Research Article An Immunohistochemistry Study …downloads.hindawi.com/journals/bmri/2013/265380.pdfResearch Article An Immunohistochemistry Study of Sox9, Runx2, and Osterix Expression

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 265380 11 pageshttpdxdoiorg1011552013265380

Research ArticleAn Immunohistochemistry Study of Sox9 Runx2 and OsterixExpression in the Mandibular Cartilages of Newborn Mouse

Hong Zhang12 Xiaopeng Zhao3 Zhiguang Zhang4 Weiwei Chen25 and Xinli Zhang2

1 Department of Orthodontics Guanghua School of Stomatology Sun Yat-Sen University Guangzhou 510055 China2Dental and Craniofacial Research Institute School of Dentistry University of California Los Angeles CA 90095 USA3Department of Oral and Maxillofacial Surgery Sun Yat-sen Memorial Hospital Sun Yat-Sen University Guangzhou 510120 China4Department of Oral and Maxillofacial Surgery Guanghua School of Stomatology Sun Yat-Sen University Guangzhou 510055 China5 Institute for Medical Biology College of Life Sciences South-Central University for Nationalities Wuhan 430074 China

Correspondence should be addressed to Zhiguang Zhang drzhangzg163com and Xinli Zhang xzhangdentistryuclaedu

Received 6 January 2013 Accepted 7 April 2013

Academic Editor Chad M Novince

Copyright copy 2013 Hong Zhang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The purpose of this study is to investigate the spacial expression pattern and functional significance of three key transcriptionfactors related to bone and cartilage formation namely Sox9 Runx2 and Osterix in cartilages during the late development ofmouse mandible Immunohistochemical examinations of Sox9 Runx2 and Osterix were conducted in the mandibular cartilagesof the 15 neonatal C57BL6N mice In secondary cartilages both Sox9 and Runx2 were weakly expressed in the polymorphic cellzone strongly expressed in the flattened cell zone and throughout the entire hypertrophic cell zone Similarly both transcriptionalfactors were weakly expressed in the uncalcified Meckelrsquos cartilage while strongly expressed in the rostral cartilage MeanwhileOsterix was at an extremely low level in cells of the flattened cell zone and the upper hypertrophic cell zone in secondary cartilagesSurprisingly Osterix was intensely expressed in hypertrophic chondrocytes in the center of the uncalcified Meckelrsquos cartilage whilemoderately expressed in part of hypertrophic chondrocytes in the rostral process Consequently it is suggested that Sox9 is a mainand unique positive regulator in the hypertrophic differentiation process of mandibular secondary cartilages in addition to Runx2Furthermore Osterix is likely responsible for phenotypic conversion of Meckelrsquos chondrocytes during its degeneration

1 Introduction

Thedevelopment of cartilages plays a pivotal role in the devel-opment and growth of the mandible Mandibular cartilagesare derived from ectomesenchymal cells of the first pharyn-geal arch but their characteristics differ Meckelrsquos cartilage isa fetal cartilaginous skeleton in the mandible Although it isclassified as primary cartilage similar to limb bud cartilage itcontains four distinct portions each having a different fateThe anterior intermediate and proximal portions convertto intramandibular symphysis sphenomandibular ligamentand the inner ear ossicles respectively The posterior portionof intramandibular Meckelrsquos cartilage facing the developingmolar buds undergoes developmental events similar to endo-chondral ossification but the degradationmechanisms of thisportion are distinct from those in endochondral ossification[1] Independent of the chondroskeleton four secondary

cartilages including the condylar coronoid angular and sym-physeal cartilage strongly influence the further developmentof the mandible These secondary cartilages differ from theprimary cartilage in embryonic origin morphological andbiochemical organization They are derived from the perios-teum of intramembranous bone after (secondary to) boneformation [2 3] Furthermore they display a unique mode ofcell proliferation and differentiation The condylar cartilageas a principle secondary cartilage does not form columns ofproliferating chondrocytes and grows multidirectionally toadapt to the mandibular fossa of the temporal bone [4]

Recent studies showed that the threemaster transcriptionfactors of Sox9 Runx2 and Osterix are involved in theformation of Meckelrsquos cartilage and mandibular condylarcartilage [3 10] Sox9 Runx2 and Osterix are key tran-scription factors which are necessary in skeletal cell fatedecision [11] Sox9 (SRY-box containing gene 9) is an essential

2 BioMed Research International

and nonredundant factor of chondrogenesis Analyses ingenetically modified mice revealed that Sox9 promotes theearly stage but suppresses the terminal stage of chondro-cyte differentiation in limb bud cartilage [12ndash14] On thecontrary the multifunctional transcription factor Runx2which is expressed in prehypertrophic and hypertrophicchondrocytes is a main positive regulator of hypertrophicdifferentiation in late chondrogenesis of the limb buds [515 16] New in vitro data demonstrated that Sox9 negativelyregulates Runx2 by enhancing Bapx1 expression which leadsto the inhibition of terminal chondrocyte differentiation[17] Osterix which acts downstream of Runx2 during boneformation is expressed in chondrocyte progenitors and pre-hypertrophic chondrocytes in rib spine and limb cartilagessuggesting that Osterix may play a critical role during theprimary cartilagematuration in combinationwith Runx2 andSox9 [6 7]

However the transcriptional control of the later develop-ment of mandibular cartilages remains poorly understoodAt birth the rostral process of intramandibular Meckelrsquoscartilage is undergoing endochondral ossification while theposterior portion of intramandibular Meckelrsquos cartilage isdegenerating [18ndash20] Meanwhile four secondary cartilagesespecially the condylar cartilage were not well documentedin terms of their developmental characteristics although theyfunctionmainly as a growth cartilage similar to limb bud car-tilage At present transcription factors are attracting increas-ing clinical attention because of their roles in the etiologyand pathogenesis of malformations and growth disordersdegenerative diseases and in regenerative and repair pro-cesses [21 22] The findings that Runx2-deficient mice lackmandibular condylar cartilage and had deformed Meckelrsquoscartilage indicate that Runx2 is essential for the formationof the mandibular cartilages [23] In many cleidocranialdysplasia (CCD) patients who were link to Runx2 deficenthowever there are no abnormal findings in the mandiblein spite of cases of condylar malformation persistent sym-physis or a narrow coronoid process being also known[24 25] These investigations provided a hint that Runx2may be just one of essential biological factors influencingthe development and growth of mandibular cartilages Thepresent study is to examine tissue distribution of Sox9 Runx2and Osterix in newborn mice mandibular cartilages usingimmunohistochemistry technique and investigate whetherthese transcription factors have similar functions to those inlimb bud cartilage which will contribute to current under-standing of mechanisms of the development of mandibleand the possible pathogenesis of some craniofacial anomaliesinvolving mandible

2 Materials and Methods

All animals were housed and handled in accordance withguidelines of the Chancellorrsquos Animal Research Committeeof the Office for Protection of Research Subjects at theUniversity of California Los Angeles CA USA

