mini review - jsir · inflammation and regeneration vol.26 no.3 may 2006 169 key words ameloblast,...

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169 Inflammation and Regeneration Vol.26 No.3 MAY 2006 Key w ey w ey w ey w ey words rds rds rds rds ameloblast, dentin, odontogenesis, tissue engineering, tooth, scaffold Mini Review Tooth-Tissue Engineering Masaki J. Honda 1,*) , Yoshinori Sumita 1) , Yoshinori Shinohara 1) , Hideaki Kagami 2) and Minoru Ueda 1,3) 1) Tooth Regeneration, Division of Stem Cell Engineering, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan 2) Department of Tissue Engineering, Nagoya University School of Medicine, Nagoya, Japan 3) Department of Oral & Maxillofacial Surgery, Nagoya University Graduate School of Medicine, Nagoya, Japan Artificial dentition, tooth transplantation, and dental implants are the traditionally prosthetic procedures for tooth replacement. While missing teeth are not life-threatening, the quality of daily life is affected. Recent progress in understanding the molecular basis of tooth development, stem cell biology, and tissue engineer- ing will provide the fundamental knowledge and basis for the realization of engineered teeth in the future. The mechanisms underlying tooth-tissue engineering are still largely unknown. We first reported techniques for the development of tissue engineered teeth in 2002. The long-term goal of our investigations remains the development of viable tissue engineered teeth. Although there are several ways that the engineering of teeth has been attempted, a complete and tissue engineered tooth has not been achieved. Engineered tooth size is smaller than that of normal teeth and only 15% of engineered tooth structures exhibited spatial organized pulp, dental and enamel. This report describes our recent investigations into the production of engineered teeth. Rec.12/12/2005, Acc.2/21/2006, pp169-174 Correspondence should be addressed to: Masaki J. Honda, Tooth Regeneration, Division of Stem Cell Engineering, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Phone: 81-3-5449-5120, Fax: 81-3-5449-5121, e-mail: [email protected] Introduction An increasing number of elderly people suffer from the loss of teeth due to periodontitis or dental caries Artificial dentition tooth autotransplantation and dental implants have been tradi tionally used as prosthetic procedures for tooth replacement Missing teeth have an impact on the quality of daily life In the s Branemark introduced one of the most successful and widely accepted dental implant systems to avoid the use of dentures The long term efficacy of dental implants is unclear due to the lack of understanding of the appropriate interfaces between implant surfaces and bone The stimulation of foreign body reactions by implant materials remains a concern As a field tissue engineering has existed for more than a de cade The ideal therapy is the regeneration of tooth substitute derived from the patient s own cells and grown in its intended location For this reason the field of regenerative medicine has significant implications for the future of clinical dentistry At present tooth tissue engineering has not advanced to a suffi

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Page 1: Mini Review - jsir · Inflammation and Regeneration Vol.26 No.3 MAY 2006 169 Key words ameloblast, dentin, odontogenesis, tissue engineering, tooth, scaffold Mini Review Tooth-Tissue

169Inflammation and Regeneration Vol.26 No.3 MAY 2006

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Mini Review

Tooth-Tissue Engineering

Masaki J. Honda1,*), Yoshinori Sumita1), Yoshinori Shinohara1),Hideaki Kagami2) and Minoru Ueda1,3)1)Tooth Regeneration, Division of Stem Cell Engineering, The Institute of Medical Science, The University ofTokyo, Tokyo, Japan2)Department of Tissue Engineering, Nagoya University School of Medicine, Nagoya, Japan3)Department of Oral & Maxillofacial Surgery, Nagoya University Graduate School of Medicine, Nagoya,Japan

Artificial dentition, tooth transplantation, and dental implants are the traditionally prosthetic procedures for

tooth replacement. While missing teeth are not life-threatening, the quality of daily life is affected. Recent

progress in understanding the molecular basis of tooth development, stem cell biology, and tissue engineer-

ing will provide the fundamental knowledge and basis for the realization of engineered teeth in the future.

