Int. J. Biol. Sci. 2009, 5 http://www.biolsci.org 226 International I n t e r n a t i o n a l Journal J o u r n a l of o f Biological B i o l o g i c a l Sciences S c i e n c e s 2009 5(3):226-243 �� Ivyspring International Publisher. All rights reserved Review A Curriculum Vitae of Teeth: Evolution, Generation, Regeneration Despina S. Koussoulakou, Lukas H. Margaritis, Stauros L. Koussoulakos University of Athens, Faculty of Biology, Department of Cell Biology and Biophysics, Athens, Greece Correspondence to: Stauros Koussoulakos, University of Athens, Faculty of Biology, Department of Cell Biology and Biophysics, Panepistimiopolis 15784, Athens ��� Greece. e-mail: skoussou@biol.uoa.gr Received: 2008.11.19 Accepted: 2009.02.21 Published: 2009.02.24 Abstract The ancestor of recent vertebrate teeth was a tooth-like structure on the outer body sur- face of jawless fishes. Over the course of 500,000,000 years of evolution, many of those structures migrated into the mouth cavity. In addition, the total number of teeth per denti- tion generally decreased and teeth morphological complexity increased. Teeth form mainly on the jaws within the mouth cavity through mutual, delicate interactions between dental epithelium and oral ectomesenchyme. These interactions involve spatially restricted expres- sion of several, teeth-related genes and the secretion of various transcription and signaling factors. Congenital disturbances in tooth formation, acquired dental diseases and odonto- genic tumors affect millions of people and rank human oral pathology as the second most frequent clinical problem. On the basis of substantial experimental evidence and advances in bioengineering, many scientists strongly believe that a deep knowledge of the evolutionary relationships and the cellular and molecular mechanisms regulating the morphogenesis of a given tooth in its natural position, in vivo, will be useful in the near future to prevent and treat teeth pathologies and malformations and for in vitro and in vivo teeth tissue regeneration. Key words: epithelial-mesenchymal interactions, teeth evolution, development and regeneration 1. Introduction A huge amount of literature is devoted to the origin, evolution, organogenesis, pathology and therapy of teeth. There have been tremendous ad- vances in recent years towards a better understanding of the regulation of teeth development [1, 2]. The immense interest in this subject is quite justified since, apart from the intrinsic scientific merit, teeth con- genital abnormalities account for 20% of all inherited disorders, whereas, oral pathology occupies a leading position in the list of human diseases [3, 4]. Teeth are highly mineralized appendages found in the entrance of the alimentary canal of both inver- tebrates and vertebrates. They are associated mainly with prehension and processing of food, but they also frequently serve other functions, such as defense, display of dominance and phonetic articulation in humans. Generally, when speaking of teeth we usu- ally refer to the dentition of vertebrates. Teeth with the basic microscopic anatomy similar to that of recent vertebrates first appeared at Ordovicium, approx. 460 million years ago. Some jawless fish developed su- perficial, dermal structures known as odontodes [5, 6] (Fig. 1). Those small tooth-like structures were located outside the mouth and served various functions, in- cluding protection, sensation and hydrodynamic ad- vantage. The encroachment of odontodes into the oropharyngeal cavity created the buccal teeth, which covered the entire surface and later were localized to the jaw margins. Dietary habits and ecological adap- tations have driven the teeth of vertebrates to acquire numerous anatomical forms and shapes, as repre- sented by incisors, canines, premolars and molars [7]. The main body of a tooth consists of a calcified tissue called the dentine, which is secreted by odon- toblasts, cells of cranial neural crest (cnc) origin [8]. Dentine is composed of collagen, dentine sialophos-