21 Tissue Preparation A total of 15 newborn C57BL6Nmice were collected in 2 hours right after being delivered and

used for this study The mandibles were then removed andimmersed in 4 paraformaldehyde (01M phosphate bufferpH 74) for 1 day at 4∘CThe specimens were decalcified with10 EDTA for 5 days at 4∘C and then embedded in paraffinusing standard procedures Sections (5 120583m) were cut in theplane parallel to the ascending ramus of the mandible allthe mandibular cartilages being in one section For generalmorphology deparaffinized sectionswere stainedwith hema-toxylin and eosin The skeletal staining with Alizarin Red-Alcian Blue was performed for preparation of gross specimenof mandible as reported previously [26]

22 Immunohistochemistry Five-micron-thick paraffin sec-tions were dewaxed in xylenes and rehydrated in ethanolbaths Endogenous peroxidases were blocked by incubat-ing sections in 3 hydrogen peroxide for 20min at roomtemperature Sections were incubated with anti-Runx2 anti-Osterix and anti-Sox9 primary antibodies (Santa CruzBiotechnology CA USA) (dilution 1 100) and biotinylatedanti-rabbit or anti-goat IgG secondary antibody (VectorLaboratories Burlingame CA) for 1 h at room temperaturePositive immunoreactivity was detected using VectastainABC kit (Vector Laboratories Burlingame CA USA) andAEC chromogenic substrate (Dako Carpiteria CA USA)with red positive staining A negative control was performedby replacing primary antibody solutions with PBS Sectionswere counterstained with hematoxylin for 30 sec followedby rinsing 5min in running water Photomicrographs wereacquired using an Olympus BX51 Image-pro Plus 60 soft-warewas used to calculate stained area and IntegratedOpticalDensity (IOD) The average optical density (mean density)represented the intensity of protein expression and wascounted in 4 random fields (times20 objective) of each cartilagearea and trabecular bone area per section The mean densityis equal to (IOD SUM)area For exact analysis three sectionswere prepared at similar plane for each sample ANOVA wasused for multiple groupsrsquo comparison and Studentrsquos 119905-testwas used for comparison between any two groups Statisticalsignificance defined as 119875 lt 005

3 Results

31 Histological Analysis of Cartilages in Newborn MouseMandible Mandibular cartilages in newborn mouse inclu-ded the portions of Meckelrsquos cartilage condylar angularand symphyseal secondary cartilage while cartilage was notpresent in the coronoid process of the newborn mouse(Figure 1(a)) On the basis of the cellular morphologicalchanges themandibular condylar and angular cartilageswerehistologically composed of four different cell zones a thinfibrous cell zone a polymorphic cell zone a wider flattenedcell zone and a broad hypertrophic cell zone occupied thelower half of the organ (Figures 1(b) and 1(c)) Almost allof the intramandibular bar of Meckelrsquos cartilage had ossifiedcompletely but a small amount ofMeckelrsquos cartilage remainedin a limited portion of the rostral region and at themylohyoidgroove between the condylar and angular processes At theposterior portion of intramandibular Meckelrsquos cartilage HEstaining pattern of the matrix changed from the intense

BioMed Research International 3

CD

PM

AG

PM

Mo

SS

RC

(a)

HY

FP

FL

(b)

AG

PM

HYHY

FPFL

(c)

PM HY R

Mo

I

(d)

ISS

RCHY

(e)

Figure 1 Histological analysis of mandibular cartilages of mice at newborn stage (a) Lingual view of mandible by Alizarin Red and AlcianBlue staining (b) hematoxylin-eosin sections of condylar cartilage and (c) hematoxylin-eosin sections of angular cartilage (AG) similarlydisplaying four different cell zones a thin fibrous cell zone (F) a polymorphic cell zone (P) a wider flattened cell zone (FL) and a broadhypertrophic cell zone (HY) (d) hematoxylin-eosin staining pattern of the matrix of the posterior portion of intramandibular Meckelrsquoscartilage (PM) changed from the intense hematoxylin to the light eosin in the resorption (R) facing the molar buds (Mo) and incisor (I) (e)hematoxylin-eosin sections of the endochondral ossification rostral process of Meckelrsquos cartilage (RC) and symphyseal secondary cartilage(SS) facing the incisor (I) Scale bar 100 120583m for (a) and (b) 250 120583m for (c) and (d) and 50 120583m for (e)


hematoxylin to the light eosin in resorption area whichindicated the degradation of Meckelrsquos cartilage matrix duringdevelopment (Figure 1(d)) Furthermore the endochondralossification rostral process of Meckelrsquos cartilage and symphy-seal secondary cartilage serve as a chondrogenic mandibu-lar symphysis of newborn mice (Figure 1(e)) The openedchondrocytic lacunae and disconnected cartilaginous matrix(arrows in Figures 1(b) 1(c) and 1(e)) were clearly foundin the resorption area of condylar angular and symphy-seal secondary cartilage in addition to rostral cartilagewhile the appearing perichondrium and the eosinophiliccartilage erode on the lateral sides (arrows in Figure 1(d))were observed in the posterior portion of intramandibularMeckelrsquos cartilage These results indicated that the degra-dation of cartilaginous matrix in the posterior portion ofintramandibular Meckelrsquos cartilage is distinct from othersamong mandibular cartilages

32 Immunohistochemical Analysis of Sox9 Rux2 and Osterixin Cartilages of the Newborn Mouse Mandible Interestinglytranscription factors Sox9 and Runx2 showed similar expres-sion level and tissue distribution patterns throughout allthe mandibular cartilages of newborn mice In secondarycartilages both Sox9 and Runx2 were weakly expressed bycells in the polymorphic cell zone strongly in the flattenedcell zone and throughout the entire hypertrophic cell zoneTo quantitatively measure changes in expression of tran-scriptional factors critical for chondrogenic differentiationSox9 (Figure 2(f)) and Runx2 (Figure 2(h)) immunohisto-chemistry in hypertrophic zones of mandibular cartilages atnewborn stage were quantitated by average optical density ofpositive stainingThe expression levels of both transcriptionalfactors in the degrading posterior portion of intramandibularMeckelrsquos cartilage (Figures 2(b) and 2(d)) exhibited a signif-icantly decrease compared with others among mandibularcartilages Meanwhile cells in the rostral cartilage (Figures2(e) and 2(g)) and cells in extramandibular Meckelrsquos cartilage(Figures 2(f) and 2(h)) similarly expressed both transcrip-tional factors more than in the degrading posterior portionof intramandibular Meckelrsquos cartilage Unexpectedly Sox9 asRunx2 was expressed in all the terminal chondrocytes of themandibular cartilages in newborn mice (Figures 2(a) 2(b)and 2(e)) contrary to the express pattern of Sox9 in limb budcartilage [8]This spatial distribution pattern indicated Sox9rsquosrequirement in the terminal stage ofmandibular chondrocytedifferentiation