The mechanisms underlying tooth-tissue engineering are still largely unknown. We first reported techniques

for the development of tissue engineered teeth in 2002. The long-term goal of our investigations remains

the development of viable tissue engineered teeth. Although there are several ways that the engineering of

teeth has been attempted, a complete and tissue engineered tooth has not been achieved. Engineered

tooth size is smaller than that of normal teeth and only 15% of engineered tooth structures exhibited spatial

organized pulp, dental and enamel. This report describes our recent investigations into the production of

engineered teeth.

Rec.12/12/2005, Acc.2/21/2006, pp169-174

*Correspondence should be addressed to:

Masaki J. Honda, Tooth Regeneration, Division of Stem Cell Engineering, The Institute of Medical Science, The University

of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Phone: 81-3-5449-5120, Fax: 81-3-5449-5121, e-mail:

[email protected]

Introduction An increasing number of elderly people suffer from the lossof teeth due to periodontitis or dental caries. Artificial dentition,tooth autotransplantation1), and dental implants have been tradi-tionally used as prosthetic procedures for tooth replacement.Missing teeth have an impact on the quality of daily life. In the1980s, Branemark introduced one of the most successful andwidely accepted dental implant systems to avoid the use ofdentures2). The long-term efficacy of dental implants is unclear

due to the lack of understanding of the appropriate interfacesbetween implant surfaces and bone. The stimulation of foreignbody reactions by implant materials remains a concern. As a field, tissue engineering has existed for more than a de-cade. The ideal therapy is the regeneration of tooth substitutederived from the patient's own cells and grown in its intendedlocation. For this reason, the field of regenerative medicine hassignificant implications for the future of clinical dentistry. Atpresent, tooth-tissue engineering has not advanced to a suffi-

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炎症・再生 Vol.23 No.1 2003170 Mini Review Tooth-Tissue Engineering

Fig.1 Strategy for tissue-engineered toothSchematic diagram of the strategy performed to produce theengineered tooth. Cell-scaffold construct is derived from iso-lated cells seeded on PGA mesh. The constructs are implantedin the omentum of nude rats, and the resultant tissues areanalyzed by histology and immunohistochemistry.

cient stage for clinical application and research in this field isongoing. This review describes several approaches for generat-ing tooth substitute based on cell-scaffold technology.

Tooth Development An understanding of tooth generation can be gained from aninsight of natural tooth development in embryogenesis. Mostorgans, including teeth, arise through interactions between twodistinct cell types, epithelium and mesenchyme3,4). In embryonicteeth, oral epithelial cells send out the first inductive signals tomesenchymal cells to initiate tooth development. Once the den-tal mesenchymal cells have received their initial instructions,they then send signals back to the dental epithelial cells. Thisreciprocal exchange continues throughout tooth development5). Tooth development is initiated as a localized thickening oforal epithelium. As the tooth grows, the dental epithelium startsto invaginate into the underlying ectomesenchyme, which in turncondenses around the protrusion, forming a tooth bud. Toothbud formation appears around E11-12 in mice6). As the dentalepithelium penetrates further, it physically folds itself aroundthe condensing mesenchyme, eventually forming a bell-shapedstructure. Ultimately, the epithelium will become the visible outerenamel of the tooth that erupts from the oral epithelium, and themesenchymal cells will have formed the dentin, dental pulp, ce-mentum, and periodontal ligament that attaches the tooth to thejawbone. Mammalian tooth development depends largely onsequential and reciprocal epithelial-mesenchymal interactions.These processes involve a series of inductive and permissive in-teractions that result in the determination, differentiation, andorganization of odontogenic tissues. It is likely that this recipro-cal interaction is critical for the production of engineered teeth.All animal experiments in these investigations were carried outaccording to the Institution's Guidelines for Laboratory Animals.

