior position of the ossify-
At each time point, pregnant rats/mice were killed by perform- ing mandible (Fig. 1a). At E16.5, a metachromatically stained
ing cervical dislocation under ether anesthesia, after which each matrix was first detected in the condylar anlage, which indicated
fetal rat/mouse was killed by carrying out cervical dislocation. The the initial formation of the condylar cartilage. The zonal architec-
heads were removed and then immersed in 4% paraformaldehyde ture was not clearly established, but the region posterior to the
(0.1 M phosphate buffer, pH 7.4) for 1 d at 4 1C. The specimens newly formed cartilage, which appeared as mesenchymal cell
were embedded in paraffin or Technovit 7100 resin (Kulzer, condensation, was termed “the embryonic zone” (Fig. 1b) as
Germany) by using standard procedures. Serial sections (2 mm previously described [15,24]. By E17.0, the condylar cartilage had
for resin, 5 mm for paraffin) were cut in the coronal plane, expanded in volume (Fig. 1c) and the architecture of each zone
perpendicular to the sagittal plane, and parallel to the long axis was established at E18.0 (Fig. 1d).
of the condylar process of the mandible. Sections were stained Serial sections at E16.0 confirmed that the condylar anlage was
with 0.1% toluidine blue (0.1 M phosphate buffer, pH 7.4) for clearly continuous with the ossifying mandible (Fig. 1e 1–9).
histologic observations. Cryosections 10-mm thick were cut in the Furthermore, serial sections at E16.5 confirmed that newly formed
coronal plane and ALP activity was detected by using the Azo-dye condylar cartilage within the condylar anlage was also continuous
method as previously described [15,17]. with the ossifying mandible (Fig. 1f 3–8).
Human samples were fixed in 10% formalin and embedded in
paraffin. Serial sections were cut at 10–20 mm in the frontal plane 3.2. ALP activity in the condylar anlage and newly formed
or transverse plane, stained with hematoxylin-eosin (H&E) or condylar cartilage
AZAN, and then examined under a light microscope.
The architecture of each zone was established at E16.0 in mice ALP activity was detected in all cells in the condylar anlage and in
or E18.0 in mice according to that present in the growing rat the continuous periosteum cells (Fig. 2a). This was further confirmed
condylar cartilage [23]: a fibrous cell (articulation) zone, a in serial sections (data not shown). At E16.5, ALP activity was also
210 S. Shibata et al. / Journal of Oral Biosciences 55 (2013) 208–216

Fig. 1. (a–d) Coronally cut rat condylar anlage at E16.0 (a), and formed condylar cartilage at E16.5 (b), E17.0 (c), and E18.0 (d). Condylar anlage, which appeared as
mesenchymal cell condensation (arrow in a), was continuous with the ossifying mandible (OM in a). Initial formation of condylar cartilage (arrow in b) was observed within
the condylar anlage and continuous with the ossifying mandible (OM in b). The embryonic zone (EZ) was observed posterior to the newly formed condylar cartilage. The
condylar cartilage was expanded in volume (arrow in c). The architecture of each zone (F, P, Fl, and H in d) became distinct at E18.0. (e) Serial sections (15 mm intervals) of
coronally cut condylar anlage at E16.0. Sections were serially cut from anterio-superior part (1) to posterio-inferior part (9). 5 corresponds to a. The condylar anlage (arrows
in 1–9) was clearly continuous with the ossifying mandible (OM). (f) Serial sections (15 mm intervals) of coronally cut, newly formed condylar cartilage at E16.5. Sections were
serially cut from anterio-superior part (1) to posterio-inferior part (10). Four corresponds to b. Newly formed condylar cartilage (arrows in 3–8) was clearly continuous with
the ossifying mandible (OM). MC: Meckel's cartilage; OM: ossifying mandible; EZ: embryonic zone; F: fibrous cell zone; P: polymorphic cell zone; Fl: flattened cell zone; and
H: hypertrophic cell zone. Technovit resin sections, toluidine blue staining. Bar ¼100 mm.

