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J Oral Maxillofac Surg65:501-507, 2007

Regulating the Role of BoneMorphogenetic Protein 4 in

Tooth BioengineeringIl-Hyuk Chung, DDS, MSD, PhD,*

Pill-Hoon Choung, DDS, MSD, PhD,† Hyun-Jeong Ryu, BS, MS,‡

Young-Ho Kang, DDS, MSD,§ Han-Wool Choung,�Jong-Hoon Chung, PhD,¶ and

Yun-Hoon Choung, DDS, MD, PhD#

Purpose: Culture of the whole organ and regulation of its development using biologic and engineeringprinciples can be used to produce structures and organs for reconstructing defects. The application ofthese bioengineering approaches in artificial tooth development may be the alternative way to replacemissing dentition.

Materials and Methods: For the artificial bioengineering of a mouse tooth, tooth buds were dissectedand transplanted into the diastema of the developing mandible. The mandiblular primordia containingtransplanted tooth buds were culture in vitro and in vivo using a bioengineering method. In addition, toregulate the development of tooth germs, bone morphogenetic protein 4 (BMP4) or its antagonist,Noggin was administered.

Results: After the period of in vitro and in vivo culture, the transplanted tooth germ in the diastemashowed tooth development with supportive structure formation. In the BMP-treated group, the bioengi-neered tooth was observed with increased maturation of cusp and enamel matrix. However, in theNoggin-treated tooth germs, the developing molar had a crater-like appearance with the immaturedevelopment of the cusp and suppressed formation of the enamel matrix.

Conclusions: This study confirmed that tooth germ transplantation in the diastema and culture withadministration of BMP4 could lead to the mature development of the dental structures. In addition, theseresults suggest the possibility of bioengineering the tooth in morphogenesis and differentiation even inthe toothless area.© 2007 American Association of Oral and Maxillofacial Surgeons

J Oral Maxillofac Surg 65:501-507, 2007

*Assistant Professor, Department of Oral and Maxillofacial Sur-

ery, Seoul National University Boramae Hospital, Seoul, Korea.

†Professor, Department of Oral and Maxillofacial Surgery, Tooth

ioengineering National Research Laboratory, BK21 and the Intel-

ectual Biointerface Engineering Center, Dental Research Institute,

chool of Dentistry, Seoul National University, Seoul, Korea.

‡Graduate Student, Department of Oral and Maxillofacial Surgery,

ooth Bioengineering National Research Laboratory, BK21 and the

ntellectual Biointerface Engineering Center, Dental Research Insti-

ute, School of Dentistry, Seoul National University, Seoul, Korea.

§Postgraduate Student, Department of Oral and Maxillofacial Sur-

ery, Tooth Bioengineering National Research Laboratory, BK21 and

he Intellectual Biointerface Engineering Center, Dental Research In-

titute, School of Dentistry, Seoul National University, Seoul, Korea.

�Student, Department of Oral and Maxillofacial Surgery, Tooth

ioengineering National Research Laboratory, BK21 and the Intel-

ectual Biointerface Engineering Center, Dental Research Institute,

School of Dentistry, Seoul National University, Seoul, Korea.

¶Professor, Department of Biosystems and Agricultural Engineer-

ing, Institute of Agricultural Science and Technology, Seoul Na-

tional University, Seoul, Korea.

#Assistant Professor, Department of Otolaryngology, School of

Medicine, Ajou University, Suwon, Korea.

This study was supported by a grant of the Korea Health 21 R&D

Project, Ministry of Health & Welfare, Republic of Korea (0405-BO01-

0204-0006) and National Research Laboratory Program (Pill-Hoon

Choung, DDS, PhD) of the Ministry of Science and Technology, Korea.

Address correspondence and reprint requests to Dr Choung:

Department of Oral and Maxillofacial Surgery, School of Dentistry,

Seoul National University, 28 Yeongun Dong, Chongno Gu, Seoul

110-744, Republic of Korea; e-mail: choungph@snu.ac.kr

© 2007 American Association of Oral and Maxillofacial Surgeons

0278-2391/07/6503-0021$32.00/0

doi:10.1016/j.joms.2006.07.004

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502 BMP4 IN TOOTH BIOENGINEERING

ased on developmental principles, the culturing of ahole organ and regulation of its development usingiological and engineering principles can be used toroduce structures and organs for reconstructing de-

ects. Using such bioengineering methods, a struc-ural relationship and cellular association within theeveloping organ can be maintained, and its develop-ent can be regulated in the artificial environment.The application of bioengineering to artificial tooth