Tooth-tissue Engineering based oncell-scaffold A recent approach to tooth-tissue engineering describes theuse of a biodegradable polymer scaffold seeded with cells disso-ciated from the porcine third molar tooth germs (bell stage)(Fig.1). PGA incisor shaped scaffold dimensions used in thisstudy were 0.8 cm by 0.8 cm by 0.5 cm. The constructs consistsof PGA (poly-glycolic acid) and tooth cells were implanted intothe omentum of nude rats (Fig.1). After 20 weeks, recognizablecomplex tooth crowns were formed with structures containingenamel, dentin, and pulp-chamber7). This study was the first touse isolated tooth cells seeded onto artificial scaffolds andstrongly suggests the existence of dental stem cells. Most teethengineered using this approach exhibit a disorganized hetero-

geneous morphology with less than 15% exhibiting normalorganization8,9). Furthermore, engineered teeth did not reach tothe expected size or shape of the scaffold7-9).

Developmental Analysis of Tissue-Engineered Teeth Although tissue-engineered tooth crowns were successfullyproduced, the developmental process is not fully understood. Theregeneration process of tissue-engineered teeth was investigatedhistologically to determine the cell types contributing to the en-gineered tooth structure (Fig.2c-f). Immunohistochemical tech-niques using anti-cytokeratin 14 and anti-collagen type I anti-bodies were used to examine the initial differentiation markersof epithelial and mesenchymal cells, respectively (Fig.2a,b)9). Two possible mechanisms underlie the in vivo regenerationprocess. In the first mechanism, regeneration of teeth may in-volve the reorganization of fully differentiated ameloblasts, od-ontoblasts and cementoblasts already present in the original tooth.The second possible mechanism is that the regeneration processinvolves a series of epithelial-mesenchymal interactions betweenprogenitor cells derived from the epithelium and mesenchyme.

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171Inflammation and Regeneration Vol.26 No.3 MAY 2006

 Until 2 weeks after implantation, no notable events or rem-nants of PGA polymer were observed in the samples. At 4-6weeks aggregated epithelial cells were observed in samples(Fig.2a,b). This was first morphological sign of tissue-engineeredodontogenesis, and involved the aggregation of CK14-reactiveepithelial cells (Fig.2a) inside collagen type I-reactive mesen-chymal tissue (Fig.2b)9). These results provide preliminary evi-dence that tissue-engineered odontogenesis involves coopera-tion between epithelial and mesenchymal progenitor cells, al-though the cell type initiating odontogenesis is unknown.

Tissue-Engineered Tooth Structure inJaw We previously reported a technique for tooth-tissue engineer-ing. This technique involved seeding dissociated tooth cells ontobiodegradable polymer scaffolds7-10). One drawback of this pro-cedure was the inability to determine whether the engineeredtooth would function when grafted into the jaw, as the donor sitewas in the omentum of the rats. This drawback was overcome byestablishing an experimental model utilizing the canine jawdirectly11). Unerupted first molars (crown formation stage) wereobtained from 8 male dogs aged 8-12 weeks. Under general an-esthesia a crestal incision was performed with a vertical releas-ing incision to expose the lateral wall of the mandibular bone. Arectangular-shaped wall was prepared and reflected laterally toexpose the first molar. The intact first molar was gently removedfrom the socket using forceps. The bony wall was then reposi-tioned. Cells were harvested from canine first molar teeth and theresulting heterogeneous cell population was seeded onto a bio-degradable polymer. The implantation was an autograft, ie, thedonor and recipient were the same animal. The constructs con-sisting of the PGA scaffolds and heterogeneous cells were placedinto the empty sockets from which the tooth had been extracted. After 24 or 26 weeks, biopsy specimens were fixed in 10%neutral-buffered formalin and scanned using a cone-beam to-mography system (CBSTAR MCT-100CB; Hitachi MedicoTechnology, Chiba, Japan) with a micro-X-ray source (60 μm,85 kV, 100 μA) directed towards the sample. After micro-CTimaging, each sample was decalcified with 10% EDTA, embed-ded in paraffin after fixation in 10% buffered formalin, andstained with hematoxylin and eosin (H-E) to observe the extentof regeneration. After 24 weeks, micro-X-ray computed tomography revealedregenerated hard tissues in the jaw (Fig.3a). A limited amountof regenerated hard tissue in two positive samples was observedin the biopsies, some of which were adjacent to regions of theoriginal mandibular bone. Histological analysis revealed that the