Int. J. Biol. Sci. 2009, 5 http://www.biolsci.org 227 phoprotein, dentine matrix protein and hydroxylapa- tite. Dentine surrounds the pulp, which is rich in fi- broblast-like cells, blood vessels and nerves. The up- per part of the dentine is usually covered by a layer of enamel, which is secreted by ameloblasts, oral epithe- lial cells. Enamel, the hardest tissue of the human body, is collagen-free. Its main proteins are amelogenin (90%), ameloblastin, enamelin and tufte- lin. The root firmly supports the tooth within an al- veolar socket by means of the periodontium. The visible part of a tooth in the oral cavity is referred to as the clinical crown [7]. Teeth are generated through highly orchestrated mutual inductive interactions between two major cell types: stomodeal ectoderm and cranial, neural crest-derived ectomesenchyme cells. In some animals the endodermal epithelium directly participates in teeth formation [9]. Morpho- logical differences between individual teeth of a den- tition arise mainly from differences in the spatiotem- poral expression of several, odontogenic genes. These genes encode transcription factors that regulate the synthesis of various signaling factors [10]. These sig- naling factors mediate inductive interactions between the odontogenic tissue layers and affect cell multipli- cation, cell death and cytodifferentiation [11]. How these inductive interactions were modified during evolution to generate the numerous anatomical fea- tures of teeth is a major interest in evolutionary biol- ogy. Interestingly, genes and signaling factors playing leading roles in teeth morphogenesis are also in- volved in the development of many other organs in various animals [10, 12]. Fig. 1. Odontodes, the ancestors of teeth, looked like placoid scales of recent sharks. Odontodes consisted of a dentine cone with a pulp cavity and covered by a hyper- mineralized tissue like enamel or enameloid. They were attached to the integument by a bony base. The plethora of molecules involved [e.g., fibro- blast growth factors (FGFs), bone morphogenetic proteins (BMPs), sonic hedgehog (SHH), wingless integrated (WNTs)] and the complexity of interactions (e.g., activation, inhibition, regulatory loops) inevita- bly lead with some frequency to homeostatic disor- ganization, which results in congenital abnormalities, such as tooth agenesis, which is the most commonly inherited disorder [3, 4]. Most human congenital teeth malformations are caused by mutations in develop- mentally regulated genes [6]. The fact that, an em- bryonic tooth bud can develop in vitro [13] indicates that the expression of teeth-related genes is not re- stricted only in vivo. Mutations that alter teeth act at many levels of control, i.e., the development of the embryonic bud, the morphogenesis of the bell stage, the production of enamel and dentin and the forma- tion of the roots [1, 2]. The mechanisms of this genetic control are surely encoded at the molecular and submolecular levels. These mechanisms are beginning to be studied. The favored animal model for such studies is the common laboratory mouse, since teeth development in mice is similar to that of man. Addi- tionally, the same set of genes functions in mouse as in man during teeth development there are only mi- nor differences in the expression patterns of these genes, and mutations in counterpart genes cause similar defective phenotypes (e.g., mouse Tabby and human EDA) [14]. Tooth damage and loss is quite frequent (7%) and adversely affects mastication, articulation, facial esthetics and psychological health. Historically, sur- geons have used several procedures to replace lost and repair damaged teeth, including tooth allotrans- plantation, autotransplantation, dental implants (metal or ceramics) and artificial dentures. Since these procedures sometimes have questionable therapeutic efficacy for various reasons (e.g., lack of biocompati- bility between implants and human tissues, lack of osteointegration, damage to surrounding tissues, etc.), regenerative medicine promises to overcome these difficulties with biological procedures [15, 16]. New strategies have been designed in an attempt to achieve biological replacement of tooth tissues and whole teeth both in vivo and in vitro [13, 15, 16, 17]. Several excellent reviews on teeth have been re- cently published e.g., [1, 2, 6, 18, 19] unfortunately, space limitations do not allow us to discuss and/or cite a significant proportion of them. In this review, we briefly present the current status of the entire field of odontology. We will review the most important aspects of teeth evolution, morphogenesis and bio- logical restoration, from the pioneering studies to the most recent developments. We aimed to provide suf-

