current tissue engineering for tooth regeneration...

2009-09-18

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Current Tissue Engineering for Tooth Regeneration

Dept. of Periodontology, SNU

College of Dentistry, SNUDept. of Periodontology

R2. Roh,Young-Hoon

Contents • Introduction

• Current approaches to engineering bio-tooth


• Major challenges in reconstructing bio-tooth

• Summary

• Comment

Introduction


Introduction


Introduction

Shape determination

Size control


Availability of dental epithelium

Directional growth and eruption

Graft rejection in the jawsBiological tooth (Bio-tooth)

Current approaches to engineering bio-tooth


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1. Recombination experiments

• Traditional method to evaluate epithelial-mesenchymalinteraction

• 3 types of recombination methods between epithelial and


3 types of recombination methods between epithelial and mesenchymal components– tissue-to-tissue, cell-to-tissue, cell-to-cell recombinants– incubated either in vitro or in vivo

• effective method for bioengineered odontogenesis





(1) Dissociated epithelial cells & mesenchymal cells (EC–MC) (2) Epithelial cells & intact dental mesenchyme (EC–MT)(3) One intact dental epithelium & dental mesenchymal cells (ET–MC)


• Difficult to separate embryonic oral/dental epithelium from underlying mesenchymal components

• Cell/tissue sources from human embryonic stages are very


Cell/tissue sources from human embryonic stages are very limited due to moral and legal issues– postnatal cells/tissues

2. Scaffold-based tooth engineering



• scaffold materials

– long-lasting porous hydroxyapatite ceramics

N t ll i l l f i t di t d ti


– Naturally occurring molecules of intermediate duration (e.g.,collagen and chitosan)

– relatively short-lasting polymers• polyglycolic acid (PGA), polylactic acid (PLA), polyglycolic

acid-poly-L-lactic acid (PGA-PLLA), polylactic polyglycolicacid (PLGA)

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• Scaffold-based tooth design have several complications

– Negative impact on sufficient epithelial–mesenchymal interactions as well as on odontogenic microenvironment


– Interrupted intrinsic positional information in cells

– Acidic products of some scaffolds ( PGA, PLGA, PLA)

– Limited nutrient delivery and metabolic waste removal

– host nonspecific inflammatory responses

3. Cell pellet engineering

• to simplify complicated operating procedures residing in scaffold-based engineering and recombination experiments– scaffold-free method named ‘‘cell pellet engineering”



• cell–cell and cell–matrix interactions in cell pellet are more sufficient and original

• cell movement and selective cell adhesion inside pellets are h h ff ld


more native than those in scaffolds

• no artificial material, no host immune response

• easy to operate, no need to separate dental mesenchymefrom epithelial components or seed cells onto scaffolds




• For optimize 3-dimensional pellet culture, needs further investigations

– in vitro pellet culture time


in vitro pellet culture time

– pellet size

– epithelial–mesenchymal ratios in cell pellets

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4. Chimeric tooth engineering

• chimera is an individual, organ, or part having more than one genetically distinct population of cells that originate from more than one zygote/individual


An example of a sheep-goat chimera: called a “geep”



Permit to create chimeric tooth within relatively short period by using dental cells from multiple teeth or individuals



Permit to create chimeric tooth within relatively short period by using dental cells from multiple teeth or individuals

5. Gene-manipulated tooth regeneration

• Tooth development is an intricate process that encompasses a series of epithelial–mesenchymal interactions guided by various homeobox genes


g

• Gene-manipulated tooth regeneration combines techniques of tooth regeneration with gene therapy


1. In vivo gene-manipulated odontogenesis– Mutations in RUNX2 can bring about the third dentition in the

patients suffering from cleidocranial dysplasia



• repressed or activated genes (esp. RUNX2 )– unexpected nondental characteristics that may be

harmful to the systemic health (esp. bone formation)


• for the lack of suitable tooth-forming cells in situ at adult– questionable that regulated genes in nondental cells can

develop into bio-tooth

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2. In vitro gene-manipulated odontogenesis


– gene transfer of growth/differentiation factor 11 (Gdf11) or bone morphogenetic protein-2 (BMP-2) can induce the differentiation of DPSCs into odontoblasts in vitro and stimulate the reparative dentin formation in the dog model

6. Engineering root and periodontal complex

• recombined tooth bud cells and bone marrow progenitor cells with biological scaffolds to generate bio-tooth and bio-bone


Young et al. Tissue engineering 2005


• hybridized tissue engineering





• cell sheet engineering


6. Engineering root and periodontalcomplex


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Major challenges in reconstructing bio-tooth


How to control shape & size of bio-teeth

• Tooth shape determination during odontogenesis

– Dental lamina stage • homeobox genes


• homeobox genes

– Physical morphological processes • Begin at cap stage • enamel knots (transient signaling centers)


• Preliminary study toward determination of bio-tooth shape– proportions of dental mesenchymal and epithelial cells



