Whole Tooth Regeneration As a Future Dental Treatment 14

Whole Tooth Regeneration as a Future Dental Treatment 14

Masamitsu Oshima and Takashi Tsuji

Abstract Dental problems caused by dental caries, periodontal disease and tooth injury compromise the oral and general health issues. Current advances for the development of regenerative therapy have been inﬂ uenced by our understanding of embryonic development, stem cell biology, and tissue engineering technology. Tooth regenerative therapy for tooth tissue repair and whole tooth replacement is currently expected a novel therapeutic concept with the full recovery of tooth physiological functions. Dental stem cells and cell-activating cytokines are thought to be candidate approach for tooth tissue regeneration because they have the potential to differentiate into tooth tissues in vitro and in vivo. Whole tooth replacement therapy is considered to be an attractive concept for next generation regenerative therapy as a form of bioengineered organ replacement. For realization of whole tooth regeneration, we have developed a novel three- dimensional cell manipulation method designated the “organ germ method”. This method involves compartmentalisation of epithelial and mesenchymal cells at a high cell density to mimic multicellular assembly

M. Oshima , Ph.D. Department of Oral Rehabilitation and Regenerative Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University , Okayama 700-8525 , Japan RIKEN Center for Developmental Biology , Kobe , Hyogo 650-0047 , Japan e-mail: [email protected] T. Tsuji , Ph.D. (*) RIKEN Center for Developmental Biology , Kobe , Hyogo 650-0047 , Japan Research Institute for Science and Technology , Tokyo University of Science , Noda Chiba , 278-8510 , Japan Organ Technologies Inc , Tokyo 101-0048 , Japan e-mail: [email protected]

© Springer International Publishing Switzerland 2015 255 L.E. Bertassoni, P.G. Coelho (eds.), Engineering Mineralized and Load Bearing Tissues, Advances in Experimental Medicine and Biology 881, DOI 10.1007/978-3-319-22345-2_14 256 M. Oshima and T. Tsuji

conditions and epithelial-mesenchymal interactions in organogenesis. The bioengineered tooth germ generates a structurally correct tooth in vitro, and erupted successfully with correct tooth structure when transplanted into the oral cavity. We have ectopically generated a bioengineered tooth unit composed of a mature tooth, periodontal ligament and alveolar bone, and that tooth unit was engrafted into an adult jawbone through bone integration. Bioengineered teeth were also able to perform physiological tooth functions such as mastication, periodontal ligament function and response to noxious stimuli. In this review, we describe recent ﬁ ndings and technologies underpinning whole tooth regenerative therapy.

Keywords Whole tooth regeneration • Tooth replacement • Dental tissue engineering • Organ germ • Tooth germ • Stem cells