observed in chondrocytes in the newly formed chondrocytes anlage (Fig. 3c). No type X collagen mRNA was detected in the
(Fig. 2b), which was similar to condylar cartilage at E17.0 (Fig. 2c). condylar anlage (Fig. 3d).
At E15.0, type I collagen mRNA was strongly detected in
osteoblasts, and weakly detected in the embryonic zone and in
3.3. In situ hybridization for collagen types in developing condylar the upper part of newly formed condylar cartilage (Fig. 3e and f).
cartilage in mice Type II collagen mRNA was strongly expressed throughout the
condylar cartilage (Fig. 3g). Type X collagen mRNA was strongly
At E14.0, type I collagen mRNA was strongly expressed in detected in the lower two-thirds of the condylar cartilage (Fig. 3h).
osteoblasts and weakly expressed in the condylar anlage (Fig. 3a At E16.0, the zonal architecture was established (Fig. 3i). Type I
and b). Type II collagen mRNA was strongly detected in the collagen mRNA was strongly expressed in osteoblasts and weakly
temporal and sphenoid bone cartilages, but not in the condylar expressed from the fibrous cell zone to the flattened cell zone in
S. Shibata et al. / Journal of Oral Biosciences 55 (2013) 208–216 211

Fig. 2. ALP activity in the rat condylar anlage at E16.0 (a) and newly formed condylar cartilage at E16.5 (b) and E17.0 (c). ALP activity (purple) was detected in
mesenchymal cells in the condylar anlage (arrow in a), in newly formed chondrocytes (arrow in b and c), and in the continuous periosteum (P). Coronally cut frozen
sections. Bar ¼ 100 mm.

the condylar cartilage (Fig. 3j). Type II collagen mRNA was expressed 3.5. Histology of developing human mandibular condylar cartilage
from the flattened cell zone to the upper hypertrophic cell zone, but
was reduced in the lower hypertrophic cell zone (Fig. 3k). Type X In human sections at 8 wks of gestation, two dense mesench-
collagen mRNA was expressed in the entire hypertrophic cell zone, ymal cell condensations were observed lateral to Meckel's carti-
which still occupied the lower two-thirds of the condylar cartilage lage in the posterior end of the future mandibular ramus in the
(Fig. 3l). frontal plane (Fig. 5a). Toward the anterior direction, these
mesenchymal cell condensations were fused to form an anlage
of the future mandibular ramus (Fig. 5b), and ossification occurred
3.4. In situ hybridization for collagen types in developing condylar in more anterior positions to form ossifying mandibular rams
cartilage in rats (Fig. 5c–f). The lateral pterygoid muscle was attached to the
position close to the upper part of the ossifying mandibular ramus
At E16.0, type I collagen mRNA was strongly expressed in (Fig. 5d–f), which indicated that the upper mesenchymal cell
osteoblasts and weakly expressed in the condylar anlage (Fig. 4a condensation was condylar anlage. This was clearly continuous
and b). Type II collagen mRNA was strongly detected in Meckel's with the ossifying mandible, but no separate anlage of condylar
cartilage, but not in the condylar anlage (Fig. 4c). No type X cartilage was observed (Fig. 5a). Meanwhile, the medial pterygoid
collagen mRNA was detected in the condylar anlage (Fig. 4d). muscle and masseter muscle were attached to the lower part of
At E17.0, type I collagen mRNA was strongly detected in the ossifying mandibular ramus (Fig. 5e and f), which indicated
osteoblasts, and weakly detected in the embryonic zone and in that the lower mesenchymal condensation was the anlage of
the upper part of newly formed condylar cartilage (Fig. 4e and f). angular cartilage (Fig. 5a).
Type II collagen mRNA was strongly expressed throughout the Tracing outlines of mesenchymal condensation (including con-
condylar cartilage (Fig. 4g). Type X collagen mRNA was first dylar anlage), ossifying matrix, and Meckel's cartilage are depicted
detected in the lower part of the condylar cartilage, but its in Fig. 5b and d.
intensity was low and the positive area only occupied the lower 3D-reconstruction further confirmed that the mandibular anlage
half of the condylar cartilage (Fig. 4h). was continuous with the ossifying mandibular ramus (Fig. 5g
At E18.0, the architecture of each zone was recognizable and h).
(Fig. 4i). Type I collagen mRNA was strongly expressed in osteo- In another human section at 8 wks of development, condylar
blasts, and weakly expressed from the fibrous cell zone to the anlage (Fig. 5i) was continuous with the ossifying mandible
flattened cell zone (Fig. 4j). Type II collagen mRNA was expressed (Fig. 5j).
from the flattened cell zone to the entire hypertrophic cell zone, Human sections at 9 wks of development showed that the
but only slightly reduced in the lower hypertrophic cell zone condylar anlage, to which the lateral pterygoid muscle was
(Fig. 4k). Type X collagen mRNA was expressed in the entire attached, was continuous with the ossifying mandibular ramus
hypertrophic cell zone, but its intensity was still not strong and the (Fig. 6a). Enlargement of cells and intercellular matrix within the
positive area only occupied the lower half of the condylar cartilage condylar anlage indicated the occurrence of condylar cartilage
(Fig. 4l). Negative controls that were created by using sense probes formation (Fig. 6b–f). This cartilage was small and could be
showed no positive reactions at any stage that we examined (data observed within only a limited region (  300 mm in width),
not shown). which indicated that it could be newly formed cartilage. This
212 S. Shibata et al. / Journal of Oral Biosciences 55 (2013) 208–216