regenerated tissues consisted of a heterogeneous composite ofbone, dentin and connective tissue (Fig.3b). At high magnifica-tion, dentin and cementum-like structures were visible, with adistinct interface between the two surfaces (Fig.3c). However,we did not observe enamel tissue, or crown and root morpholo-gies. Based on our results, further study is needed to addressthese problems.

Novel culture system for odontogenicepithelial cells The final goal of this study is to establish a way to obtaincultured cells that has a capacity to produce tooth. So far, thereis only one report to show the technique to produce tooth usingcultured cells10). The culture period is six days and the method isthat mixture of epithelial and mesenchymal cells during cultureis applied. Therefore, there is still no report to produce tooth incombination with each cultured cells after epithelium and mes-enchyme were separated in respectively. The development ofnew technology to produce tooth from the combination of cul-tured in separately, epithelial and mesenchymal cells will benecessary to accomplish for clinical application. On the other hand, one of the tissue composing tooth, enamelorgan development is regulated by reciprocal interactions be-tween epithelial ameloblasts and mesenchymal odontoblasts3,4).Although odontogenic epithelial cells differentiate into variouscell types during enamel formation, the mechanisms responsiblefor this differentiation are not well understood. Moreover, theculture of a variety of connective tissue types has been achievedwhile culture of odontogenic epithelial cells has not. A way toexpand epithelial cells to produce tooth is therefore needed, as isa more complete understanding of the mechanism underlyingdifferentiation during enamel formation. The establishment ofporcine ameloblast primary-culture systems12-14) and ameloblast-like cell lines15,16) has been previously reported. The approach usedinvolves the transformation and immortalization of epithelial cellswith the transforming genes of simian virus 40 (SV40)16). Todate, it has proved difficult to maintain primary odontogenicepithelial cells in cultures for prolonged periods due to their fi-nite lifespan. The experimental approach developed in this study uses 3T3-J2 cells as a feeder layer for the odontogenic epithelial cells. Theaim is to establish and characterize a long-term-cultured odon-togenic epithelial cell lineage that maintains the primary pheno-type of odontogenic epithelial cells17). Enamel organs were harvested as described previously14). Thirdmolar tooth buds (bell stage) were removed from 6-month-oldpigs. The released cells from enamel organ were cultured intoD-MEM medium (Gibco BRL, Grand Island, NY, USA) supple-

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Fig.2 Developmental process of tissue-engineeredtooth

a: The epithelial cells are beginning to stain with anti-cytokeratinK14 antibody, and have an appearance similar to the dentallamina formed during tooth development (black arrow). Bar:20 μm.b: The mesenchymal cells are beginning to stain with anti-collagen type I antibody (black arrow). Bar: 20 μm.c: The epithelium has increased in size (black arrow). The stel-late reticulum-like structure has grown and stains with hema-toxylin and eosin (H-E) (asterisk). Bar: 50 μm.d: A layer consisting of two types of epithelial cell (black arrows)is elongated adjacent to the mesenchymal tissue (blackarrowhead) and stains with H-E. Bar: 20 μm.e: Pre-mineralizing stage. Typical pre-ameloblasts (black arrow)and odontoblasts (black arrowhead) stained with H-E 10 weeksafter implantation. Bar: 20 μm.f: A basement membrane, indicated by blue Azan staining(black arrow), has formed between the pre-ameloblasts (blackarrowhead) and odontoblasts (asterisk). Bar: 20 μm.g: The onset of mineralization 12 weeks after implantation,stained with H-E (black arrow). Hertwig's root sheath is visible(black arrowhead) and columnar odontoblasts are clearly iden-tified (asterisk). Bar: 20 μm.h: At 15 weeks after implantation, the dentin tissue (asterisk)has formed a dental root-like tissue including dentin andHertwig's epithelial root sheath-like tissues (black arrow),stained with H-E. Bar: 100 μm.