14.1 Introduction treat tooth loss (Brenemark and Zarb 1985 ; Burns et al. 2003 ). Although these artifi cial therapies Oral functions regarding mastication, swallowing have been widely applied to rehabilitation of tooth and speech are an important aspect of good health loss, it is anticipated that further technological and quality of life (Proffi t et al. 2004 ). The tooth developments based on biological fi ndings are is an ectodermal organ whose development is needed to restore tooth physiological functions regulated by reciprocal epithelial-mesenchymal (Tucker and Sharpe 2004 ). interactions (Jussila et al. 2013 ; Tucker and Recent regenerative therapies have been devel- Sharpe 2004 ; Ikeda and Tsuji 2008 ) and contains oped based on our understanding of embryonic distinctive hard tissue structure composed of development, stem cell biology and tissue engi- enamel, dentin and cementum (Avery 2002 ; Nanci neering technology (Korbling and Estrov 2003 ; 2012 ). Teeth also have soft connective tissues Brockes and Kumar 2005 ; Watt and Hogan 2000 ; such as pulp and periodontal ligament (PDL) that Langer and Vacanti 1999; Atala 2005 ). One con- include peripheral nerve fi bres and blood vessels cept in regenerative therapies rely on the cell to maintain tooth homeostasis and physiological transplantation of purifi ed tissue-derived stem functions (Avery 2002 ; Nanci 2012 ). Tooth physi- cells or embryonic stem (ES) or induced pluripo- ological functions are exerted effi ciently by the tent stem (iPS) cells. These stem cell transplanta- characteristic three-dimensional multicellular tion, which targets structural and functional structure that establishes functional synergy with diseases such as malignant diseases, neurological the maxillofacial region (Avery 2002 ; Nanci disorders, myocardial infarction, and hepatic dys- 2012). Tooth loss due to dental caries, periodontal function, has been attempted to repair damaged disease and traumatic injury causes fundamental tissues (Copelan 2006 ; Lindvall and Kokaia 2006 ; problems for oral functions and associated gen- Segers and Lee 2008 ; Wang et al. 2003 ). In den- eral health issues (Proffi t et al. 2004 ). To restore tistry, basic research of stem/progenitor cells have the occlusal function or aesthetic condition after provided new insights concerning tooth tissue- tooth loss, several dental therapies that replace the derived stem cells, including dental pulp stem tooth with artifi cial materials such as fi xed dental cells (DPSCs), stem cells from human exfoliated bridges and removable dentures have been per- deciduous teeth (SHED) and stem cells from api- formed conventionally (Rosenstiel et al. 2001 ; cal papilla (SCAP) that have been isolated from Pokorny et al. 2008 ). Recently, osseointegrated the dental pulp tissue (Huang et al. 2009 ; Gronthos dental implants that can restore function without et al. 2000 ; Miura et al. 2003 ; Sonoyama et al. affecting the healthy teeth have been adopted to 2008 ). These stem cells are thought to be a poten- 14 Whole Tooth Regeneration as a Future Dental Treatment 257 tial resource for stem cell-mediated tissue repair, a lost or damaged tooth with a bioengineered including dentin or pulp regeneration, based on tooth reconstructed from stem cells and with the their high proliferation and multi-differentiation potential to generate a functional tooth unit capacity (Mantesso and Sharpe 2009 ; Yen and including the whole tooth and periodontal tissue Sharpe 2008 ). Also, periodontal ligament-derived surrounding the alveolar bone (Purnell 2008 ; stem cells (PDLSCs), which can differentiate into Volponi et al. 2010 ; Sharpe and Young 2005 ). It is all periodontal cell types after transplantation, anticipated that whole tooth replacement therapy have also been identifi ed, and have been attempted will be established in the near future as a success- to develop cell sheet-engineering using PDLSCs ful biological treatment that will provide essen- for clinical use in periodontal tissue regeneration tial functional recovery of lost teeth to satisfy (Seo et al. 2004 ). Although these treatments con- aesthetic and physiological requirements tribute to partial tissue repair, many researchers (Volponi et al. 2010; Sharpe and Young 2005 ) anticipate the development of further therapeutic (Fig. 14.1). Over the past three decades, many technologies using dental stem cells that can approaches for replacing lost teeth have been regenerate lost teeth (Mantesso and Sharpe 2009 ; studied, including three-dimensional bioengi- Yen and Sharpe 2008 ). neered teeth and tooth germ generation using Organ replacement regenerative therapy, biodegradable materials and cell aggregation unlike stem-cell transplantation, holds great methods (Sharpe and Young 2005 ; Duailibi et al. promise for the replacement of dysfunctional 2006 ). Recently, the fi rst studies of fully func- organs via a regenerative strategy by reconstruct- tioning bioengineered tooth replacement with the ing a fully functional bioengineered organ using correct tooth structure, masticatory performance, three-dimensional cell manipulation in vitro responsiveness to mechanical stress and percep- (Atala 2005; Seo et al. 2004 ). It is expected that tive potential following transplantation into a bioengineering technology will eventually enable tooth-loss region were reported (Nakao et al. the reconstruction of fully functional organs 2007; Ikeda et al. 2009 ; Oshima et al. 2011 ). In in vitro through the proper arrangement of sev- this chapter, we describe novel technologies for eral cell components. In the dental fi eld, tooth whole tooth replacement therapy that have the regenerative therapy involves the replacement of potential to provide functional recovery and