Runx2 and Osterix are involved in the formation ofMeckelrsquos cartilage and mandibular condylar cartilage [3 10]Thus we correlated the expression patterns of Runx2 (Figures2(c) 2(d) and 2(g)) and Osterix (Figure 3) in mandibularcartilages at newborn stages Results showed that Osterix wasat an extremely low level in part of cells of the flattened cellzone and the upper hypertrophic cell zone in condylar car-tilage and angular cartilage independent on Runx2 UnlikeSox9 the spatial pattern of Osterix in condylar cartilageand angular cartilage was consistent with that in limb budcartilage [7 9] Notably Osterix was intensely expressed onlyin hypertrophic chondrocytes of the center of the uncalcifiedMeckelrsquos cartilage containing the strong basophilic matrix

while it was entirely absent in hypertrophic chondrocytes inthe resorption area containing the light eosinophilic matrix(Figures 3(b) 1(c) and 1(d)) Additionally the expressionlevel of Osterix in the hypertrophic chondrocytes of Meckelrsquoscartilage (Figure 3(b)) was significantly higher comparedwith that in condylar cartilage and angular cartilage whichhave only few positive cells in the flattened cell zone and theupper hypertrophic cell zone (Figures 3(a) 3(b) and 3(d))Moreover Osterix wasmoderately expressed in part of hyper-trophic chondrocytes in the rostral process (Figure 3(c))while it was absent in extramandibular Meckelrsquos cartilage Atpresent themechanisms ofOsterix regulation of chondrocytedifferentiation and function are still under investigationwhereas the significantly intense immunohistochemistry ofOsterix in hypertrophic chondrocytes of the center of theuncalcified Meckelrsquos cartilage provided evidence of Osterixrsquosrole in the degradation of the posterior portion of uncalcifiedintramandibular Meckelrsquos cartilage

33 Comparison of Expression Intensity of Runx2 and Osterixin the Chondrocytes with That in the Osteoblasts of theNewborn Mouse Mandible Since Runx2 and Osterix areindispensable for osteoblast differentiation and known to beexpressed in osteoblasts we first confirmed the positive stain-ing of osteoblasts using the same sections of cartilages withRunx2 and Osterix positive staining which also validatedour IHC approach to be highly reliable Then we comparedthe expression intensity of the two key transcriptional factorsrelated to bone formation in chondrocytes with that inmandibular osteoblasts to further confirm the significanceof both during the development of mandibular cartilagesSimilar to the condylar subchondral bone in 56-day-oldrats [27] Runx2 protein which was expressed in secondaryhypertrophic chondrocytes was not localized in the cellsgathering in the erosive front of all themandibular secondarycartilages (Figure 4(a)) but in some osteoblasts surroundingthe trabecular bone and some osteocytes buried in thetrabecular bone in the mandible (Figure 4(b)) Thus wequantitatively analyzed the expression of Runx2 protein inosteoblasts and osteocytes (Figure 4(e)) comparing withthat in condylar cartilage and the posterior portion ofintramandibular Meckelrsquos cartilage In the present study theexpression of Runx2 protein in condylar hypertrophic chon-drocytes was the most intense (Figures 2(h) and 4(e)) beingstatistically significant difference from that in osteoblastswhich indicated an important role of Runx2 in secondarychondrocyte maturation in addition to that in chondrocytematuration of growth cartilage and osteoblast differentiationExpectedly Osterix was localized in some osteoblasts andbone marrow cells in sub-chondral bone area (Figure 4(c))Furthermore more positive osteoblasts and osteocytes werevisualized in the trabecular bone area (Figure 4(d)) Inter-estingly the immunohistochemistry of Osterix in hyper-trophic chondrocytes of the center of the uncalcifiedMeckelrsquoscartilage is still significantly more intense than that in theosteoblasts (Figure 4(f))This pointed out that Osterix highlylikely performed a regulatory effect on the degradation ofthe posterior portion of uncalcified intramandibularMeckelrsquoscartilage


(a)

PM

AG

(b)

(c)

PM

AG

(d)

SSI

RC

(e)

Sox9lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

CD AG SS RC PM EMMandibular cartilage regions

0010203040506070809

1

Rela

tive p

ositi

vity

of im

mun

osta

inin

g

(f)

SSI

RC

(g)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast


00102030405060708

Rela

tive p

ositi

vity

of im

mun

osta

inin

g

Runx2

(h)

Figure 2 Immunohistochemistry of Sox9 andRunx2 inmandibular cartilages ofmice at newborn stage Sox9 (a b and e) andRunx2 (c d andg) showed similar expression patterns throughout all themandibular cartilages In condylar cartilage (a and c) angular cartilage (b and d) andsymphyseal secondary cartilage and rostral cartilage (e and g) both Sox9 and Runx2 were strongly expressed by cells entire hypertrophic cellzone Scale bar 100 120583m for (a b c and d) and 50 120583m for (e and g) Results of Sox9 (f) and Runx2 (h) immunohistochemistry in hypertrophiczones of mandibular cartilages including condylar (CD) angular (AG) and symphyseal secondary cartilage (SS) and rostral process (RC)posterior Meckelrsquos (PM) and extramandibular Meckelrsquos (EM) cartilage were quantitated by average optical density of positive staining per200 field (lowastlowast119875 lt 0001) The expression levels of both transcriptional factors in the posterior portion of uncalcified intramandibular Meckelrsquoscartilage (b and d) were significantly reduced than in other cartilages


(a) (b)

(c)

lowastlowast

lowastlowast

lowastlowast

lowastlowast


0010203040506070809

Rela

tive p

ositi

vity

of im

mun

osta

inin

g

Osterix

lowast

(d)

Figure 3 Immunohistochemistry of Osterix in mandibular cartilages of mice at newborn stage Osterix was at a extremely low level incondylar cartilage (in the red box of (a)) and angular cartilage (in the blue box of (b)) while intensely expressed in the center of the Meckelrsquoscartilage containing the strong basophilic matrix (in the yellow box of (b) and Figure 1(c)) Further Osterix was moderately expressed inthe rostral process (c) Scale bar 25120583m for (a1015840 b1015840 and b10158401015840) and 50 120583m for (c) Results of Osterix (d) immunohistochemistry in mandibularcartilages including condylar (CD) angular (AG) and symphyseal secondary cartilage (SS) and rostral process (RC) posterior Meckelrsquos(PM) and extramandibular Meckelrsquos (EM) cartilage were quantitated by average optical density of positive staining per 200 field (lowast119875 lt 005lowastlowast

119875 lt 0001) Immunohistochemistry of Osterix in the posterior portion of intramandibular Meckelrsquos cartilage was significantly stronger andhad more positive cells than in other cartilages

4 Discussion

The majority of in vivo studies on cartilage differentiationare carried out using the appendicular skeleton as a modelsystem with the implicit assumption that chondrogenesis isequivalent throughout the body However Eames directlytested that the programs of chick head chondrogenesis areunique by comparing the neural crest-derived pharyngealarch skeleton to that of the mesoderm-derived limb dueto the fact that each skeleton forms from unique embry-onic populations [28] Meckelrsquos cartilage and mandibularsecondary cartilages are markedly distinguished from limbbud cartilage in their embryonic origin The mechanismsthat regulate the diverse developmental programs inMeckelrsquoscartilage and mandibular secondary cartilages remain to bediscovered The present study investigated the expression ofthe essential transcription factors related to chondrogenesisin these cartilages during the later development of mandibu-lar cartilages