Fig.3 Tissue-engineered dentin and cementumstructures in canine jaw

a: Two-dimensional coronal images of a biopsy using micro-CT. The images confirm the presence of stable regeneratedhard tissue with signs of radiopacity 24 weeks after transplan-tation (white arrow). The tissue density is similar to that of theoriginal bone (white arrowhead).b: H-E stained section of regenerated tissue 24 weeks afterimplantation. Bar: 100 μm.c: Detail of part b showing regenerated calcified tissue. A highermagnification view showing two types of calcified tissue; thefirst type is cementum-like and includes cementoblast-like cells(white arrowhead), and the second type is dentin-like (whitearrow). The characteristic well-organized structure of the den-tin tubule-like tissues in the calcified tissue is visible.

mented with 10% fetal bovine serum (GIBCO-BRL). These cellsformed a mixed population of epithelial- and fibroblast-like cells(Fig.4a). Before the cells reached confluence, the medium wasreplaced with LHC-9 (Biofluids, Bethesda, MD, USA) withoutfetal bovine serum. LHC-9 medium is selective for epithelialcells12,14). After the substitution of D-MEM for LHC-9 media,the fibroblasts died as expected and were lost from the culture,leaving only the epithelial cells (Fig.4b). 3T3-J2 cells (1 × 104 cell/cm2; a gift from Dr. H Green, HarvardMedical School) were used as a feeder layer and also inhibited

Mini Review Tooth-Tissue Engineering

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173Inflammation and Regeneration Vol.26 No.3 MAY 2006

Fig.4 Establishment of novel culture technique forodontogenic epithelial cell

a: Phase-contrast micrographs of the cells after 1 week of cul-ture. The epithelial cells (e) have formed colonies among thestrongly proliferating pulp cells (p). Bar: 50 μm.b: Odontogenic epithelial cells were cultured in LHC-9 me-dium. The epithelial cells are cuboidal or polygonal in appear-ance (arrowhead). Bar: 50 μm.c: Secondary sub-cultured cells on the top of the layer of 3T3-J2 cells after 14 days of cultivation. Most cells are cuboidal orpolygonal (arrowhead) in appearance. Bar: 50 μm.d: Polygonal cells can be seen after 14 days at the tenth pas-sage. The epithelium cells had a consistent characteristicmorphology throughout the 10 passages. Bar: 50 μm.

Fig.5 RT-PCR analysisa: Expression of amelogenin from the second to the tenth pas-sage was analyzed using RT-PCR. The expected 310-bpamelogenin product was generated. Lane 1, no template, nega-tive control; lanes 2-10, second to tenth passage cells; lane11, 3T3-J2 cells.b: Expression of ameloblastin from the second to the tenthpassage was analyzed using RT-PCR. The expected 380-bpamelogenin product was generated. Lane 1-9, second to tenthpassage cells.

the growth of any contaminating fibroblasts (Fig.4c). When theepithelial cell had reached confluence, the 3T3-J2 feeder cellshad disappeared (Fig.4d). The cultures were examined usingphase-contrast microscopy during 20 passages over 6 months. The use of 3T3-J2 feeder cells was successful in overcomingthe limitations of odontogenic epithelial cell culture. The pas-saged cells expressed the ameloblast-specific markers amelogeninand ameloblastin (Fig.5). Our findings imply that the proliferat-ing odontogenic epithelial cells were ameloblasts or ameloblastprogenitors. There are several reports of stem cells in the dentalmesenchyme18-22), and embryonic stem cells are available to toothengineers as a cell source23). However, an intact E10 dental epi-thelium is required for tooth production. If these stem cells canbe obtained from the dental mesenchyme or other stem cells,and be combined with the cultured odontogenic epithelial cells,it could be possible to develop a tooth.

