Regenerated Teeth

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Editorial

135ISSN 1746-075110.2217/RME.10.101 © 2011 Future Medicine Ltd Regen. Med. (2011) 6(2), 135–139

“We hope that the rapid scienc and technological advancement will provide new

informaon and soluons that will allow regenerated teeth to become a roune

treatment for individuals with missing teeth.”

Thimios A Mitsiadis

Author for correspondence:

Instute of Oral Biology,

ZZM, Faculty of Medicine,

University of Zurich,

8032 Zurich, Switzerland

Tel.: +41 446 343 390

Fax: +41 446 344 310

[email protected]

Petros PapagerakisDepartment of Orthodoncs

& Pediatric Denstry, Center for

Organogenesis, Center for

Computaonal Medicine &

Bioinformacs, Dental & Medical

Schools, University of Michigan,

Ann Arbor, 48109 MI, USA

Regenerated teeth: the uture o tooth replacement?

Despite the considerable progress o dental treat-ment and tooth decay prevention, elderly peoplerequently encounter the problem o tooth loss,aecting their quality o lie. Restorative pros-

thetics in the orm o implants provide a com-mon solution to this problem. Dental implanttechnology or tooth replacement was used inseveral ancient civilizations. Indeed, dentalimplants dating rom 2500 BC were ound inEgypt, and tooth replacement was documentedrom the Mayan culture in 600 AD [1]

Dental implants are still used today orthe unctional replacement o missing teeth.However, despite their long history, there areseveral limitations in unctionality and longevity o the implants. Indeed, dental implants cannotrepresent the ideal solution or tooth replacement

since the physiology and plasticity o naturally ormed teeth is not respected. The tooth inter-acts actively with the alveolar bone through theperiodontal ligament (PDL). The mechani-cal stress during mastication is supported andmodulated by this highly specialized tissue thatoccupies the space between the tooth root andthe alveolar bone. PDL is not ormed arounddental implants, making the bone tissue vulner-able when excessive orces are applied during mastication [2]. Alternative strategies are being tested to create a unctional biocompatible type

o replacement or dental implants and eortsare being made to regenerate parts or even theentire tooth organ.

A new concept: brand new teethmade in the laboratoryThe rapid progress made in stem cell, mate-rial and molecular biology sciences over thelast 20 years has allowed scientists working on teeth to imagine alternative and innovativestrategies or tooth replacement [3,4]. Recently,scientists have implemented a new concept

o tooth replacement, where new whole teeth

could be generated experimentally using stemcells (reerred to as BioTeeth, meaning living teeth) [3]. Regenerated tooth (RegTooth) is a more appropriate term, since it is possible to dis-

tinguish between naturally and experimentally ormed teeth. The rationale or the generationo a new whole tooth is simple and consists o recreating and mimicking the molecular andcellular events that occur during the initiation o odontogenesis. This procedure might be a bet-ter alternative to the use o dental implants ortooth replacement, since it involves the regrowthand eruption o new teeth in the mouth o the patients, ater experimental manipulationin vitro. However, a regenerated tooth has sev-eral challenges that need to be solved prior toany clinical trial/application. The reactivation

o the odontogenic program using stem cells isnot obvious and does not guarantee the successo new tooth ormation in an adult mouth. Itis possible to regenerate several human dentaltissues (e.g., dentin and PDL) ater experimen-tal manipulation [3–5]. However, the regener-ated tissues were not identical to their naturally ormed counterparts. With the continuousprogress o science it might be possible in theuture to regenerate more complex dental struc-tures (e.g., enamel) or even the entire tooth.Scientists working in that eld are conronted

daily with new challenges and limitations thatmight postpone the generation o brand new teeth in the laboratory or many years.