Fig. 14.1 Concepts of dental regenerative therapy. Recent approaches to developing technologies for tooth regenerative therapy have included tissue repair and whole-tooth replacement 258 M. Oshima and T. Tsuji entirely replace the current dental treatments genes e.g. Msx1 , Msx2 , Lhx8 and Barx1 and based on artifi cial materials. secretory molecules including fi broblast growth factors (FGFs) and bone morphogenetic pro- teins (BMPs) (Ikeda and Tsuji 2008 ; Thesleff 14.2 Developmental Process 2003 ; Bei 2009; Nakatomi et al. 2010 ). At ED of Tooth Formation 11.5, oral epithelium invaginates into the mesenchymal region, and then tooth bud is formed Ectodermal organs, including the teeth, hair and by the condensation of mesenchyme that is salivary glands, arise from their respective organ derived from neural crest cells. At ED 13.5– germs through the reciprocal epithelial- 14.5, a transient epithelial signalling centre mesenchymal interactions in the developing called the fi rst enamel knot, which expresses embryo (Thesleff 2003) (Fig. 14.2). The mecha- several signalling molecules, including Shh, nism of tooth formation is also regulated by Wnts, BMPs and FGFs, is thought to regulate reciprocal epithelial and mesenchymal interac- individual cell fates and to orchestrate epithelial tions, particularly those involved in stem cell, morphogenesis through the epithelial-mesen- signalling molecule and transcription factor chymal interactions (Ikeda and Tsuji 2008 ). The pathways (Jussila et al. 2013; Thesleff 2003). epithelial and mesenchymal cells in the tooth During early craniofacial development in mice, germ differentiate into the respective progenitor the dental lamina fi rst thickens, followed by epi- cells, including ameloblasts, odontoblasts and thelial thickening at the sites of future teeth and dental follicle cells at ED 16.5–18.5. These pro- subsequent epithelial budding to the neural genitor cells secrete a collagenous extracellular crest-derived ecto-mesenchyme. Tooth-forming matrix that mineralises into the enamel and den- fi elds are specifi ed at embryonic day (ED) tin tissue at the epithelium–mesenchyme inter- 10–11 through the expression of homeobox face (Fukumoto and Yamada 2005 ). Tooth

Fig. 14.2 Schematic representation of the developmental epithelial and mesenchymal cells in the tooth germ stages of a tooth. The tooth germ is formed from the differentiate into tooth tissue-forming cells including dental lamina, which consists of an invaginated epithelium ameloblasts, odontoblasts and dental follicle cells (ED that is developed from the immature oral epithelium and 18). Ameloblasts and odontoblasts accumulate enamel condensed mesenchyme cells that is derived from neural and dentin, respectively, at the boundary surface between crest cells (ED 11–12). The ﬁ rst enamel knot, which acts the epithelium and mesenchyme. On the other hand, the as a developmental signalling centre forms within the dental follicle cells differentiate into periodontal tissues, dental epithelium (ED 13–15). The secondary enamel including the periodontal ligament, cementum and knots are formed; these tissues play an important role in alveolar bone regulating the cusp position and number (ED 16). The 14 Whole Tooth Regeneration as a Future Dental Treatment 259 morphogenesis, including tooth size and shape, achieved their goal of reconstructing complex are thought to be regulated by signalling mole- organs which is in stark contrast to the stem cell cules such as the BMPs and FGFs emanating transplantation therapies used to repair the dam- from the secondary enamel knots, which aged tissues. The ultimate goal of regenerative regulate the cusp pattern of the mature natural therapy in the future is to develop organ replace- tooth in the early bell stage to form the tooth ment regenerative therapies that will restore lost crown (Thesleff 2003 ). After tooth crown for- or damaged tissues following disease, injury or mation, tooth eruption into the oral cavity begins aging with a fully functioning bioengineered involving tooth-root elongation, and dental fol- complex organ (Atala 2005 ; Seo et al. 2004 ). To licle cells form the periodontal tissues including realize the reconstruction of bioengineered cementum, PDL and alveolar bone onto root- organs, one biological approach is based on reca- dentin surface (Ikeda and Tsuji 2008 ; Foster pitulating organogenesis by mimicking the recip- et al. 2007 ; Yang et al. 2009 ). rocal epithelial-mesenchymal interactions that occur in the organ developmental process, thereby developing fully functional bioengi- 14.3 Technological Development neered organ from a bioengineered organ germ for Whole Tooth by using immature stem cells via three- Regeneration by Using dimensional cell manipulation technology a Novel Three-Dimensional in vitro (Ikeda and Tsuji 2008 ; Sharpe and Young Cell Manipulation Method 2005 ). For whole tooth regeneration, an attractive concept has been proposed in which a bioengi- Stem cell transplantation therapy is now consid- neered tooth germ would be transplanted into the ered an effective approach to restore partial organ tooth loss region and would develop into a func- functions at local damaged sites. However, cur- tioning mature tooth (Ikeda et al. 2009 ) (Fig. rent bioengineering technologies have not yet 14.3, upper panel). Furthermore, it is expected