The accumulated studies confirmed that Sox9 accelerateschondrocyte differentiation in proliferating chondrocytes butinhibits the terminal stages of chondrocyte differentiationin limb bud cartilage [29 30] However few investigationsfocused on themechanism of Sox9 in secondary chondrocytedifferentiation [31] Our findings clearly demonstrated thatthe key transcription factor Sox9 was strongly expressedat the whole hypotrophic cell zone of condylar cartilageand angular cartilage in newborn mice which was differentfrom the expression pattern of Sox9 in limb bud cartilage(Figure 5(a)) [8] Moreover Rabie et al have demonstratedthat Sox9 were expressed at the hypertrophic cell zone ofthe condylar cartilage in 36-day-old rats and continued to beexpressed throughout the examined period until day 52 [32]Conversely in limb bud cartilage both of the Sox9 transcriptsand protein were absent or at very diminished levels inhypertrophic chondrocytes [8 9] The recent data demon-strated that Sox9 is a major negative regulator of cartilagevascularization bone marrow formation and endochondral


(a) (b)

(c) (d)

lowastlowast

CD PM OB0

0102030405060708

Rela

tive p

ositi

vity

of im

mun

osta

inin

g

Runx2

Mandibular chondrocytes and osteoblasts

(e)

lowastlowast

CD PM OB0

010203040506070809

Rela

tive p

ositi

vity

of im

mun

osta

inin

g


Osterix

(f)

Figure 4 Immunohistochemistry of Runx2 and Osterix in mandibular osteoblasts of mice at newborn stage Runx2 protein which wasexpressed in secondary hypertrophic chondrocytes was not localized in the cells gathering in the erosive front of mandibular secondarycartilages (a) but in some osteoblasts surrounding the trabecular bone and some osteocytes buried in the trabecular bone in the mandible(b)The expression of Runx2 protein in condylar hypertrophic chondrocytes was significantly stronger than that in osteoblasts (e)MeanwhileOsterix was localized in some osteoblasts and bonemarrow cells in subchondral bone area (c) while more positive osteoblasts and osteocyteswere visualized in the trabecular bone area (d) The immunohistochemistry of Osterix in hypertrophic chondrocytes of the center of theuncalcified Meckelrsquos cartilage is still significantly more intense than that in the osteoblasts (f) (lowastlowast119875 lt 0001) CD condylar cartilage PM theposterior portion of intramandibular Meckelrsquos cartilage and OB osteoblasts Scale bar 25120583m for (a b c and d)

ossification [33] Despite this observation our investiga-tions indicate that Sox9 downregulation is not necessaryin the terminal stage of secondary cartilage developmentWe speculated that the transcription factor Sox9 may be amain positive regulator in the secondary cartilage terminalmaturation contrary to its function in later differentiation oflimb bud cartilage based on strong expression of Sox9 in themandibular secondary hypertrophic cell zone

In the present study surprisingly transcription factorsSox9 and Runx2 were similarly expressed at mandibularsecondary cartilages in newborn mice suggesting that Sox9and Runx2 may coregulate secondary chondrocyte differ-entiation In avian secondary cartilage formation Buxtonreported that Runx2-expressing preosteoblasts exit from thecell cycle and rapidly differentiate into hypertrophic chon-drocytes which is correlated with the up-regulation of Sox9


Epiphysis

Columnarchondrocyte

Prehypertrophicchondrocyte

Upper hypertrophicchondrocyte

Lower hypertrophicchondrocyte

Terminal chonodrocyte

Runx2

Sox9Osterix

(a) Limb bud cartilage

Fibrous cell

Polymorphic cell

Flattened cell

Upper hypertrophic cell

Lower hypertrophic cell

Osterix

Sox9Runx2

(b) Condylar cartilage

Osterix

Runx2

Sox9

(c) Posterior portion of intramandibular meckelrsquos cartilage

Figure 5 Schematic representations of the expression pattern of three key transcription factors in the different types of cartilage duringthe newborn stage The expression pattern of Sox9 Runx2 and Osterix in limb bud cartilage is based on previous reports of Kim et al [5]Kaback et al [6] Nishimura et al [7] Ng et al [8] and Dy et al [9] Furthermore the expression patterns of Sox9 (red) Runx2 (blue) andOsterix (yellow) in condylar cartilage andMeckelrsquos cartilage are based on the present findings Long arrows indicate the expressing cell zonesof transcription factors in cartilage (a) limb bud cartilage (b) condylar cartilage and (c) the posterior portion of intramandibular Meckelrsquoscartilage

[31] In addition Buxton described two routes to chondrocytehypertrophy and had postulated that precursors expressingSox9 differentiate into prehypertrophichypertrophic chon-drocytes mediated by the up-regulation of Runx2 in pri-mary cartilage formationWhereas preosteoblasts expressingRunx2 differentiate into prehypertrophichypertrophic chon-drocytes mediated by the upregulation of Sox9 in secondarycartilage formation [31] Mammalian mandibular secondarycartilages are a heterogeneous tissue containing cells at var-ious stages of chondrocyte maturation [34] Moreover thesesecondary cartilages manifest a unique zone-like packing ofmaturing chondrocytes [35] Shibata and Yokohama-Tamakirecently demonstrated that the mandibular secondary car-tilage anlages are derived from Runx2 mRNA expressingmandibular anlage [10] Thus our observations on theoverlapping expression of Sox9 and Runx2 at mandibularsecondary cartilages in newborn mice support Buxtonrsquos pro-posed concept in principle The up-regulation of Sox9 fromthe polymorphic cell zone to the hypertrophic cell zonemightact as a trigger for subsequent mammalian secondary chon-drocyte differentiationThis can be interpreted as evidence ofa unique differentiation pathway the formation of secondary

hypertrophic chondrocytes from osteoblast precursors withthe help of the positive regulator Sox9

More unexpectedly our finding that Sox9 and Runx2were coexpressed in the hypertrophic cell zone of the rostralregion is not in line with the analyses of endochondralossification in limb bud cartilage The previous studiesrevealed that Sox9 inhibits the hypertrophic chondrocyte dif-ferentiation through suppression of Runx2 in endochondralossification of limb bud cartilage [17] Furthermore Sox9protein needs to be degraded to allow chondrocyte terminalmaturation in limb bud cartilage [13] However Eames et alhad proposed that a unique combination of Sox9 and Runx2may drive the expression of themajormarker of hypertrophicchondrocytes Col10 based on the analysis of Sox9 andRunx2functions in primary cartilage differentiation of the aviancranial skeleton [36] In the present study the overlappingexpression pattern of Sox9 and Runx2 in the hypertrophiccell zone of the rostral region of Meckelrsquos cartilage providesclear evidence that Runx2 can drive the chondrocyte terminaldifferentiation in the presence of Sox9 protein Additionallyour data that Sox9 and Runx2 were similarly expressed lessin the hypertrophic cell zone of the posterior portion of


intramandibular Meckelrsquos cartilage has reinforced the notionthat degeneration of Meckelrsquos cartilage represents a differentprocess from endochondral ossification