Int. J. Biol. Sci. 2009, 5 http://www.biolsci.org 228 ficient information for even the casual reader that may not be quite familiar with the subject. 2. Origin and Evolution of Teeth 2.1 The ancestors of teeth were dermal ap- pendages In a few organisms there is substantial evidence to suggest that teeth may have derived from both ec- toderm and endoderm [20, 21]. In most cases, teeth evolved from scale-like epidermal structures, the odontodes, which ���migrated��� into the mouth after enough mutations. This process is visible in modern sharks, which have placoid scales on the skin that grade into the teeth on the jaws. In certain cases, however, dermal denticles did not transform into teeth and underwent independent evolution [22]. Natural selection has favored toothed organisms, which have a major advantage in their ability to cap- ture and process food. Teeth can be classified into three types, based on where they are formed: jaw, mouth and pharyngeal. The close relationship be- tween past and present teeth can be demonstrated by a phylogenetic analysis. Using this type of analysis, amelogenin appears to have been duplicated from SPARC (SPARC, secreted protein, acidic, rich in cys- teine), 630,000,000 years ago, i.e., long before the Cambrian explosion [23, 24]. 2.2. During evolution the number of teeth per dentition decreased Variations in tooth number may represent an important factor for mammalian diversification. The evolutionary pathway from fish to reptiles to mam- mals is characterized by a reduction in the number of teeth (from polyodonty to oligodonty) and of their generations (from polyphyodonty to di- and/or mo- nophyodonty) as well as an increase in morphological complexity of the teeth (from homodonty to hetero- donty) [7, 25]. Some organisms (e.g., killer whales, rats, elephants) develop their dentition only once in their life others (e.g., turtles, birds, toothless whales, anteaters) have lost their dentition and are character- ized by adontia. Adontia in many organisms is con- sidered to be secondary, since the embryo possesses tooth germs that undergo apoptosis before birth [26, 27]. Region-specific tooth loss has been a common trend in vertebrate evolution. Some organisms re- tained a high number of teeth, however: Opossum (50 teeth), sirenoids (possess 44 molars) and some dol- phins (bearing more than 200 relatively similar teeth, having thus lost heterodonty and returned to homo- donty). Interestingly, some teeth that were lost during evolution reappeared in an atavistic sense [28], thus violating the ���law��� of irreversibility in evolution. If we could understand the mechanism of spontaneous re-acquisition of lost properties, we might be able to apply this knowledge to the clinical, biological resto- ration of lost teeth. Along those lines, understanding the rules of polyphyodonty will surely support tooth regenerative efforts. How and why evolutionary tooth loss occurs is not known, but several interesting hy- potheses have been proposed. For example, there could be a loss of a tooth-type-specific initiation mes- sage, attenuation of the inductive and/or inhibitory signal or a reduction in the concentration of required proteins. In support of this last idea, the lack of ca- nines and premolars in the mouse upper diastema has been attributed to the weak expression of the PAX9 gene [29, 30]. Changes in the number and morphology of teeth may reflect a significant factor in the generation of new species in mammals. The most common feature is the loss of various teeth, perhaps as a result of a mu- tation in tooth-related genes. For example, rodents lack lateral incisors, canines and premolars. Sheep have lost their upper incisors and the canines. An analysis of mutant mice phenotypes has clearly indi- cated that specific mutations (e.g., GLI2-/-,GL��3+/-) cause phenotypes that resemble several ungulates that lack all upper incisors [3, 29]. It is worth noting that in placental mammals teeth tend to disappear over the course of evolution in an order that is oppo- site the order of their appearance during eruption [7, 9]. A reaction/diffusion model of morphogenesis has been used to explain this phenomenon. According to this model, repeated structures (e.g., vertebrae, pha- langes, feathers, color patterns, teeth) arise as a result of the coordination of two molecules, an activator and an inhibitor. Two well known examples of such in- teracting molecules are FGF8/BMP4 [31] and ecto- din/BMP4 [32]. Teeth located at a distance from the center of the morphogenetic field tend to disappear due to field attenuation [33]. 2.3. Evolution favored an increase in teeth com- plexity Diet and mastication are regarded as central factors in teeth evolution. There is a strong correlation between teeth form (e.g., cardiform, villiform, incisor, canine, molariform) and feeding habits. During evo- lution, mammals, which originated from reptile-like ancestors, (Diapsida), developed in each side of their skull two openings (temporal fenestrae) behind the orbit that are still present in a modified form in mod- ern mammals. This opening has been used as a rigid place for the attachment of powerful masticatory muscles. This evolutionary event allowed a much more efficient exploitation of the food caloric energy