evelopment may provide an alternative approach toeplacing missing dentition.1 Tooth development oc-urs within developing embryos through sequentialnd reciprocal interactions between different cellularayers.2-5 For artificial development of the tooth, bio-ogical principles of the developmental process muste maintained in the artificial environment. In addi-ion, creation of a replacement tooth critically reliesn the regulation of its development according tongineering principles.Various transcription factors and signaling mole-

ules participate in epithelial–mesenchymal signaling,ncluding bone morphogenetic proteins (BMPs), fi-roblast growth factors, and hedgehog and Wnt fam-

ly molecules.3-5 Among these molecules, BMPs haveeen extensively evaluated for odontogenic initiationnd morphogenesis. BMP4 and BMP2 are believed toe key signals participating in the epithelial–mesen-hymal interactions during tooth development. In ad-ition, the BMP4 expression sites in the maxillary andandibular processes suggest a role in early tooth

evelopment.6-8 At the initial stage of murine toothormation, both BMP2 and BMP4 are expressed in theental lamina, whereas BMP4 alone is expressed inhe underlying mesenchyme. As tooth developmentroceeds to the bud stage, BMP4 expression shifts tohe mesenchyme, whereas BMP2 and BMP4 are ex-ressed in the dental epithelium.3-5 The region form-

ng the enamel knot, an organizing center for estab-ishing the tooth form, exhibits elevated BMP2 andMP7 expression. At the cap stage, BMP4 is alsoxpressed in the enamel knot and the dental mesen-hyme, and localized BMP2 expression occurs in theesenchymal cells deep in the dental papilla. At the

ell stage, the presumptive ameloblasts express BMP4nd the odontoblasts express BMP2, BMP4, andMP7.6,8 The application of BMP4-soaked beads to

solated dental mesenchymal tissue induces gene ex-ression patterns that also have been observed inondensing mesenchyme in vivo and in in vitro re-ombination studies.9,10 Moreover, the induction po-ential of tooth development shifts from the epithe-ium to the mesenchyme at embryonic day (E) 11.5,oinciding with the timing of the shift of BMP4 ex-ression from the tooth germ epithelium to the dentalapilla.12 In addition, BMP4 expression in the odon-

oblasts and ameloblasts at the bell stage and later has t

een reported. These findings suggest that BMP4 isnvolved in the differentiation of these cells and in theeposition of the matrices of dental hard tissue, den-in, and enamel.11

In this study, artificial development of the toothnd the role of BMP4 on tooth morphogenesis andell differentiation were examined using bioengineer-ng methods. Toward this end, dissected tooth germs

ere transplanted into the diastema of mandibles andultured with beads soaked with either BMP4 or itsntagonist, noggin. The developing teeth were exam-ned after the organ cultures were obtained.

aterials and Methods

PREPARATION OF EMBRYOS, MANDIBLES,AND TOOTH GERMS

Mice from the CD-1 strain and their embryos weresed. At E11.5 and E13.5, the embryos were removedrom the fetal membranes. The heads were removed,nd the primordial mandibles of the first brachial archere dissected. The molar tooth germs were thenissected.

TRANSPLANTATION OF THE TOOTH GERMS INTOTHE DIASTEMA AND IN VITRO ORGAN CULTURE

The dissected tooth germs of the E11.5 and E13.5ice were transplanted into the diastema of a mandi-

le at the same developmental stage (Fig 1), and theMP4 or noggin-soaked beads were then implantedround the transplanted tooth germs. For bead im-lantation, the Affi-Gel blue agarose beads (75 to 150m in diameter; Bio-Rad, Hercules, CA) were incu-ated in either 50 �g/mL of recombinant humanMP4 (Genetics Institute, Cambridge, MA) or 50g/mL of noggin (R&D systems, Minneapolis, MN) at7°C for 30 minutes. In addition, the Affi-Gel bluegarose beads were incubated with bovine serumlbumin (BSA; 100 �g/mL) as a negative control underimilar conditions.

The primordial mandibles containing the trans-lanted tooth germs were cultured according to therowell method.12,13 The culture medium was cre-ted using Dulbecco’s Modified Eagle Medium withlutaMax-1 (Gibco-BRL, Detroit, MI) supplementedith 10% heat-inactivated fetal calf serum and 20

U/mL of penicillin-streptomycin. The tissues wereultured in a standard incubator at 37°C in a humid-fied atmosphere of 5% CO2 for 2 days.