Fig. 3. Mouse condylar anlage and condylar cartilage in the coronal plane at E14.0 (a–d), E15.0 (e–h), and E16.0 (i–l). Toluidine blue staining (a, e, and i), in situ hybridization
for collagen types I (b, f, and j), II (c, g, and k), and X (d, h, and l) in dark fields except for d (light field). (a) Condylar anlage (CA) was clearly identified in the posterior position
of the ossifying mandible (OM). Temporal bone cartilage (TBC) and sphenoid bone cartilage (SBC) were seen. (b) Type I collagen mRNA was strongly expressed in osteoblasts
in the ossifying mandible (arrow) and weakly expressed in the condylar anlage (arrowhead). (c) Type II collagen mRNA was strongly detected in the temporal cartilage (TBC)
and sphenoid bone cartilage (SBC), but not in the condylar anlage (arrowhead). (d) No type X collagen mRNA was detected in the condylar anlage (CA) and in the ossifying
mandible (OM). (e) The bone collar (BC) had formed around the newly formed condylar cartilage (CC). The embryonic zone (EZ), which consisted of mesenchymal cells, was
observed. (f) Type I collagen mRNA was strongly detected in osteoblasts in the bone collar (BC), and was weakly detected in the embryonic zone (arrowhead) and in the
upper part of the condylar cartilage (arrow). (g) Type II collagen mRNA was strongly expressed throughout the condylar cartilage (arrow) and in the temporal cartilage (TBC)
and sphenoid bone cartilage (SBC). (h) Type X collagen mRNA was strongly detected in the lower two-thirds of the condylar cartilage (arrow) and in the f sphenoid bone
cartilage (SBC). (i) The zonal architecture consisting of a fibrous cell zone (F), a polymorphic cell zone (P), a flattened cell zone (Fl) and a hypertrophic cell zone (H) was
established. (j) Type I collagen mRNA was strongly expressed in osteoblasts in the bone collar (BC), and weakly expressed from the fibrous cell zone to the flattened cell zone
in the condylar cartilage (arrowheads). (k) Type II collagen mRNA was expressed in the temporal bone cartilage (TBC) and in condylar cartilage from the flattened cell zone to
the upper hypertrophic cell zone (arrows), but was reduced in the lower hypertrophic cell zone (arrowheads). (l) Type X collagen mRNA was expressed in the entire
hypertrophic cell zone (arrow). Bar ¼ 100 mm.