Normally Osterix is present at an extremely low level inprehypertrophic chondrocytes of limb bud cartilage com-pared to osteoblasts [9] To our knowledge the present studyis the first to demonstrate the expression of Osterix protein inmandibular cartilages Osterix protein is faintly expressed inprehypertrophic chondrocytes of secondary cartilages sim-ilar to limb bud cartilage which suggests that Osterix playssimilar roles during the two types of cartilage developmentBy contrast Osterix protein is intensely expressed in hyper-trophic chondrocytes in the central zone of the bars of intra-mandibular Meckelrsquos cartilage while Osterix protein is notpresent in cells around light eosinophilic matrix dynamicallychanged from the strong basophilic matrix in the front of thedegrading Meckelrsquos cartilageThe light eosinophilic matrix infront of the degrading Meckelrsquos cartilage might display thecalcified cartilage matrix [37] A great amount of in vitro datademonstrated that the chondrocytes of Meckelrsquos cartilage cantransdifferentiate to osteogenic cells as characterized by pro-duction of type I collagen [38ndash40] Furthermore the previousin vivo investigations revealed that the extracellular matrix ofintramandibular portion of the Meckelrsquos cartilage is replacedgradually by type I collagen secreted by chondrocytes duringthe development ofMeckelrsquos cartilage [41]We speculated thatOsterix may be relevant to phenotypic conversion ofMeckelrsquoschondrocytes The enhanced expression of Osterix in maturechondrocytes might be an explanation of type I collagensynthesis by chondrocytes in Meckelrsquos cartilage Furtherstudies are needed to elucidate the exact role of Osterixduring the late development of Meckelrsquos cartilage On theother hand the disparity in the expression pattern betweenOsterix and Runx2 in chondrocytes in the present studysuggested that Osterix might perform its regulation andfunction in mandibular cartilage development independentof Runx2 Moreover with respect to the more remarkableexpression of Runx2 in the condylar cartilage and Osterixin intramandibular degrading Meckelrsquos cartilage relative tothose in osteoblasts in the present study we speculated thatRunx2 or Osterix could need much more intense expressionin the chondrocytes than in the osteoblasts in order to playa functional role during the development of mandibularcartilages

Cartilage is a complex and developmentally importanttissue type Transcriptional factors are crucial to the devel-opment of cartilages The differential expression of key tran-scriptional factors in several types of cartilages will dictatethe distinct cellular events during the development of thecartilages The present data provide insights into the similarroles that master transcriptional factors Sox9 and Runx2 playduring the later development of mandibular cartilages whichis different from that in limb bud cartilage It is necessaryto investigate in further detail whether the differences incellular events between ectomesenchymal chondrocytes andmesodermal chondrocytes involve the derivation of the cellsFurthermore Osterix is likely responsible for phenotypicconversion of Meckelrsquos chondrocytes during its degener-ation based on its intensive expression in hypertrophic

chondrocytes of the degrading Meckelrsquos cartilage of newbornmice Human mandibular anomaly appears to be a commonmalformation and appears inmultiple congenital birth defectsyndromes ranging from agnathia (agenesis of the jaw) tomicrognathia to patterning malformations These malfor-mations are particularly devastating as our faces are ouridentity [42] The regeneration of complex facial structuresrequires precision and specificity A much more thoroughunderstanding of the mechanism of master transcriptionalfactors in mandibular chondrogenesis lay the importantfoundation for the application of targeted interventions atthe molecular level endogenous tissue engineering and cell-based therapies in mandibular anomalies

5 Conclusions

Our study demonstrated similar tissue distribution of Sox9and Runx2 in newborn mice mandibular cartilages whichis distinguished from that in limb bud cartilage It is spec-ulated that Sox9 is a main and unique positive regulatorin the hypertrophic differentiation process of mandibularsecondary cartilages in addition to Runx2Moreover the dis-tinct expression pattern of osterix in degenerating posteriorportion of Meckelrsquos cartilage suggests that Osterix may berelevant to phenotypic conversion of Meckelrsquos chondrocytes

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgment

The authors thank the support of the National NaturalScience Foundation of China (no 10972242)

References

[1] Y Sakakura Y Hosokawa E Tsuruga K Irie M Nakamuraand T Yajima ldquoContributions of matrix metalloproteinasestoward Meckelrsquos cartilage resorption in mice immunohis-tochemical studies including comparisons with developingendochondral bonesrdquo Cell and Tissue Research vol 328 no 1pp 137ndash151 2007

[2] S Shibata K Fukada S Suzuki and Y Yamashita ldquoImmuno-histochemistry of collagen types II and X and enzyme-histo-chemistry of alkaline phosphatase in the developing condylarcartilage of the fetal mouse mandiblerdquo Journal of Anatomy vol191 no 4 pp 561ndash570 1997

[3] S Shibata N Suda S Suzuki H Fukuoka and Y YamashitaldquoAn in situ hybridization study of Runx2 Osterix and Sox9 atthe onset of condylar cartilage formation in fetal mousemandiblerdquo Journal of Anatomy vol 208 no 2 pp 169ndash177 2006

[4] M S Kim S Y Jung J H Kang et al ldquoEffects of bisphospho-nate on the endochondral bone formation of the mandibularcondylerdquo Anatomia Histologia Embryologia vol 38 no 5 pp321ndash326 2009

[5] I S Kim FOtto B Zabel and SMundlos ldquoRegulation of chon-drocyte differentiation by Cbfa1rdquo Mechanisms of Developmentvol 80 no 2 pp 159ndash170 1999


[6] L A Kaback D Y Soung A Naik et al ldquoOsterixSp7 regulatesmesenchymal stem cell mediated endochondral ossificationrdquoJournal of Cellular Physiology vol 214 no 1 pp 173ndash182 2008

[7] RNishimuraMWakabayashi KHata et al ldquoOsterix regulatescalcification anddegradation of chondrogenicmatrices throughmatrixmetalloproteinase 13 (MMP13) expression in associationwith transcription factor Runx2 during endochondral ossifica-tionrdquo The Journal of Biological Chemistry vol 287 no 40 pp33179ndash33190 2012

[8] L J Ng S Wheatley G E O Muscat et al ldquoSOX9 binds DNAactivates transcription and coexpresses with type II collagenduring chondrogenesis in the mouserdquo Developmental Biologyvol 183 no 1 pp 108ndash121 1997

[9] P Dy W Wang P Bhattaram et al ldquoSox9 directs hypertrophicmaturation and blocks osteoblast differentiation of growth platechondrocytesrdquo Developmental Cell vol 22 no 3 pp 597ndash6092012