Fig. 14.3 Regenerative strategy of whole-tooth the organ germ method or by transplanting bioengineered replacement. Fully functioning teeth can be regenerated tooth units with a periodontal ligament and alveolar bone in vivo by transplanting bioengineered tooth germs that developed from bioengineered tooth germs reconstituted from epithelial and mesenchymal cells via 260 M. Oshima and T. Tsuji that it will be possible to transplant a bioengi- considered to be the cause of the low frequency neered mature tooth unit, including tooth, PDL of tooth formation and the irregularity of the and alveolar bone, which will engraft through resulting tooth tissue structures e.g. the enamel- physiological bone integration of the recipient’s dentin complex and the cell arrangement of ame- jaw (Oshima et al. 2011 ) (Fig. 14.3 , lower panel). loblast/odontoblast lineages (Honda et al. 2003 ; Transplantation of a bioengineered tooth unit has Young et al. 2002 ). Fully generation of proper also been proposed as a viable option to repair tooth structure using scaffolds requires the for- the large resorption defects in the alveolar bone mation of complex junctions between the after tooth loss (Oshima et al. 2011 ). To enable enamel, dentin and cementum that result from these whole tooth regenerative strategies, it will accurate spatiotemporal cell gradients of amelo- be important fi rst to develop techniques for the blasts, odontoblasts and cementoblasts as well as manipulation of cells in three dimensions in order natural tooth development (Volponi et al. 2010 ; to reconstruct bioengineered tooth using com- Nakao et al. 2007 ). pletely dissociated epithelial and mesenchymal cells in vitro. To date, two conventional approaches and a novel cell manipulation method 14.3.2 Cell Aggregation Method are currently being examined, as described below. The cell aggregation method is known as a typi- cal bioengineering protocol employed for the 14.3.1 Biodegradable Scaffold reconstitution of a bioengineered organ germ to Method reproduce the epithelial-mesenchymal interactions that occur during organogenesis (Purnell Scaffold technology represented by three- 2008 ; Volponi et al. 2010 ). Previous studies have dimensional tissue engineering has contributed reported that transplanting bioengineered cell to the large-scale tissue regeneration for dam- aggregates derived from the ectodermal origin aged tissues through seeding target cells on such as hair follicle and mammary gland, and degradable materials such as natural molecules demonstrated in the in vivo regeneration of each and synthetic polymers. This method has shown organ with the proper tissue structure and cellular high utility in three-dimensional tissue engineer- arrangements (Zheng et al. 2005 ; Shackleton ing technology, and these preparations have been et al. 2006 ). In the dental fi eld, many researchers used in clinical applications including bone and have isolated the dental epithelial and mesenchy- cartilage regenerative therapies (Quarto et al. mal tissues from embryonic tooth germs using 2001 ; Cao et al. 1997 ; Caplan and Bruder 2001 ). stereomicroscopic guidance, and dissociating Previous studies using collagen/gelatine sponges such tissues with surgical and enzymatic treat- or polyglycolic acid/poly-L-lactate-co-glycolide ments to obtain single stem cells. It has been copolymers (PLA/PLGA) have reported the par- reported that bioengineering cell aggregates re- tial generation of tooth tissue structure through pelleted with dental epithelial and mesenchymal seeding epithelial and mesenchymal cells iso- stem cells using the cellular centrifugation have lated from porcine tooth germ (Honda et al. the potential for tooth germ formation after 2003 , 2007 ; Young et al. 2002 ; Iwatsuki et al. in vivo transplantation (Hu et al. 2006 ; Yamamoto 2006; Duailibi et al. 2004 ; Yelick and Vacanti et al. 2003 ). Even when bioengineered cell aggre- 2006 ; Sumita et al. 2006). This scaffold-based gate were mixed with epithelial and mesenchy- method may be practical for controlling tooth mal stem cells isolated from tooth germ, the shape and size; however, the fundamental prob- correct tooth structure could be generated by the lems regarding the regeneration of tooth itself self-reorganisation through the cell rearrange- have not been resolved. The