KIDNEY TRANSPLANTATION AND IN VIVOORGAN CULTURE

For the study of tooth development under a phys-ological environment, the diastema area containing

he transplanted tooth germs and some of the sur-

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CHUNG ET AL 503

ounding mesenchymal tissues were dissected fromhe in vitro cultured primordial mandibles and trans-erred to an adult mouse kidney under the innerapsule. The explants were then left to develop for 2o 3 weeks in vivo. After the in vivo culture period,he mice were sacrificed, and the developing mandib-lar explants were removed from the kidney.

HISTOLOGICAL EXAMINATION

Subsequently, developing mandibular explantsere fixed in 4% paraformaldehyde, dehydrated, and

hen embedded in paraffin. Then 5-�m-thick sectionsere prepared and stained with hematoxylin and

osin and Masson trichrome. The development andormation of the dental tissues were evaluated byptical microscopy. In some cases, the developingeeth were dissected from the cultured diastema andxamined. All experiments involving animals wereerformed in accordance with the regulations andith the approval of the Dental Research Institute of

eoul National University Dental College.

esults

Tooth germs cultured in the diastema showed pro-iferation and differentiation that progressed to theate bell stage. The crown pattern of the developingooth was established by a folding of the inner dentalpithelium. Dentin and enamel formation were ob-erved at the crest of the folded inner dental epithe-ium, the cusp-developing site. In addition, the devel-pment of supporting structures and the alveolarone, and formation of the neurovascular bundles,ere observed around the developing tooth (Fig 2).he tooth germs cultured with BSA-soaked beads de-eloped into the late bell stage. After cusp develop-

IGURE 1. The dissected mandibular primordia and the tooth germsf the mandible (M, molar tooth germ of E13.5; T, developing ton, Embryonic mandible with the transplanted tooth germs (black arro

hung et al. BMP4 in Tooth Bioengineering. J Oral Maxillofac Su

ent, dentin and enamel formation were observed in c

oth E11.5 and E13.5 tooth germs. In some E13.5ooth germ cases, second molar formation was ob-erved along with first molar formation (Fig 3).

In the BMP4-soaked beads implant group, the tootherms developed into the late bell crown stage,hich is characterized by hard tissue formation. The

11.5 tooth germs showed crown development withhe formation of enamel and dentin matrix. Formationnd development of the first and the second molarsere frequently observed in this group (Fig 4A). The

ooth germs, which were prepared from the E13.5ice and cultured with BMP4-soaked beads, also

eached the crown stage. The developing teeth werehe first and second molars. In 46 of the 56 cases82%), the BMP4-treated tooth buds showed the de-elopment of second molars along with that of firstolars. The first molars showed mature crown forma-

ion with cusp development along the folded internalental epithelium. In the developing cusp, there wasn obvious ameloblast and odontoblast differentiationlong with deposition of enamel and dentin matrix. In9 of the 56 cases (88%), the BMP4-soaked beads

mplanted tooth buds showed mature cusp develop-ent with the deposition of the enamel and dentinatrix, as described earlier. The developing secondolar was smaller than the first molar; however, its

usp development was evident at the postnatal devel-pment stage. At the cervical loop, Hertwig’s epithe-

ial root sheath grew around the dental papilla be-ween the dental papilla and the dental follicle. Afterhis growth, the dental root developed with forma-ion of the dentin matrix (Fig 4B).

The tooth germs transplanted into the diastemahat were cultured with noggin-soaked beads exhib-ted tooth development. In the E11.5 tooth germs,

13.5 mouse. A, The molar tooth germs observed on the lateral sideB, Embryonic tooth germs dissected from the embryonic mandible.e diastema.

7.

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504 BMP4 IN TOOTH BIOENGINEERING

fter the proliferation and development of the trans-lanted tooth germs. Odontoblast and ameloblast dif-

erentiation occurred, and the dentin and enamel ma-rix formed in the entire region of the crown.owever, the developing cusp exhibited flat occlusal

urfaces, and the first molar developed without for-ation of the second molar (Fig 5A). The E13.5 tooth

uds cultured with noggin-soaked beads showedooth development. However, the crown of the de-eloping tooth had an immature, crater-like appear-nce. The occlusal region of the crown was relativelyat with immature cusps, and there was no differen-iation of the inner dental epithelial cells into amelo-lasts. In contrast, those on the lateral surface of theegion proximal to the tooth crown differentiatedormally. Odontoblast differentiation was observed,