• Proportions of dental mesenchymal and epithelial cells

– dental pulp stem cells (DPSC) & apical bud cells (ABC) from dental epithelial stem cell niche of rat incisors


f p f

– DPSC/ABC cell ratios 1:10, 1:3, 1:1, 3:1, 10:1


• Scaffolds to regulate shape and size of bio-teeth– widely used but controversial issue


How to find postnatal epithelial cells necessary for making of bio-teeth

• Epithelial–mesenchymal interactions are a prerequisite– for initiation of odontogenesis– for whole-tooth reconstruction

• Mesenchymal stem cells


• Mesenchymal stem cells– Stem cells from human exfoliated deciduous teeth (SHED)– Adult dental pulp stem cells (DPSC)– Stem cells from the apical part of the papilla (SCAP)– Stem cells from the dental follicle (DFSC)– Periodontal ligament stem cells (PDLSC)– Bone marrow derived mesenchymal stem cells (BMSC)

Bluteau et al. stem cells for tooth engineering. Eur Cells & Materials. 2008

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• In search of epithelium-originated dental stem cells

– no information available for dental EpSC in humans


– dental epithelial cells (ameloblasts and ameloblasts precursors) are eliminated soon after tooth eruption

– insufficient donor tissues (mainly impacted third molar germs)

– low ex vivo expansive potential of epithelial cells


• Epithelial stem cells from labial i l l f d i i


cervical loop of rodent incisor

Harada et al. 1999



How to apply the odontogenic molecularcascades to tooth regeneration

• Current knowledge on molecular regulation of tooth morphogenesis

1. At beginning, signaling molecules from dental epithelium• BMP2 BMP4 FGF8 FGF9 sonic hedgehog (Shh) lymphoid enhancing


• BMP2, BMP4, FGF8, FGF9, sonic hedgehog (Shh), lymphoid enhancing factor 1 (Lef1), Wnt10a, Wnt10b

2. At the bud stage, signaling molecules from dental mesenchyme• BMPs, FGFs, Activin, Msx1, Pax9, Lef1, Dlx1, Barx1, Lhx6 and -7, Gli1, -

2, and -3, Cbfa1

3. At the cap stage, signaling molecules from enamel knot • Shh,BMP2, BMP4, BMP7, FGF4, and FGF9

Current knowledge on molecular regulation of tooth morphogenesis

• Pattern of dentition1. Proximal–distal patterning

• presumptive molar field - Fgf8, Fgf9B 1 (B H lik h b 1)


– Barx1 (BarH-like homeobox 1)– Dlx2 (distal-less homeobox 2)

• Presumptive incisor field - BMP4– Msx1 and Msx2

(homeobox, msh-like 1 & 2)

Tucker et al. Nature review genetics. 2004


• Pattern of dentition2. Rostral–caudal patterning

• FGF8


– Lhx6 and Lhx7

– Gsc (goosecoid)

Tucker et al. Nature review genetics. 2004

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• Tooth number– size of the tooth field is proportional to the number of teeth

• EDA - TNF (tumour necrosis factor) ligandIf th l l f EDA i lli i i d th i f th l t th fi ld


• If the level of EDA signalling is increased, the size of the molar tooth fieldexpands, and a supernumerary tooth develops distal to the first molar

Tucker et al. Dev. Biol. 2004


• Shape of the resulting tooth– TNF family of signalling molecules - Eda

• level of activation of the receptor, rather than the quantity of the ligand


• Ligand concentration is rate limiting for tooth number but not cusp number

Mustonen, T. et al. Dev. Biol. 2003

How to make bio-teeth grow in jaws

• The development of a bioengineered organ germ method

Nakao et al. Nature method. 2007


How to make bio-teeth grow in jaws

• The development of a bioengineered organ germ method

Nakao et al. Nature method. 2007


How to make bio-teeth erupt from jaws

• Current studies focus on the reconstruction of enamel organ but neglect the role of true dental follicle cells

• Tooth eruption mostly relies on normal development of


Tooth eruption mostly relies on normal development of dental follicles

• Making bio-teeth erupt and guiding into accurate occlusalposition may be the most difficult work in future tooth regeneration


• The effect of removing the true dental follicle on premolar eruption in the dog

Larson, EK et al Archs oral biol Vol 39, No. 4 1994


• Tooth eruption : evidence for the central role of the dental follocle

Donald RC. et al J Oral Pathol 9, 189, 1980

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• Alternative 2-step implantation-transplantation strategy


Hu et al. Tissue engineering 2006

Summary

• Current approaches to engineering bio-tooth

1. Recombination experiments2 S ff ld b d h i i


2. Scaffold-based tooth engineering3. Cell pellet engineering4. Chimeric tooth engineering5. Gene-manipulated tooth regeneration6. Engineering root and periodontal complex

Summary

• Major challenges in reconstructing bio-tooth

1. To control shape & size of bio-teeth 2. To find postnatal epithelial cells necessary for making of


bio-teeth 3. To apply the odontogenic molecular cascades to tooth

regeneration 4. To make bio-teeth grow in jaws 5. To make bio-teeth erupt from jaws

Comment


Thank you for your attention


Thank you for your attention

current tissue engineering for tooth regeneration...

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