cartilage was also continuous with the ossifying mandibular In another human section at 9 wks of development, although
ramus at its upper-medial side (Fig. 6b–d). The 3D reconstruction condylar cartilage was not clearly formed, slight expansion of the
confirmed that the newly formed cartilage was continuous with intercellular matrix within the condylar anlage implied cartilage
the ossifying mandibular ramus at its upper-medial side (Fig. 6i formation (Fig. 6k), which continued to the ossifying mandibular
and j). ramus (Fig. 6l).
S. Shibata et al. / Journal of Oral Biosciences 55 (2013) 208–216 213

Fig. 4. Rat condylar anlage and condylar cartilage in the coronal plane at E16.0 (a–d), E17.0 (e–h), E18.0 (i–l). Toluidine blue staining (a, e, and i), in situ hybridization for
collagen types I (b, f, and j), II (c, g, and k), and X (d, h, and l) in dark fields except for d (light field). (a) Condylar anlage (CA) was clearly identified in the posterior position of
the ossifying mandible (OM). Meckel's cartilage (MC) was also seen. (b) Type I collagen mRNA was strongly expressed in osteoblasts in the ossifying mandible (arrow),
weakly expressed in the condylar anlage (arrowhead), and not expressed in Meckel's cartilage (MC). (c) Type II collagen mRNA was strongly detected in Meckel's cartilage
(arrow), but not in the condylar anlage (arrowheads). (d) Type X collagen mRNA was not detected in the condylar anlage (CA) and ossifying mandible (OM). (e) The bone
collar (BC) had formed around the newly formed condylar cartilage (CC) adjacent to the ossifying mandible (OC). The embryonic zone (EZ) and Meckel's cartilage (MC) were
observed. (f) Type I collagen mRNA was strongly detected in osteoblasts in the bone collar (BC), weakly detected in the embryonic zone (arrowhead) and in the upper part of
condylar cartilage (arrow), and not detected in Meckel's cartilage (MC). (g) Type II collagen mRNA was strongly expressed throughout the condylar cartilage (arrow) and in
Meckel's cartilage (MC). (h) Type X collagen mRNA was slightly expressed in the lower part of the condylar cartilage (arrow) and not in Meckel's cartilage (MC). (i) The
architecture of each zone (F, P, Fl, and H) was observed. Temporal bone cartilage (TBC) and sphenoid bone cartilage (SBC) were seen. (j) Type I collagen mRNA was strongly
expressed in osteoblasts in the bone collar (BC), and weakly expressed from the fibrous cell zone to the flattened cell zone (arrowheads). (k) Type II collagen mRNA was
expressed in temporal bone cartilage (TBC) and in sphenoid bone cartilage (SBC). It was also expressed in condylar cartilage from the flattened cell zone to the entire
hypertrophic cell zone (arrows), but only slightly reduced in the lower hypertrophic cell zone (arrowhead). (l) Type X collagen mRNA was moderately expressed in the
hypertrophic cell zone (arrow). F: fibrous cell zone; P: polymorphic cell zone; Fl: flattened cell zone; and H: hypertrophic cell zone. Bar ¼ 100 mm.
214 S. Shibata et al. / Journal of Oral Biosciences 55 (2013) 208–216