presence of residual ment of epithelial and mesenchymal cells (Song scaffold material after in vivo transplantation is et al. 2006). Although this technique partially 14 Whole Tooth Regeneration as a Future Dental Treatment 261 replicated tooth organogenesis, further improve- tion. The bioengineered tooth germ generates a ments in the frequency of bioengineered tooth structurally correct tooth after transplantation in development and correct tissue formation has an organ culture in vitro as well as following been required. placement into a subrenal capsule in vivo (Nakao et al. 2007 ) (Fig. 14.4a ). Tooth morphology is defi ned by not only the crown size and tooth 14.3.3 Three-Dimensional Cell- length as macro-morphological feature but also Manipulation Method: the cusp numbers/position as micro- The “Organ Germ Method” morphological feature. These morphological regulations are determined in the tooth-forming To achieve precise replication of the develop- fi eld by specifi c gene expression in immature oral mental processes in organogenesis, an in vitro epithelium and neural crest-derived mesenchyme three-dimensional novel cell manipulation in the embryonic jaw. It is considered that macro- method designated as a bioengineered organ patterning, including the number and size of teeth germ method has been recently established are spatiotemporally regulated by the patterned (Nakao et al. 2007 ). We investigated the possibil- signalling molecules in accordance with an ity of developing a bioengineered tooth germ activator- inhibitor model. The tooth micro- using completely dissociated single stem cells patterning, including the position and number of from epithelial and mesenchymal tissues of inci- cusps in the teeth, are also thought to be involved sor or molar tooth germ at cap stage in ED 14.5 in the secondary enamel knots. Thus, the pattern- mice. The bioengineered cell aggregate reconsti- ing of signalling molecules based on the reaction- tuting by epithelial or mesenchymal cells alone diffusion mechanism, which is regulated by the generated keratinized-epithelium or bone, respec- reciprocal activation and inhibition of cell prolif- tively, on subrenal capsule transplantation. eration in epithelial and mesenchymal tissues, is Bioengineered tooth germ that reconstituted the important for the determination of tooth morpho- cell compartmentalization between epithelial and genesis (Thesleff 2003 ). Bioengineered tooth mesenchymal cells at a low cell density (0.5– germs were reconstructed using various cell-to- 1.0 × 108 cells/ml), or which did not form cell cell contact lengths between the epithelial and compartmentalization (i.e. mixed cell aggregate) mesenchymal cell layers, and thereby the crown at high-cell density (5.0 × 108 cells/ml), was also widths and cusp numbers of generated bioengi- unable to generate a correct tooth structure neered teeth were dependent on the contact (Nakao et al. 2007 ). The most signifi cant break- length of the bioengineered tooth germs (Ishida through using our method is the achievement of et al. 2011 ). three-dimensional cell compartmentalization of Moreover, an attractive technology for gener- epithelial and mesenchymal cells at a high cell ating a bioengineered tooth unit composed of a density in a collagen gel. This bioengineered mature tooth, PDL and alveolar bone through tooth germ can achieve initial tooth development in vivo transplantation into the subrenal capsule with the appropriate cell-to-cell compaction has been developed (Fig. 14.4b, c ). Multiple between epithelial and mesenchymal cells bioengineered tooth units surrounded by alveo- in vitro organ culture. Bioengineering tooth germ lar bone could also be generated through trans- reproducing by this method successfully repli- planting several tooth germs in a size-control cates the multicellular assembly, including ame- device (Oshima et al. 2011 ). Each bioengineered loblasts, odontoblasts, pulp cells and dental tooth had the correct toot structure including the follicle cells, based on the epithelial- mesenchymal pulp cavities and the partitioned periodontal tis- interactions as well as natural tooth development, sue structure (Fig. 14.4b ). Hence, it would be and could allow for large-scale organ reconstruc- possible to accomplish the multiple teeth 262 M. Oshima and T. Tsuji