[10] S Shibata and T Yokohama-Tamaki ldquoAn in situ hybridizationstudy of Runx2 Osterix and Sox9 in the anlagen of mousemandibular condylar cartilage in the early stages of embryoge-nesisrdquo Journal of Anatomy vol 213 no 3 pp 274ndash283 2008

[11] L Zou X Zou H Li et al ldquoMolecular mechanism of osteo-chondroprogenitor fate determination during bone formationrdquoAdvances in Experimental Medicine and Biology vol 585 no 7pp 431ndash441 2006

[12] W Bi J M Deng Z Zhang R R Behringer and B de Crom-brugghe ldquoSox9 is required for cartilage formationrdquo NatureGenetics vol 22 no 1 pp 85ndash89 1999

[13] H AkiyamaM C Chaboissier J FMartin A Schedl and B deCrombrugghe ldquoThe transcription factor Sox9 has essential rolesin successive steps of the chondrocyte differentiation pathwayand is required for expression of Sox5 and Sox6rdquo Genes ampDevelopment vol 16 no 21 pp 2813ndash2828 2002

[14] H Akiyama J P Lyons Y Mori-Akiyama et al ldquoInteractionsbetween Sox9 and 120573-catenin control chondrocyte differentia-tionrdquo Genes amp Development vol 18 no 9 pp 1072ndash1087 2004

[15] M Mikasa S Rokutanda H Komori et al ldquoRegulation of Tcf7by Runx2 in chondrocytematuration and proliferationrdquo Journalof Bone andMineralMetabolism vol 29 no 3 pp 291ndash299 2011

[16] M Ding Y Lu S Abbassi et al ldquoTargeting Runx2 expression inhypertrophic chondrocytes impairs endochondral ossificationduring early skeletal developmentrdquo Journal of Cellular Physiol-ogy vol 227 no 10 pp 3446ndash3456 2012

[17] S Yamashita M Andoh H Ueno-Kudoh T Sato S MiyakiandH Asahara ldquoSox9 directly promotes Bapx1 gene expressionto repress Runx2 in chondrocytesrdquo Experimental Cell Researchvol 315 no 13 pp 2231ndash2240 2009

[18] H Sugito Y Shibukawa T Kinumatsu et al ldquoIhh signalingregulates mandibular symphysis development and growthrdquoJournal of Dental Research vol 90 no 5 pp 625ndash631 2011

[19] F Tsuzurahara S Soeta T Kawawa K Baba andMNakamuraldquoThe role of macrophages in the disappearance of Meckelrsquoscartilage during mandibular development in micerdquo Acta Histo-chemica vol 113 no 2 pp 194ndash200 2011

[20] Y Sakakura Y Hosokawa E Tsuruga K Irie and T YajimaldquoIn situ localization of gelatinolytic activity during developmentand resorption of Meckelrsquos cartilage in micerdquo European Journalof Oral Sciences vol 115 no 3 pp 212ndash223 2007

[21] B Rath-Deschner N Daratsianos S Duhr et al ldquoThe signifi-cance of RUNX2 in postnatal development of the mandibularcondylerdquo Journal of Orofacial Orthopedics vol 71 no 1 pp 17ndash31 2010

[22] S Matsushima N Isogai R Jacquet et al ldquoThe nature and roleof periosteum in bone and cartilage regenerationrdquo Cells TissuesOrgans vol 194 no 2ndash4 pp 320ndash325 2011

[23] S Shibata N Suda S Yoda et al ldquoRunx2-deficient mice lackmandibular condylar cartilage and have deformedMeckelrsquos car-tilagerdquo Anatomy and Embryology vol 208 no 4 pp 273ndash2802004

[24] K Ishii I L Nielsen and K Vargervik ldquoCharacteristics of jawgrowth in cleidocranial dysplasiardquoThe Cleft Palate-CraniofacialJournal vol 35 no 2 pp 161ndash1666 1998

[25] B L Jensen ldquoCleidocranial dysplasia craniofacial morphologyin adult patientsrdquo Journal of Craniofacial Genetics and Develop-mental Biology vol 14 no 3 pp 163ndash176 1994

[26] X L Zhang K Ting C M Bessette et al ldquoNell-1 a keyfunctional mediator of Runx2 partially rescues calvarial defectsin Runx2+- micerdquo Journal of Bone and Mineral Research vol26 no 4 pp 777ndash791 2011

[27] A B M Rabie G H Tang and U Hagg ldquoCbfa1 coupleschondrocytes maturation and endochondral ossification in ratmandibular condylar cartilagerdquoArchives of Oral Biology vol 49no 2 pp 109ndash118 2004

[28] B F Eames and J A Helms ldquoConserved molecular programregulating cranial and appendicular skeletogenesisrdquo Develop-mental Dynamics vol 231 no 1 pp 4ndash13 2004

[29] M Wuelling and A Vortkamp ldquoTranscriptional networks con-trolling chondrocyte proliferation and differentiation duringendochondral ossificationrdquo Pediatric Nephrology vol 25 no 4pp 625ndash631 2010

[30] V Lefebvre and P Smits ldquoTranscriptional control of chondro-cyte fate and differentiationrdquo Birth Defects Research C vol 75no 3 pp 200ndash212 2005

[31] P G Buxton B Hall C W Archer and P Francis-WestldquoSecondary chondrocyte-derived lhh stimulates proliferation ofperiosteal cells during chick developmentrdquo Development vol130 no 19 pp 4729ndash4739 2003

[32] A B M Rabie T T She and U Hagg ldquoFunctional appliancetherapy accelerates and enhances condylar growthrdquo AmericanJournal of Orthodontics and Dentofacial Orthopedics vol 123no 1 pp 40ndash48 2003

[33] THattori CMuller SGebhard et al ldquoSOX9 is amajor negativeregulator of cartilage vascularization bone marrow formationand endochondral ossificationrdquoDevelopment vol 137 no 6 pp901ndash911 2010

[34] J Chen A Utreja Z Kalajzic T Sobue D Rowe and SWadhwa ldquoIsolation and characterization ofmurinemandibularcondylar cartilage cell populationsrdquo Cells Tissues Organs vol195 no 3 pp 232ndash243 2012

[35] G Shen and M A Darendeliler ldquoThe adaptive remodelingof condylar cartilagemdasha transition from chondrogenesis toosteogenesisrdquo Journal of Dental Research vol 84 no 8 pp 691ndash699 2005

[36] B F Eames P T Sharpe and J A Helms ldquoHierarchy revealedin the specification of three skeletal fates by Sox9 and Runx2rdquoDevelopmental Biology vol 274 no 1 pp 188ndash200 2004

[37] K Ishizeki H Saito T Shinagawa N Fujiwara and TNawa ldquoHistochemical and immunohistochemical analysis ofthe mechanism of calcification of Meckelrsquos cartilage duringmandible development in rodentsrdquo Journal of Anatomy vol 194no 2 pp 265ndash277 1999

[38] K Ishizeki N Takahashi and T Nawa ldquoFormation of the sphe-nomandibular ligament by Meckelrsquos cartilage in the mouse


possible involvement of epidermal growth factor as revealed bystudies in vivo and in vitrordquo Cell and Tissue Research vol 304no 1 pp 67ndash80 2001