IGURE 2. Cultured diastema area containing the transplanted toothtructures were observed (E13.5 tooth germ; hematoxylin and eosin;erms showed development to the late bell stage. The developingell-stage development (E, enamel; D, dentin; E11.5 tooth germ; hem

hung et al. BMP4 in Tooth Bioengineering. J Oral Maxillofac Su

IGURE 3. Tooth germs cultured with BSA-soaked beads. A, E11.evelopment (E, enamel; D, dentin; Masson trichrome; original magnifiolars. The developing molars showed enamel and dentin matrix for

hung et al. BMP4 in Tooth Bioengineering. J Oral Maxillofac Surg 200

nd the dentin matrix was formed in the whole regionf the crown. At the cervical loop, the dental rooteveloped after Hertwig’s epithelial root sheath. In 37f the 52 cases (72%), the noggin-treated tooth budshowed immature cusp development, as describedarlier. In addition, the developing tooth originatingrom the noggin-treated tooth buds was the first mo-ar, without formation of the second molar. Formationf the first molar without the second molar was ob-erved in the noggin-treated tooth buds in 43 of the2 cases (84%) (Fig 5B). Although formation of theecond molar was observed in 9 cases of the noggin-oaked beads implant group, the developing secondolars were immature and smaller than those of theMP4-treated group. The incidences of mature cuspevelopment and second molar formation were sig-

A, Tooth development (black arrow) and formation of the supportingmagnification �10) Alv b, alveolar bone. B, The transplanted toothowed formation of the dentin and enamel matrix, compatible withn and eosin; original magnification �40).

7.

germs showed enamel and dentin matrix formation following cusp�40). B, E13.5 tooth germs showed formation of the first and second

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CHUNG ET AL 505

ificantly different in the noggin- and BMP4-treatedroups (Tables 1, 2).

iscussion

Odontogenesis is the process by which tooth fieldsre specified and developed into a mature dentaltructure. Tooth formation in mice usually beginsfter 11.5 days of intrauterine development, with aigh proliferation of oral epithelium and neoforma-ion of dental lamina. Subsequently, the dental laminarows into the underlying mesenchyme of the firstranchial arch, which forms the epithelial buds (budtage: E13.5). After this stage, the epithelial compo-ent undergoes specific foldings during the cap stageE14.5) and bell stage (E15.5), which eventually giveise to the ameloblasts that deposit enamel.3,4 Thisdontogenesis is regulated by a series of interactionsetween morphologically distinct tissues. Moreover,ynergistic and antagonistic effects of signaling mole-ules are involved in the localized tissue responses athe different stages of tooth development.4-6

For the artificial development of a tooth from aoothless area, developing tooth germs were trans-lanted into the diastema and cultured. In addition, toegulate the development of the tooth germs, BMP4r noggin was administrated during the culture pe-iod. The expression of BMP in the maxillary andandibular processes6,8 and the BMP receptors ALK3

IGURE 4. Tooth germs cultured with the BMP4-soaked beads. A, Ehe late bell stage (hematoxylin and eosin; original magnifcation �40)E, enamel; D, dentin; Masson trichrome; original magnification �100ransplanted tooth germs (hematoxylin and eosin; original magnificatioormation of the enamel and dentin matrix (E, enamel; D, dentin; hemental epithelium and differentiation of the ameloblasts were observe

4) The developing teeth dissected from the diastema area after the in

hung et al. BMP4 in Tooth Bioengineering. J Oral Maxillofac Su

BMPR 1A) and ALK6 (BMPR 1B) in the inner and I

uter dental epithelium14,15 are suggestive of its rolen the tooth development. Two known antagonists ofMP are chordin and noggin. These proteins bindMPs in the extracellular space to prevent the activa-ion of BMP receptors.16 In tooth development, nog-in binds BMP4 with a high affinity and can eliminatets activity by blocking its binding to cell surfaceeceptors. This activity transformed the tooth identityrom incisor to molar when the E11.5 tooth germsrom the incisor development site were cultured withoggin.17 This suggests that BMP4 in the dental epi-helium at the initiation stage is involved in determin-ng a tooth’s identity.

As a BMP antagonist, noggin evidently affects theusp formation in the tooth germs and results in arater-like appearance of the dental crown. The tran-ition from the bud to cap stage is a determining stepn tooth morphogenesis. In cusp development, therimary enamel knot is a prerequisite for tooth devel-pment to proceed to the cap stage and acts as aignaling center.18-20 Among the numerous growthactors, BMP4 is intensively expressed on the enamelnot and acts as a signal to control the shape of theooth.21 Based on these findings, the antagonistic ef-ect of noggin on BMP also inhibited enamel knotunction and caused immature cusp formation duringooth development. In addition, histological examina-ion showed inhibited proliferation of the inner den-al epithelium into ameloblasts in the occlusal region.

ooth germs. (1) The transplanted tooth germ showed development toe developing tooth showed formation of the enamel and dentin matrix13.5 tooth germs. (1) The first and second molars originated from the). (2) The developing tooth showed mature cusp development with theand eosin; original magnification �40). (3) Proliferation of the inner

namel, D, dentin; Masson trichrome; original magnification �100).nd in vivo culture.