a 3D-cell manipulation by an organ germ method Cell manipulation in collagen gel Organ culture day 14m HE staining

Scale bar:400µm Scale bar :500 µm

bcSingle bioengineered tooth unit Bioengineered tooth unit Photograph Micro CT HE analysis

Scale bar :200 µm Multiple bioengineered tooth units Photograph Micro CT

Scale bar : 500 µm Scale bar : 50 µm

Scale bar : 200 µm

Fig. 14.4 The organ germ method: a three-dimensional isolated from incisor and molar tooth germ, developed in cell-processing system. ( a) Dissociated mesenchymal in vitro organ culture over 14 days (right panels ). (b ) cells at a high density are injected into the centre of a Transplantation of this bioengineered tooth germ into a collagen drop. Tooth germ-derived epithelial cells and subrenal capsule for 30 days ( upper panels ). Multiple mesenchymal cells are injected into the collagen drop at a bioengineered tooth units surrounded by alveolar bone high density ( left panel ). At 1 day of organ culture, the can also be generated by transplanting several tooth germs bioengineered tooth germ with the appropriate ( lower panels ). (c ) A bioengineered tooth unit composed compartmentalization between the epithelial and of a mature tooth, periodontal ligament and alveolar bone, mesenchymal cells and cell-to-cell compaction was was generated with the natural tooth structure such as formed (centre -left panel). Bioengineered molar tooth enamel ( E ), dentin ( D ), the periodontal ligament ( PDL ) germs, which were reconstituted using dissociated cells and alveolar bone replacement in the case of edentulous jaw by 14.4 Functional Whole-Tooth using this regenerative transplantation method Replacement Using (Oshima et al. 2011 ). These technologies have the Bioengineered Tooth the great promise to achieve functional tooth regeneration and also indicate a substantial Regenerated organs should exhibit organ- advance in bioengineered organ replacement intrinsic functions in cooperation with the sur- regenerative therapy. rounding environment, including vascular and 14 Whole Tooth Regeneration as a Future Dental Treatment 263 nervous system in the host. Previous studies in quently affect the resorption of the alveolar bone Xenopus showed that regenerated secondary overlying the tooth germ during tooth eruption organs can restore the physiological function of (Dawson 2006 ; Wise and King 2008). So far, it organs such as heartbeat and neural innervation has been reported that a transplanted natural (Sedohara et al. 2003; Tyler and Baker 2003 ). It tooth germ erupted in a murine toothless dia- is, thus, expected to demonstrate the organ stema region (Nakao et al. 2007 ; Ohazama et al. replacement by using a bioengineered organ in 2004 ). Recently, we have demonstrated that a mammalians. Oral functions such as mastication, bioengineered tooth germ can develop the correct pronunciation, and facial aesthetics have an tooth structure in an oral cavity and also success- important contribution to the quality of life fully erupt 37 days after transplantation (Nakao because they facilitate both oral communication et al. 2007; Ikeda et al. 2009 ). Bioengineered and general health. These functions are exerted tooth erupts autonomously through the regulation by the teeth, masticatory muscles and of bone remodelling in the eruption pathway that temporomandibular joint under the control of the faithfully reproduces the molecular mechanisms central nervous system (Proffi t et al. 2004 ; involved in natural tooth eruption. Subsequently, Dawson 2006 ). For the realisation of tooth the erupting bioengineered tooth not only reached replacement regenerative therapy, a regenerated the occlusal plane but also maintained occlusal tooth developing from bioengineered germ or a function with the opposing natural tooth at 49 transplanted bioengineered mature tooth unit days after transplantation (Ikeda et al. 2009 ) (Fig. must be capable of properly engrafting into the 14.5a ). lost tooth region in an adult oral environment and Transplantation of a bioengineered mature acquiring full functionality, including suffi cient organ would lead to the immediate performance masticatory performance, biological integration of the full functions in vivo and greatly affect via periodontal attachment and afferent respon- on the survival outcomes in numerous diseases siveness to noxious stimulations in the maxillofa- (Gridelli and Remuzzi 2000). In the case of a cial region (Proffi t et al. 2004 ; Dawson 2006 ). transplanted bioengineered mature tooth unit comprising mature tooth, PDL and alveolar bone, the most critical consideration is whether 14.4.1 Successful Transplantation that unit can be engrafted into the tooth loss of a Bioengineered Tooth region through bone integration, which involves Germ or a Bioengineered natural bone remodelling in the recipient. A Mature Tooth Unit for Whole- bioengineered tooth unit transplanted at a posi- Tooth Replacement tion reaching the occlusal plane with the opposing upper fi rst molar was successfully engrafted The critical issue regarding the successful tooth after 40 days and then maintained the periodon- regenerative therapy via the transplantation of tal ligament originating from the bioengineered bioengineered tooth germ into the tooth loss tooth unit through successful bone integration region is whether the germ can erupt and occlude and regeneration (Oshima et al. 2011 ) (Fig. properly with the opposing tooth in an adult oral 14.5b, c). The hardness of the enamel and den- environment. Natural tooth eruption is autono- tin components in bioengineered teeth were mously regulated by biological mechanism that equivalent to that of natural teeth upon analyse involves the tooth germ cell components and the using the Knoop hardness test (Ikeda et al. surrounding alveolar/jawbone area (Wise et al. 2009 ; Oshima et al. 2011 ). These approaches 2002 ; Wise and King 2008 ). In the tooth develop- demonstrated that the potential to successfully mental process, dental follicle cells, which encir- recover masticatory performance and natural cle around the developing tooth germ, generate tooth tissue through state-of-the-art bioengi- the cementum, PDL and alveolar bone and subse- neering technology. 264 M. Oshima and T. Tsuji

a Bioengineered tooth Bioengineered tooth HE analysis of a eruption occlusion bioengineered tooth Day 16