Future Perspective Although tissue engineering is considered one of the most

powerful future approaches to repair or replace injured tissue ororgans, a 3-dimensional functional organ has not been generatedfrom any stem cell type. Four key milestones must be reached toestablish successful tooth-tissue engineering. First, the cell sourcemust be identified and readily obtained from patients. Second,engineered teeth must develop in the environmental of the adultjaw. Moreover, the teeth must erupt from the gingiva. Third, theshape and size of engineered teeth must be predictable and con-trollable and undistinguishable from the patient's own teeth.Fourth, periodontium is necessary to support regenerated toothin the jaw. In fact, a method to produce hybrid tooth and bonewas recently reported24). Although there are many hurdles to be overcome and ques-tions to be answered, tooth-tissue engineering provides a sub-stantial potential benefit to the quality of life of people requiringnew teeth. Continued progress in this field will ensure that tooth-tissue engineering is realized in the next few decades.

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Acknowledgements The authors would like to acknowledge to Drs. JP. Vacanti, PC. Yelick,JD. Bartlett, and CS. Young for scientific advice. This work was supportedby a Grant for Scientific Research from the Japanese Ministry of Educa-tion, Culture, Sports, Science and Technology (Kakenhi Kiban B 16390578to MH and Houga 16659548 to MH), the HITACHI Medical Corporation,

and DENICS International.

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14)Matsumura T, Tabata MJ, Wakisaka S, Sakuda M, KurisuK: Ameloblast-lineage cells of rat tooth germs proliferateand scatter in response to hepatocyte growth factor in cul-ture. Int J Dev Biol, 42: 1137-1142, 1998.

15) Chen LS, Counwenhoven RI, Hsu D, Luo W, Snead ML:Maintenance of amelogenin gene expression by transformedepithelial cells of mouse enamel organ. Arch Oral Biol, 37:771-778, 1992.

16) DenBesten PK, Gao C, Li W, Mathews CH, Gruenert DC:Development and characterization of an SV40 immortal-ized porcine ameloblast-like cell line. Eur J Oral Sci, 107:276-281, 1999.

17) Honda MJ, Shimodaira T, Ogaeri T, Shinohara Y, Hata K,Ueda M: A Novel Culture System for Porcine OdontogenicEpithelial Cells Using a Feeder Layer. Archives of OralBiology, 2006, in press

18) Gronthos S, Mankani M, Brahim J, Robey PG, Shi S: Post-natal human dental pulp stem cells (DPSCs) in vitro and invivo. Proc Natl Acad Sci USA, 97: 13625-13630, 2000.

19)Miura M, Gronthos S, Zhao M, Lu B, Fisher LW, RobeyPG, Shi S: SHED: stem cells from human exfoliated de-ciduous teeth. Proc Natl Acad Sci USA, 100: 5807-5812,2003.

20) Seo BM, Miura M, Gronthos S, Bartold PM, Batouli S,Brahim J, Young M, Robey PG, Wang CY, Shi S: Investi-gation of multipotent postnatal stem cells from human peri-odontal ligament. Lancet, 364: 149-155, 2004.

21)Mina M, Braut A: New insight into progenitor/stem cells indental pulp using Col1a1-GFP transgenes. Cells TissuesOrgans, 176: 120-133, 2004.

22) Nakashima M, Mizunuma K, Murakami T, Akamine A: In-duction of dental pulp stem cell differentiation into odonto-blasts by electroporation-mediated gene delivery of growth/differentiation factor 11 (Gdf11). Gene Ther, 9: 814-818,2002.

23) Ohazama A, Modino SA, Miletich I, Sharpe PT: Stem-cell-based tissue engineering of murine teeth. J Dent Res, 83:518-522, 2004.

24) Young CS, Abukawa H, Asrican R, Ravens M, Troulis MJ,Kaban LB, Vacanti JP, Yelick PC: Tissue-engineered hy-brid tooth and bone. Tissue Eng, 11: 1599-1610, 2005.

Mini Review Tooth-Tissue Engineering

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