Fig. 5. (a–f) Serial sections (200 mm intervals) of human embryos at 8 wks of gestation (Br-4) cut along the frontal plane and stained with H&E (a, d, and e) or AZAN (b, c, in
Meckel's f). Sections were serially cut from posterior part (a) to anterior part (f). Two mesenchymal cell condensations were observed lateral to Meckel’s cartilage (MC) in the
posterior end of future mandibular ramus (arrows in a). The upper condensation and lower condensation can be regarded as condylar anlage (CA) and angular anlage (AA),
respectively. In the anterior direction, these mesenchymal cell condensations were fused to form an anlage of the future mandibular ramus (arrow in b) and ossification
occurred in more anterior positions to form the ossifying mandibular ramus (OMR in c–f). An anlage of the articular disc was also observed (arrowheads in a–d), but
disappeared at the point where masseteric, inferior alveolar, and lingual nerves were branched (MN, IAN, and LN in e). The lateral pterygoid muscle (LPM) was attached to
the upper part of the ossifying mandibular ramus (arrows in d–f). The medial pterygoid muscle (MPM) and masseter muscle (MM) were attached to the lower part of the
ossifying mandibular ramus (arrowheads in e and f). The tracing outline of mesenchymal condensation including condylar anlage (black), Meckel’s cartilage (blue), and
ossifying matrix (red) are described as dotted lines in b and d. (g and h) 3D reconstruction models of the future mandibular ramus region viewed from the medio-posterior
direction (g) and the latero-posterior (h) direction. Mesenchymal condensation (green) was formed lateral to Meckels cartilage (blue), and ossifying matrix (red) was formed
within the mesenchymal condensation. Condylar anlage (arrow) was continuous with the ossifying matrix. (i and j) Serial sections (800 mm intervals) of embryo at 8 wks of
gestation (Ca-2) cut along the horizontal plane stained with H&E. Condylar anlage (arrow in i) was continuous with ossifying matrix (arrow in j). MC: Meckel's cartilage.
Bar ¼100 mm.

4. Discussion findings were based on frontally cut, paraffinized serial sections,


whereas we used coronally cut serial resin sections. These differ-
4.1. Origin of mandibular condylar cartilage in mice, rats, and ences, especially the differences in the cutting plane, likely caused
humans the discrepancy between our results and theirs. We believe that
our methods provide a greater advantage, since coronally cut
In rat fetuses, we first detected a metachromatically stained matrix sections can directly demonstrate the positional relationship
in the condylar anlage at E16.5, which indicated the initial formation between ossifying mandible and condylar anlage/cartilage.
of condylar cartilage at this stage. Serial sections confirmed that the Although Duterloo and Jansen [9] described the existence of a
newly formed condylar cartilage was continuous with the ossifying mesenchymal cell condensation of the condylar blastema at E16.0,
mandible. its connection to the ossifying mandible was not confirmed. By
Duterloo and Jansen [9] reported that the initial formation of using coronally cut serial sections, we clearly identified the
condylar cartilage in rat fetuses occurs at E17.0, and that there is condylar anlage in the posterior position of the ossifying mandible
no direct connection with the ossifying mandible. Based on their and observed its continuation with the ossifying mandible
findings, they asserted the blastema theory in which condylar at E16.0.
cartilage is derived from a separate blastema and is not an original We further observed that mesenchymal cells in the condylar
part of the ossifying mandible. The proposed schematic diagram in anlage at E16.0 showed ALP activity, which indicated that these cells
their paper was still adopted in a recently published textbook [6]. have pre-osteoblastic characteristics. Furthermore, chondrocytes in
Our present findings as well as the findings of Durterloo and the newly formed cartilage at E16.5 also showed ALP enzymatic
Jansen [9] are in complete opposition with each other. Their activity, which indicated that the newly formed condylar cartilage
S. Shibata et al. / Journal of Oral Biosciences 55 (2013) 208–216 215