Day 37

Day 49

Scale bar:200 µm Scale bar:200 µm Scale bar:100 µm

b Bioengineered tooth unit HE analysis of a Higher magnification occlusion bioengineered tooth unit NT BT

Scale bar:200 µm

Scale bar:500 µm

c Bone regeneration by the transplantation of a bioengineered tooth unit Control Transplantation

Fig. 14.5 Transplantation of a bioengineered tooth germ bioengineered tooth unit also had the correct tooth or a bioengineered tooth unit. (a ) Bioengineered tooth structure. NT natural tooth, BT bioengineered tooth, AB germ erupted ( arrowheads , left panels) and reached the alveolar bone, PDL periodontal ligament (centre and right occlusal plane with the opposing lower ﬁ rst molar at 49 panels ). (c ) Three- dimensional superposition of micro-CT days after transplantation ( arrowheads , centre panels ). images of natural dentition ( gray , double dotted line ), a The bioengineered tooth also formed a correct tooth transplanted bioengineered tooth unit ( right panel) and a structure comprising enamel, dentin, dental pulp, and no transplantation control ( left panel ) at day 0 in an alveolar bone ( right panels ). (b ) The bioengineered tooth extensive bone defect (red , straight line), and at 45 days unit was engrafted through bone integration and occluded after transplantation ( green , dotted line) are represented. with the opposing upper ﬁ rst molar at 40 days after The superior edges of the recipient alveolar bone are transplantation ( arrowheads , left panels ). The engrafted indicated by each line 14 Whole Tooth Regeneration as a Future Dental Treatment 265

14.4.2 Biological Response critical dental functions and subsequently lead of Bioengineered Tooth to the restoration and re-establishment of func- to Mechanical Stress tional teeth within the maxillofacial region (Ikeda et al. 2009 ; Oshima et al. 2011 ). Full oral functions are essential for the cooperation with teeth and the maxillofacial region via the biological connection of periodontal liga- 14.4.3 Perceptive Potential ments. Tooth loss and periodontal disease cause for Noxious Stimulation fundamental problems for oral and general in Bioengineered Tooth health issues (Proffi t et al. 2004 ; Dawson 2006 ). The structural properties of periodontal tissue The afferent nervous system is established by the contribute to the physiological function of the growth of axons that navigate and establish con- tooth, including the absorption of occlusal load- nections with developing target organs during ing, the maintenance of alveolar bone height embryogenesis (Luukko et al. 2005 ). The teeth are and orthodontic tooth movement involving bone the peripheral organs innervated by the sensory remodelling (Foster et al. 2007 ). Although nerves and sympathetic or parasympathetic nerves autologous tooth transplantation can success- in the maxillofacial region, and that afferent ner- fully restore physiological tooth functions, it vous system contribute to the regulation of tooth has been known that the remaining healthy peri- physiological functions and the perception of nox- odontal tissue around extracted tooth root is ious stimulations (Luukko et al. 2005 ). It is thus required for the accomplishment of highly sur- thought that neuronal regeneration, which is asso- vival rate and the prevention of ankyloses ciated with the re-entry of nerve fi bres subsequent (Tsukiboshi 1993 ). In contrast, the absence of a to the transplantation of a tooth germ or an autolo- PDL in a dental implant is associated with defi - gous tooth, is required for the achievement of bio- cits in important tooth functions requiring the logical dental regenerative therapy (Kjaer et al. coordination of the teeth and the maxillofacial 1999 ). Current dental implants, which are directly components through the connection of PDL connected to the alveolar bone as an osseointegra- (Avery 2002 ; Pokorny et al. 2008 ; Lindhe et al. tion, cannot perceive noxious stimulations such as 2008 ). Thus, biological regenerative therapy excessive occlusal loading and trauma because of using functional periodontal tissue has been the absence of neuronal innervation in the peri- shown to be effective in restoring normal tooth odontal tissue (Brenemark and Zarb 1985 ; Burns functions as an alternative to artifi cial therapy. et al. 2003). Therefore, it is expected that tooth To analyse the movement of bioengineered regenerative therapy will achieve the functional tooth, we have experimentally developed an recovery of the neuronal perceptive potential for orthodontic tooth movement model. noxious stimulation. We have demonstrated that Experimental tooth movement consisted of a sensory and sympathetic nerve fi bres can inner- horizontal orthodontic force of about 10–15 g vate both the pulp tissue and the PDL region with applied continuously to the bioengineered tooth the reconstruction of blood vessels in an engrafted in a buccal direction using a nickel-titanium bioengineered tooth (Ikeda et al. 2009 ; Oshima wire with a diameter of 0.012 inch. (Ikeda et al. et al. 2011) (Fig. 14.6). Bioengineered teeth also 2009; Oshima et al. 2011 ). We have demon- represented the perceptive potential for nocicep- strated that bioengineered teeth successfully tive pain stimulation including pulp injury and underwent the functional tooth movement orthodontic movement, and they can properly equivalent to that of natural teeth through the transduce these peripheral stimulation to the proper localisation of osteoclasts and osteo- superfi cial layers of the medullary dorsal horn blasts in response to mechanical stress (Ikeda through c-Fos immunoreactive neurons in the cen- et al. 2009 ; Oshima et al. 2011 ). These fi ndings tral nervous system (Ikeda et al. 2009 ; Oshima indicate that a bioengineered tooth can replicate et al. 2011 ). These fi ndings indicate that bioengi- 266 M. Oshima and T. Tsuji