[39] K Ishizeki T Kagiya N Fujiwara K Otsu and H HaradaldquoExpression of osteogenic proteins during the intrasplenictransplantation of Meckelrsquos chondrocytes a histochemical andimmunohistochemical studyrdquo Archives of Histology and Cytol-ogy vol 72 no 1 pp 1ndash12 2009

[40] K Ishizeki ldquoImaging analysis of osteogenic transformationof Meckelrsquos chondrocytes from green fluorescent protein-transgenic mice during intrasplenic transplantationrdquo Acta His-tochemica vol 114 no 6 pp 608ndash619 2012

[41] Y Harada and K Ishizeki ldquoEvidence for transformation ofchondrocytes and site-specific resorption during the degrada-tion of Meckelrsquos cartilagerdquo Anatomy and Embryology vol 197no 6 pp 439ndash450 1998

[42] Y Chai and R E Maxson Jr ldquoRecent advances in craniofacialmorphogenesisrdquo Developmental Dynamics vol 235 no 9 pp2353ndash2375 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


and nonredundant factor of chondrogenesis Analyses ingenetically modified mice revealed that Sox9 promotes theearly stage but suppresses the terminal stage of chondro-cyte differentiation in limb bud cartilage [12ndash14] On thecontrary the multifunctional transcription factor Runx2which is expressed in prehypertrophic and hypertrophicchondrocytes is a main positive regulator of hypertrophicdifferentiation in late chondrogenesis of the limb buds [515 16] New in vitro data demonstrated that Sox9 negativelyregulates Runx2 by enhancing Bapx1 expression which leadsto the inhibition of terminal chondrocyte differentiation[17] Osterix which acts downstream of Runx2 during boneformation is expressed in chondrocyte progenitors and pre-hypertrophic chondrocytes in rib spine and limb cartilagessuggesting that Osterix may play a critical role during theprimary cartilagematuration in combinationwith Runx2 andSox9 [6 7]

However the transcriptional control of the later develop-ment of mandibular cartilages remains poorly understoodAt birth the rostral process of intramandibular Meckelrsquoscartilage is undergoing endochondral ossification while theposterior portion of intramandibular Meckelrsquos cartilage isdegenerating [18ndash20] Meanwhile four secondary cartilagesespecially the condylar cartilage were not well documentedin terms of their developmental characteristics although theyfunctionmainly as a growth cartilage similar to limb bud car-tilage At present transcription factors are attracting increas-ing clinical attention because of their roles in the etiologyand pathogenesis of malformations and growth disordersdegenerative diseases and in regenerative and repair pro-cesses [21 22] The findings that Runx2-deficient mice lackmandibular condylar cartilage and had deformed Meckelrsquoscartilage indicate that Runx2 is essential for the formationof the mandibular cartilages [23] In many cleidocranialdysplasia (CCD) patients who were link to Runx2 deficenthowever there are no abnormal findings in the mandiblein spite of cases of condylar malformation persistent sym-physis or a narrow coronoid process being also known[24 25] These investigations provided a hint that Runx2may be just one of essential biological factors influencingthe development and growth of mandibular cartilages Thepresent study is to examine tissue distribution of Sox9 Runx2and Osterix in newborn mice mandibular cartilages usingimmunohistochemistry technique and investigate whetherthese transcription factors have similar functions to those inlimb bud cartilage which will contribute to current under-standing of mechanisms of the development of mandibleand the possible pathogenesis of some craniofacial anomaliesinvolving mandible

2 Materials and Methods

All animals were housed and handled in accordance withguidelines of the Chancellorrsquos Animal Research Committeeof the Office for Protection of Research Subjects at theUniversity of California Los Angeles CA USA

21 Tissue Preparation A total of 15 newborn C57BL6Nmice were collected in 2 hours right after being delivered and

used for this study The mandibles were then removed andimmersed in 4 paraformaldehyde (01M phosphate bufferpH 74) for 1 day at 4∘CThe specimens were decalcified with10 EDTA for 5 days at 4∘C and then embedded in paraffinusing standard procedures Sections (5 120583m) were cut in theplane parallel to the ascending ramus of the mandible allthe mandibular cartilages being in one section For generalmorphology deparaffinized sectionswere stainedwith hema-toxylin and eosin The skeletal staining with Alizarin Red-Alcian Blue was performed for preparation of gross specimenof mandible as reported previously [26]

22 Immunohistochemistry Five-micron-thick paraffin sec-tions were dewaxed in xylenes and rehydrated in ethanolbaths Endogenous peroxidases were blocked by incubat-ing sections in 3 hydrogen peroxide for 20min at roomtemperature Sections were incubated with anti-Runx2 anti-Osterix and anti-Sox9 primary antibodies (Santa CruzBiotechnology CA USA) (dilution 1 100) and biotinylatedanti-rabbit or anti-goat IgG secondary antibody (VectorLaboratories Burlingame CA) for 1 h at room temperaturePositive immunoreactivity was detected using VectastainABC kit (Vector Laboratories Burlingame CA USA) andAEC chromogenic substrate (Dako Carpiteria CA USA)with red positive staining A negative control was performedby replacing primary antibody solutions with PBS Sectionswere counterstained with hematoxylin for 30 sec followedby rinsing 5min in running water Photomicrographs wereacquired using an Olympus BX51 Image-pro Plus 60 soft-warewas used to calculate stained area and IntegratedOpticalDensity (IOD) The average optical density (mean density)represented the intensity of protein expression and wascounted in 4 random fields (times20 objective) of each cartilagearea and trabecular bone area per section The mean densityis equal to (IOD SUM)area For exact analysis three sectionswere prepared at similar plane for each sample ANOVA wasused for multiple groupsrsquo comparison and Studentrsquos 119905-testwas used for comparison between any two groups Statisticalsignificance defined as 119875 lt 005

3 Results

31 Histological Analysis of Cartilages in Newborn MouseMandible Mandibular cartilages in newborn mouse inclu-ded the portions of Meckelrsquos cartilage condylar angularand symphyseal secondary cartilage while cartilage was notpresent in the coronoid process of the newborn mouse(Figure 1(a)) On the basis of the cellular morphologicalchanges themandibular condylar and angular cartilageswerehistologically composed of four different cell zones a thinfibrous cell zone a polymorphic cell zone a wider flattenedcell zone and a broad hypertrophic cell zone occupied thelower half of the organ (Figures 1(b) and 1(c)) Almost allof the intramandibular bar of Meckelrsquos cartilage had ossifiedcompletely but a small amount ofMeckelrsquos cartilage remainedin a limited portion of the rostral region and at themylohyoidgroove between the condylar and angular processes At theposterior portion of intramandibular Meckelrsquos cartilage HEstaining pattern of the matrix changed from the intense


CD

PM

AG

PM

Mo

SS

RC

(a)

HY

FP

FL

(b)

AG

PM

HYHY

FPFL

(c)

PM HY R

Mo

I

(d)