Fig. 6. (a–h) Serial sections (200 mm intervals) of human embryo at 9 wks of gestation (OY-2) cut along the frontal plane and stained with H&E (a, c, e, and g) or AZAN (b, d, f,
and h). Sections were serially cut from posterior part (a) to anterior part (h). e and f are enlargements of the rectangular areas in c and d, respectively. (a–h) Condylar anlage
(CA in a), to which the lateral pterygoid muscle (LPM in a–d, g, and h) was attached, was continuous with the ossifying mandibular ramus (OMR in a–d, g, and h). The
expansion of cells and intercellular matrix within the condylar anlage (arrows in e and f) indicated the condylar cartilage formation (CC in b–d). At the posterior position, this
cartilage was attached to the ossifying mandibular ramus along its medial side (CC in b and c, arrow in e), but was attached to its upper side at the anterior position (CC in d,
arrows in f), which indicated that the cartilage covered the ossifying matrix from its upper-medial side. (i and j) 3D reconstruction models of the future mandibular ramus
region viewed from the medio-posterior direction (i) and the latero-posterior direction (j). Newly formed condylar cartilage (arrow) covered the ossifying matrix (red) from
the upper-medial side. The green line indicates the outline of mesenchymal condensation. (k and l) Serial sections (800 mm intervals) of a human embryo at 9 wks of
gestation (PE) cut along the horizontal plane and stained with AZAN. Slight expansion of the intercellular matrix indicated cartilage formation within the condylar anlage
(arrow in k), which was continuous with ossifying matrix (arrow in l). MC: Meckel's cartilage. The tracing outlines of mesenchymal condensation (black), condylar cartilage
(blue), Meckel's cartilage (blue), and ossifying matrix (red) are depicted as dotted lines in b. Bar ¼ 100 mm.

was derived from periosteum-like tissue as described in previous cartilage could not be classified as secondary cartilage in terms of
studies on mice [15,28]. Therefore, we propose that the periosteum this definition. The present findings confirmed that the condylar
theory, in which condylar cartilage derives from ALP-positive cartilage could be classified as secondary cartilage in terms of the
periosteum-like tissues that are continuous with the ossifying narrowest definition.
mandible, can also be applied to rat condylar cartilage.
Both the blastema theory [7,8] and the periosteum theory [11]
have been proposed for humans, but no consensus has been 4.2. Direct comparison between mice and rats of expression of
reached on which to use. These previous studies did not present collagen types in developing mandibular condylar cartilage
complete serial sections. However, the serial sections and 3D
reconstruction models in the present study indicated that both In the present study, type I collagen mRNA was weakly expressed
the condylar anlage at 8 wks of gestation and newly formed in the mesenchymal cells of the condylar anlage at E14.0 in mice and
condylar cartilage at 9 wks of gestation were continuous with at E16.0 in rats. It was subsequently expressed in chondrocytes that
the ossifying mandible. These findings suggest that the periosteum were located in the embryonic zone and the upper layer of cartilage
theory applies to human mandibular condylar cartilage as well as up to E16.0 in mice and E18.0 in rats. Therefore, the type I collagen
that of rats and mice, although we did not perform enzyme- mRNA expression pattern was similar between both types of
histochemical or immunohistochemical analyses. Schematic mod- rodents. Previous studies on developing condylar cartilage in mice
els of the blastema theory and the periosteum theory are sum- demonstrated the same results [4,16]. In addition, a similar expres-
marized in Fig. 7. sion pattern of type I collagen was also reported in postnatal mice/
One most narrow definition of secondary cartilage is that it rats [3,29], thereby indicating fibrocartilaginous characteristics of
arises from the periosteum of membrane bone after (secondary to) this cartilage. This also supports the periosteum theory as described
bone formation. If the blastema theory holds true, the condylar above.
216 S. Shibata et al. / Journal of Oral Biosciences 55 (2013) 208–216

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Osterix, and Sox9 in the anlagen of mouse mandibular condylar cartilage in
No potential conflicts of interest exist. the early stages of embryogenesis. J Anat 2008;213:274–83.
[26] Shibata S, Suda N, Yoda S, Fukuoka H, Ohyama K, Yamashita Y, Komori T.
Runx2-deficient mice lack mandibular condylar cartilage and have deformed
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Acknowledgments [27] Shibata S, Dias RA, Hashimoto-Uoshima M, Abe T, Yanagishita M. Immuno-
histochemical localization of syndecan-1 in the dental follicle of postnatal
This work was supported by a Grant-in-Aid for Scientific Research mouse teeth. J Periodontol 2007;78:1322–8.
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(No. 22592044) from the Ministry of Education, Culture, Sports,
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Science, and Technology of Japan. mouse embryos. J Anat 1997;190:239–60.
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immunoreactivity of the mandibular condylar cartilage in a naturally aging
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