Fig. 14.6 Neuronal Innervation of a bioengineered tooth. by using the immunostaining for neurofi lament H. D The nerve innervation of the dental pulp and periodontal dentin, P pulp, PDL periodontal ligament, AB alveolar ligament in the natural and bioengineered teeth detected bone neered teeth can indeed restore the proprioceptive regeneration and tooth replacement therapy potential to recognize and respond to noxious (Purnell 2008 ; Volponi et al. 2010 ). These dental stimulation within the maxillofacial region. stem cells, such as DPSCs, SHED, SCAP, PDLSCs and dental-follicle stem cells, can differentiate into several dental-cell lineages and 14.5 Conclusion and Future also contribute to the turnover and supply of vari- Consideration ous progenitor cells (Gronthos et al. 2000 ; Miura et al. 2003 ; Sonoyama et al. 2008 ; Seo et al. Current regenerative technology has progressed 2004 ). Although these linages would be valuable remarkably, and many patients can be benefi tted cell sources for stem-cell transplantation therapy by the contributions of the tooth regenerative aimed toward dental tissue regeneration (Egusa therapy for dental disorders. To address the desir- et al. 2012 , 2013 ), the tooth inductive potential able future clinical applications of whole-tooth cells, which can replicate an epithelial- replacement therapy, one of the major research mesenchymal interaction for whole-tooth regen- hurdles remaining is the identifi cation of appro- eration, has not yet been identifi ed. Pluripotent priate cell sources (Ikeda and Tsuji 2008 ). Of stem cells including ES cells and iPS cells are course, the cell source may be optimised by using also candidate cell sources that are capable of dif- the patient’s own cells for regenerative therapy to ferentiating into endodermal, ectodermal and avoid immunological rejection. In the dental mesodermal cell types (Yan et al. 2010 ). Recently, fi eld, recent studies of stem-cell biology have led iPS cells have been established from several oral to the identifi cation of candidate cell sources tissues such as pulp, PDL, gingiva and oral based on tooth organogenesis for tooth tissue mucosa, and they have represented the ability to 14 Whole Tooth Regeneration as a Future Dental Treatment 267 differentiate into dental epithelial and mesenchy- References mal cells (Egusa et al. 2010 ; Arakaki et al. 2012 ; Otsu et al. 2012 ). Meanwhile, the identifi cation Arakaki M, Ishikawa M, Nakamura T et al (2012) Role of of master genes for reprogramming non-dental epithelial-stem cell interactions during dental cell differentiation. J Biol Chem 287:10590–10601 cells to differentiate into dental epithelial and Atala A (2005) Tissue engineering, stem cells and clon- mesenchymal cells is considered to an important ing: current concepts and changing trends. 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