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506 BMP4 IN TOOTH BIOENGINEERING

ears to be caused by site-specific proliferation of thenner dental epithelial cells. Subsequently, dentin-orming odontoblasts and enamel-forming amelo-lasts differentiate at the dental epithelium–mesen-hyme interface. During the differentiation stageMP2, BMP4, and BMP7 are expressed in the innernamel epithelium as epithelial signals regulating theifferentiation of the underlying mesenchymal cells

nto odontoblasts, and are expressed by the amelo-lasts during their terminal differentiation. Of these,MP4 is the most intensively expressed in the amelo-lasts.8,23 The BMP4 expression level in the odonto-lasts decreases with advancing development,hereas it persists in the ameloblasts.2

Therefore, BMP4s are present at the right time andocation to act as signals for dental hard tissue forma-

IGURE 5. The tooth germs cultured with noggin-soaked beads. A, Ehe bell stage (hematoxylin and eosin; original magnification �40).namel matrix. The developing cusp was relatively flat (E, enamel; Derms. (1) The developing tooth was the first molar with an immaturelhe occlusal region of the crown was flat with immature cusps, wheifferentiated normally. In the occlusal regions, the inner dental epi

E, enamel; D, dentin; Masson-trichrome; original magnification �100ad a crater-like appearance with immature development of cusps on

hung et al. BMP4 in Tooth Bioengineering. J Oral Maxillofac Su

Table 1. EFFECT OF BMP4 AND NOGGIN ONCUSP DEVELOPMENT

Treatment

Number ofTransplantedTooth Buds

Cusp Development (%)

Mature Immature

BMP4 56 49 (88.0) 7 (12.0)Noggin 52 15 (28.0) 37 (72.0)

hung et al. BMP4 in Tooth Bioengineering. J Oral Maxillofacurg 2007.

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ion. The binding of noggin to BMP4 receptors stopshe differentiation of inner dental epithelial cells intomeloblasts and suppresses formation of the enamelatrix. These results are in agreement with a previ-

us experimental study in which treatment of thentisense oligodeoxynucleotide against BMP4 sup-ressed formation of a molar cusp in in vitro culturedooth germs.23 In contrast, thick layers of dentin andnamel matrix separating ameloblasts from the odon-oblasts were observed in the BMP4-treated tootherms.In mice, development of the second molar was

iscernible at E14.0,24 and the anterior extremities ofhe upper and lower second molars could be detectedy E16.0.25 At E17.0, the dental epithelium of the

ooth germs. (1) The transplanted tooth germs showed development tocrown of the developing tooth showed formation of the dentin and

n; Masson-trichrome; original magnification �100). B, E13.5 toothoped cusp (hematoxylin and eosin; original magnification �40). (2)ose on the lateral surface of the region proximal to the tooth crowncell remained undifferentiated, and enamel deposition was absenthe first molar originated from the transplanted tooth germ. The crownclusal view.

7.

Table 2. EFFECT OF BMP4 OR NOGGIN ON THESECOND MOLAR FORMATION OF E13.5TOOTH BUDS

Treatment

Number ofTransplantedTooth Buds

Second MolarFormation (%)

Normal Abnormal

BMP4 56 46 (82.0) 10 (18.0)Noggin 52 9 (16.0) 43 (84.0)

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econd molars was separated from those representinghe first molar. In addition, anterior-posterior elonga-ion and the development of the molar tooth germsere detected.25 The development of second molarsas evident in the BMP4-treated group, which was

ompatible with the postnatal dentin and enamel dep-sition period. However, in most cases of the nog-in-treated tooth germs, the first molars withoutormation of the second molars arose from theransplanted tooth germs. This suggests that nogginnhibits initiation and proliferation of the second

olar from the transplanted tooth germs.Our findings confirm that tooth germ transplanta-

ion in the diastema and culture with the administra-ion of BMP4 can lead to the mature development ofental structures. In addition, these results suggesthe possibility of bioengineering tooth morphogene-is and differentiation even in toothless areas.22

eferences1. Modino S, Sharp P: Tissue engineering of teeth using adult stem

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defects. Acta Odontol Scand 58:191, 20006. Vainio S, Karavanova J, Jowetta A, et al: Identification of BMP-4

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