Generation of specialized dentalstructures in vivo

Teeth are ormed rom speciic embryoniccells, grow and nally erupt into the oral cav-ity. Through a series o epithelial–mesenchymalinteractions, cells o the oral epithelium andcranial neural crest-derived mesenchymal cells(CNCCs) give rise to complex mineralized

structures that orm the tooth organ [6]. CNCCs



Regen. Med. (2011) 6(2)136 future science group

Editorial Mitsiadis & Papagerakis

orm the dental ollicle and dental pulp, whilethe oral epithelium gives rise to the inner den-tal epithelium. Subsequently, dental pulp cellsdierentiate into odontoblasts and inner dentalepithelium into ameloblasts. Odontoblasts areresponsible or dentin matrix synthesis, whilst

ameloblasts produce the enamel matrix. Oncethe mineralization o the crown is completedthe tooth starts to erupt in the oral cavity, whilethe root continues to develop. Hertwig’s epi-thelium root sheet, a derivative rom the outerdental epithelium and the inner dental epithe-lium, initiates radicular dentin ormation anddetermines the root shape. Root development will be accomplished together with the organi-zation o innervation, vascularisation and rootanchoring to the surrounding alveolar bone.This latest process will be accomplished mainly

by the relationship o three main tissues in theperiodontium: cementum, alveolar bone andPDL. PDL contains a great variety o cells andextracellular matrix. The cellular componentsinclude osteoblasts, broblasts, cementoblasts,osteoclasts, cementoclasts, endothelial cells andepithelial rests o Malassez [4,6].

“Strategies are being tested to create a

funconal biocompable type of

replacement for dental implants and

eorts are being made to regenerate parts

or even the enre tooth organ.”

Partial dental tissue regeneration could beachieved more easily and rapidly than whole-tooth regeneration. In act, methods thatenhance tertiary dentin repair and regenera-tion o the entire dental pulp tissue are being evaluated with variable degrees o success [7–9].In addition, periodontal tissue regeneration isprogressing rapidly with the application o bio-degradable scaolds and growth actors [10]. By contrast, ew studies exist on the regeneration o enamel, which is the hardest human tissue and

represents the visible part o the teeth, protecting them rom abrasion and bacterial attack.

Strategies for building the RegToothThere are two main approaches in constructing a new whole tooth. The rst implies the in vivo implantation o tooth germ cells that were previ-ously generated rom various populations o stemcells or dental progenitor cells and grown in vitro or some time. Organotypic culture is the mostappropriate o the techniques or the develop-ment o the teeth in vitro. The other approach

consists o implanting into the jaw tooth-shaped

polymer scaolds that are lled with in vitro expanded stem cells or dental progenitor cellpopulations. Ideally, this implant should repro-duce the 3D structure required or the trans-planted cells to support their dierentiation andavoid xenograt rejection [3].

One o the challenges in regenerative dentistry is to nd cells that can replace the clonogenicCNCCs. The identication and characterizationo human adult stem cells (or progenitor cells)o dental origin will contribute to regeneratesuccessully tooth primordia. Dental stem cellscould be removed rom a patient, expanded andput back into the same individual when toothregeneration becomes necessary, thereby remov-ing the need or immunosuppression [3]. Thesecells can be isolated rom either primary or per-manent teeth. Dental stem cells can be extracted

rom the apical papilla o shed primary teeth,exoliated deciduous teeth and the dental pulp(DPSCs). These stem cells have the potentialto dierentiate into various cell types, such aschondrocytes, adipocytes, osteoblasts, myocytes,neuronal cells and cardiomyocytes [3,4]. Thecomposition o the culture medium in whichDPSCs are grown can dictate their dierentia-tion into odontoblasts, osteoblasts and chondro-blasts. However, adult stem cells are present atlow requency (e.g., roughly one stem cell per100,000 bone marrow cells), making both iso-lation and expansion o DPSCs problematic [4].

Recently, dental pulp cells were reprogrammedinto induced pluripotent stem (iPS) cells [11].

Limited inormation is available regarding theregenerative potential o dental epithelial stemcells (DESCs), which could give rise to amelo-blasts. Ameloblasts are not present in eruptedteeth. Undierentiated wisdom teeth present a potential source o human dental epithelium [12].The Epithelial Rests o Mallasez (ERM) con-stitute another source o DESCs [Papagerakis P,

Unpublished Data]. ERM are localized in theperiodontal ligament o adult teeth and exhibit

stem cell properties. ERM can dierentiate intoenamel-secreting cells when co-cultured withDPSCs [Papagerak is P, Unpublished Data] [13]. Thus,DESCs isolated rom ERM can provide a via-ble source o ameloblast progenitors or enamelregeneration. Alternatively, epithelial stem cellso nondental origin (e.g., hairs and skin) couldbe used or the ormation o enamel.