ISS

RCHY

(e)









(a)

PM

AG

(b)

(c)

PM

AG

(d)

SSI

RC

(e)

Sox9lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast


0010203040506070809

1

Rela

tive p

ositi

vity

of im

mun

osta

inin

g

(f)

SSI

RC

(g)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast


00102030405060708

Rela

tive p

ositi

vity

of im

mun

osta

inin

g

Runx2

(h)



(a) (b)

(c)

lowastlowast

lowastlowast

lowastlowast

lowastlowast


0010203040506070809

Rela

tive p

ositi

vity

of im

mun

osta

inin

g

Osterix

lowast

(d)



4 Discussion




(a) (b)

(c) (d)

lowastlowast

CD PM OB0

0102030405060708

Rela

tive p

ositi

vity

of im

mun

osta

inin

g

Runx2


(e)

lowastlowast

CD PM OB0

010203040506070809

Rela

tive p

ositi

vity

of im

mun

osta

inin

g


Osterix

(f)





Epiphysis

Columnarchondrocyte





Runx2

Sox9Osterix


Fibrous cell

Polymorphic cell

Flattened cell



Osterix

Sox9Runx2


Osterix

Runx2

Sox9











5 Conclusions




Acknowledgment


References





















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


CD

PM

AG

PM

Mo

SS

RC

(a)

HY

FP

FL

(b)

AG

PM

HYHY

FPFL

(c)

PM HY R

Mo

I

(d)

ISS

RCHY

(e)









(a)

PM

AG

(b)

(c)

PM

AG

(d)

SSI

RC

(e)

Sox9lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast


0010203040506070809

1

Rela

tive p

ositi

vity

of im

mun

osta

inin

g

(f)

SSI

RC

(g)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast


00102030405060708

Rela

tive p

ositi

vity

of im

mun

osta

inin

g

Runx2

(h)



(a) (b)

(c)

lowastlowast

lowastlowast

lowastlowast

lowastlowast


0010203040506070809

Rela

tive p

ositi

vity

of im

mun

osta

inin

g

Osterix

lowast

(d)



4 Discussion




(a) (b)

(c) (d)

lowastlowast

CD PM OB0

0102030405060708

Rela

tive p

ositi

vity

of im

mun

osta

inin

g

Runx2


(e)

lowastlowast

CD PM OB0

010203040506070809

Rela

tive p

ositi

vity

of im

mun

osta

inin

g


Osterix

(f)





Epiphysis

Columnarchondrocyte





Runx2

Sox9Osterix


Fibrous cell

Polymorphic cell

Flattened cell



Osterix

Sox9Runx2


Osterix

Runx2

Sox9











5 Conclusions




Acknowledgment


References





















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








(a)

PM

AG

(b)

(c)

PM

AG

(d)

SSI

RC

(e)

Sox9lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast


0010203040506070809

1

Rela

tive p

ositi

vity

of im

mun

osta

inin

g

(f)

SSI

RC

(g)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast


00102030405060708

Rela

tive p

ositi

vity

of im

mun

osta

inin

g

Runx2

(h)



(a) (b)

(c)

lowastlowast

lowastlowast

lowastlowast

lowastlowast


0010203040506070809

Rela

tive p

ositi

vity

of im

mun

osta

inin

g

Osterix

lowast

(d)



4 Discussion




(a) (b)

(c) (d)

lowastlowast

CD PM OB0

0102030405060708

Rela

tive p

ositi

vity

of im

mun

osta

inin

g

Runx2


(e)

lowastlowast

CD PM OB0

010203040506070809

Rela

tive p

ositi

vity

of im

mun

osta

inin

g


Osterix

(f)





Epiphysis

Columnarchondrocyte





Runx2

Sox9Osterix


Fibrous cell

Polymorphic cell

Flattened cell



Osterix

Sox9Runx2


Osterix

Runx2

Sox9











5 Conclusions




Acknowledgment


References





















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a)

PM

AG

(b)

(c)

PM

AG

(d)

SSI

RC

(e)

Sox9lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast


0010203040506070809

1

Rela

tive p

ositi

vity

of im

mun

osta

inin

g

(f)

SSI

RC

(g)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast


00102030405060708

Rela

tive p

ositi

vity

of im

mun

osta

inin

g

Runx2

(h)



(a) (b)

(c)

lowastlowast

lowastlowast

lowastlowast

lowastlowast


0010203040506070809

Rela

tive p

ositi

vity

of im

mun

osta

inin

g

Osterix

lowast

(d)



4 Discussion




(a) (b)

(c) (d)

lowastlowast

CD PM OB0

0102030405060708

Rela

tive p

ositi

vity

of im

mun

osta

inin

g

Runx2


(e)

lowastlowast

CD PM OB0

010203040506070809

Rela

tive p

ositi

vity

of im

mun

osta

inin

g


Osterix

(f)





Epiphysis

Columnarchondrocyte





Runx2

Sox9Osterix


Fibrous cell

Polymorphic cell

Flattened cell



Osterix

Sox9Runx2


Osterix

Runx2

Sox9











5 Conclusions




Acknowledgment


References





















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b)

(c)

lowastlowast

lowastlowast

lowastlowast

lowastlowast


0010203040506070809

Rela

tive p

ositi

vity

of im

mun

osta

inin

g

Osterix

lowast

(d)



4 Discussion




(a) (b)

(c) (d)

lowastlowast

CD PM OB0

0102030405060708

Rela

tive p

ositi

vity

of im

mun

osta

inin

g

Runx2


(e)

lowastlowast

CD PM OB0

010203040506070809

Rela

tive p

ositi

vity

of im

mun

osta

inin

g


Osterix

(f)





Epiphysis

Columnarchondrocyte





Runx2

Sox9Osterix


Fibrous cell

Polymorphic cell

Flattened cell



Osterix

Sox9Runx2


Osterix

Runx2

Sox9











5 Conclusions




Acknowledgment


References





















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b)

(c) (d)

lowastlowast

CD PM OB0

0102030405060708

Rela

tive p

ositi

vity

of im

mun

osta

inin

g

Runx2


(e)

lowastlowast

CD PM OB0

010203040506070809

Rela

tive p

ositi

vity

of im

mun

osta

inin

g


Osterix

(f)





Epiphysis

Columnarchondrocyte





Runx2

Sox9Osterix


Fibrous cell

Polymorphic cell

Flattened cell



Osterix

Sox9Runx2


Osterix

Runx2

Sox9











5 Conclusions




Acknowledgment


References





















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Epiphysis

Columnarchondrocyte





Runx2

Sox9Osterix


Fibrous cell

Polymorphic cell

Flattened cell



Osterix

Sox9Runx2


Osterix

Runx2

Sox9











5 Conclusions




Acknowledgment


References





















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






5 Conclusions




Acknowledgment


References





















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article An Immunohistochemistry Study …downloads.hindawi.com/journals/bmri/2013/265380.pdfResearch Article An Immunohistochemistry Study of Sox9, Runx2, and Osterix Expression