Thereore, dental mesenchymal stem cells thatinteract in vitro with dental epithelial stem cells(ater recombination) might be able to orm thevarious mesenchymal and epithelial cell popula-

tions in a regenerated tooth. A study in rodents



137future science group www.futuremedicine.com

Regenerated teeth: the uture o tooth replacement? Editorial

has shown that bone marrow cells have stemcell properties and can substitute or CNCCsduring tooth regeneration [14]. However, thisexperimental approach was partly successulsince a truncated ‘tooth’ (i.e., absence o roots) was obtained, which resembled an odontoma

(a dental pathological condition) more than a normal tooth.

Tooth-like structures have also been producedusing heterogeneous dental cell populations inbiodegradable polymer scaolds. Disaggregatedand reaggregated dental epithelial and mesen-chymal cells are able to interact and recapitulateodontogenesis and ormation o tooth-specicstructures in animal models. However, theseimplants exhibited a mosaic o organized anddisorganized enamel, dentin and pulp tissues,and did not embrace the size and shape o the

scaolds[15]

.

Challenges of dentaltissue regeneration Enamel regeneration: structure,color & timeEnamel ormation (amelogenesis) is the result o a series o complex, dynamic and programmedcellular, chemical and physiological events [16].These events allow categorization o enamelormation in three distinct stages (i.e., thesecretory, transition and maturation stages).The secretory stage is characterized by active

protein synthesis and secretion by the amelo-blasts. Ameloblasts also deposit enamel crystalsat oblique angles while they are moving in thedirection o the uture cusps to accommodateexpansion o the enamel surace. The matura-tion stage is characterized by removal o enamelorganic materials and growth o hydroxy apatitecrystals in thickness, as well as by regulatedmovement o ions into and out o the enamelmatrix [17,18]. Ions arrive to ameloblasts romthe blood vessels ater traversing a distance o 50–100 µm (i.e., 2–3 cell layers). Stem cells

destined to orm enamel must reproduce thesethree stages and the cellular movement thatoccurs during enamel apposition. Organicmaterial removal, vascularization and exten-sive ion transport must also be achieved in theregenerated enamel.

Besides their involvement in crystal orma-tion, ions also contribute to the color o theteeth [19]. Enamel in humans is characterizedby a big diversity o colors (i.e., a rich spec-trum o yellow, grey and white tonalities), a variety that can be oten observed in teeth o

the same individual. Tooth color variations are

also observed in other animal species, such as inmice, where the enamel o the incisors exhibitsa deep yellow color, while molars have a whitecolor. However, little inormation exists onhow ions control color variability in humanenamel adding another diculty in the enamel

regeneration process.Time represents another great challenge o

enamel regeneration. It is well known that the whole process o enamel ormation in humanpermanent teeth may take more than 5 yearsand might be regulated by complex interactionso clock genes [20]. This long-term physiologicalprocedure may be discouraging or individu-als with missing enamel who look orward toimmediate treatment outcomes.

“The rapid progress made in stem cell,

material and molecular biology sciences

over the last 20 years has allowed sciensts

working on teeth to imagine alternave and

innovave strategies for tooth replacement.”

Thereore, regeneration o enamel requiresproduction and secretion o the right amounto proteins, at the right place, at the right time.Otherwise, the eort to regenerate enamel willail (as happens in several mutations aect-ing enamel ormation) [21]. Thus, regenerationo human enamel is a very dicult – almostimpossible – task at the present. Much more

must be discovered beore enamel regenerationbecomes a routine procedure in dentistry. Thisalso holds true or whole tooth regeneration.

The right shape & size ofthe RegToothIt is imperative that the RegTooth, which willdevelop in the patient’s mouth ater transplanta-tion, acquires the correct morphology and size.Tooth shape is primarily determined during early odontogenesis. The ormation o the vari-ous tooth shapes might be either the result o

prepatterned CNCC, or a consequence o theresponse o CNCCs to signals originated by the oral epithelium. Tissue recombination andtransplantation experiments indicated that sig-nals rom the oral epithelium infuence CNCCsto adopt a dental identity [6].

Several secreted signaling molecules, such asBMPs, FGFs, Wnts and Shh, are expressed inthe epithelium and unction as morphogens thatcontrol the generation o diverse tooth shapes.For example, BMP4 expression is linked withthe incisors’ shape, while FGF8 is linked with

the shape o molars. BMP4 activates expression



Regen. Med. (2011) 6(2)138 future science group

Editorial Mitsiadis & Papagerakis

o Msx1 and Msx2 in the mesenchyme o utureincisors. Similarly, Islet1 is expressed only in theepithelium o the incisors and its expression isregulated by BMP4. By contrast, FGF8 acti-vates Dlx1, Dlx2 and Barx1 expression in themesenchyme o uture molars [6].

Furthermore, the shape o maxillary andmandibular teeth diers and is controlled by genes such as Dlx , Barx1 and Pitx1 [6]. Forexample, Pitx1 deletion aects only the man-dibular molars, which are smaller and have ewercusps [22].

Alteration o the odontogenic signaling cas-cade might also lead to modication o toothsize. For example, smaller teeth were reported inmice ater deletion o Wnt signaling [6].

Root & eruption of the RegTooth

Successul tooth regeneration requires the or-mation o roots with appropriate shape andlength. For example, short roots could beproblematic in retaining the regenerated teethin place. Similarly, it is important that root-related structures (e.g., PDL) o the regener-ated teeth remain unctional or long periods,thereby avoiding pathological maniestationssuch as tooth ankylosis. The time and orien-tation o tooth eruption in adults also has tobe controlled. Both proper root ormation andtooth eruption are time-consuming processes,thus making the choice o a RegTooth or tooth

replacement problematic.Ideally, autologous tissues must be used or

the implantation o regenerated teeth in a givenpatient, thus avoiding immunological rejec-tion [3]. Gene therapy-based strategies must beapplied or the regeneration o teeth in indi-viduals with mutations that aect their denti-tion (e.g., amelogenesis imperecta, ectodermaldysplasia) [6].

ConclusionTooth regeneration provides an attractive alter-

native to existing tooth restoration therapies.This concept relies on the in vitro recreation

o the genetic odontogenic program using stemcells. Numerous genes control embryonic toothdevelopment and dene the various dental ter-ritories (i.e., incisors, canines, premolars andmolars) in the mouth, as well as the number,shape, size and color o the teeth. A stem cell

strategy or tooth regeneration must combinestem cell populations with adequate signaling molecules. Cell-based therapies are in theirinancy and many issues need to be addressedbeore any clinical application. The exist-ing challenges include the need to determineconsistent protocols to control the size, shapeand color o teeth, as well as to considerably shorten the time o enamel and root ormationand tooth eruption. Furthermore, the use o culture-expanded stem cell populations needsto take into account the possibility o genetic

and epigenetic instability.

“Sciensts … are confronted daily with

new challenges and limitaons that

might postpone the generaon of brand

new teeth in the laboratory for

many years.”

Although the prospect o tooth regenerationusing stem cells is very attractive, it is not likely that they will replace routine clinical dentalpractices in the near uture. We hope that therapid scientic and technological advancement

will provide new inormation and solutions that will allow regenerated teeth to become a routinetreatment or individuals with missing teeth.

Financial & competing interests disclosure

The authors have no relevant aliations or nancial

involvement with any organization or entity with a nan-

cial interest in or nancial confict with the subject matter

or materials discussed in the manuscript. This includes

employment, consultancies, honoraria, stock ownership or

options, expert testimony, grants or patents received or

pending, or royalties.

No writing assistance was utilized in the production o this manuscript.

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Regenerated Teeth