The evolutionary histories of dentin and bone are closely intertwined and must be considered together(Sire and Kawasaki, 2012). The earliest appearance of mineralized tissue in vertebrates occurred as an external body armor in Heterostraci, extinct jawless ostracoderms. These jawless fish had a dermal exoskeleton comprising osseous plates ornamented with odontodes made of dentin with an enameloid cap (Huysseune and Sire, 1998, Keating et al., 2018, Ørvig, 1951, Sire and Kawasaki, 2012). Dentin and bone were maintained for more than 460 million years of evolution, as were the cells producing them (odontoblasts and osteoblasts, respectively). The regulatory gene networks and matrix components (type I collagen fibrils, carbonated hydroxyapatite and water) of these closely linked tissues have also remained mostly unchanged (Rosa et al., 2021, Sire and Kawasaki, 2012). Odontoblasts are derived from the neural crest while osteoblasts are mostly of mesodermal origin. However, neural crest-derived cells can also differentiate into osteoblasts to form some of the craniofacial bones, like the mandible (Sire and Huysseune, 2003, Urano-Morisawa et al., 2017). Osteoblasts differentiate from their progenitor cells through the transcription factor cbfa/runx2. However, it has been shown that runx2 can induce the trans-differentiation of odontoblasts into osteoblasts. Furthermore, down-regulation of runx2 is required during odontoblast differentiation to obtain functional odontoblasts capable of dentinogenesis (Komori, 2010, Miyazaki et al., 2008). These observations point to the very close association between osteoblasts and odontoblasts.

Mammals have a single dentin type, which is a clearly defined and well-characterized tissue (Hall, 2015). Mammalian dentin is distinct from bone, and is easily recognized by its location, the cells responsible for producing its matrix and its structural details at various length scales. Mammalian dentin, called orthodentin, is deposited by odontoblasts and comprises an acellular matrix of mineralized collagen type I fibrils, water and non-collagenous proteins (Nanci, 2005). Structurally, orthodentin is traversed by numerous tubules 1–2 µm in diameter, each containing an odontoblastic cell process belonging to one of the odontoblasts that line the pulp cavity (Marshall et al., 1997). The dentinal tubules are surrounded by a matrix of intertubular dentin consisting of mineralized collagen fibrils that are arranged randomly within planar layers orthogonal to the tubules (Bertassoni et al., 2012, Marshall et al., 1997, Weiner and Wagner, 1998). In the crown part of the teeth the tubules are surrounded by a highly mineralized, collagen-poor cuff called peri-tubular dentin, which is embedded within the inter-tubular dentin. Mammalian bones, on the other hand, are deposited by osteoblasts, and their basic structural unit is the lamella, comprising arrays of aligned mineralized collagen fibrils which form discrete layers. The fibrils in successive layers vary in orientation, forming a plywood-like structure (Gebhardt, 1905, Raguin et al., 2020, Weiner and Wagner, 1998).

The dentin in the teeth of almost all vertebrates except fish have only one type of dentin – orthodentin (the only exception being the aardvark, Orycteropus afer, a mammal with teeth which contain osteodentin (Berkovitz and Shellis, 2017)). However, the teeth of the fish species so far studied (∼100 species out of more than 33,000 extant fish species (Texereau et al., 2018) have exhibited a remarkable variety of dentin types, which include orthodentin, osteodentin, pseudoosteodentin, vasodentin, trabecular dentin and plicidentin. Furthermore, considerable variation can be seen even within these dentin types (Ørvig, 1967).

Dentin histo-type characterization has been studied intensively in chondrichthyan (cartilaginous) fish. Three different types of dentin have so far been found in the teeth of modern sharks. These include orthodentin, osteodentin and pseudoosteodentin (dentin which consists of a core of osteodentin encased by orthodentin) (Jambura et al., 2020, Li et al., 2022). On the other hand, the extant holocephalans (Chimaeroidei), the other sub class of the chondrichthyans, have lost the ability to form individual teeth. Their dentition consists of several dental plates, and these are entirely composed of continuously growing dentin which consists of osteodentin (also called trabecular dentin) that surrounds hyper-mineralized pleromin, in which the mineral phase is magnesium-rich whitlockite (Iijima and Ishiyama, 2020, Johanson et al., 2020, Smith et al., 2020, Smith et al., 2019).

Many different dentin types and sub-types have also been found in osteichthyes (bony fish), both extant and extinct (Deng et al., 2022, Germain et al., 2019, Germain and Meunier, 2017, Meunier and Germain, 2018, Texereau et al., 2018). A recent study compared the structure of two species belonging to the Serrasalmidae, the piranha (Pygocentrus nattereri) which is a predator using its teeth to tear its prey, and the pacu (Colossoma macropomum) which is a durophagous feeder, using its teeth to crush hard shells (Velasco-Hogan et al., 2021). While the authors found differences in material properties and external morphology which are adapted to their different functions, the dentin of both piranha and pacu is orthodentin. Some authors (e.g. (Li et al., 2022)) have speculated that osteodentin may have a functional or structural advantage over orthodentin in sharks and bony fishes, and is better suited to a durophagous diet. However, functional (e.g. dietary) differences do not seem to be an obvious taxonomic reason for dentin type, as the teeth of durophagous species (e.g. A. lupus) as well as carnivorous species (such as the pike Esox lucius) both have osteodentin, while the teeth in some durophagous species (e.g. the gilthead seabream S. aurata) and carnivorous fish species (e.g. the mackerel icefish C. gunnari) have orthodentin in their teeth. Interestingly, the Australian lungfish Neoceratodus forsteri (Osteichthyes: Dipnoi), does not have individual teeth and has a dentition consisting of continuously growing tooth plates. These tooth plates consist of osteodentin which is surrounded by enamel and bone (Kemp and Barry, 2006).

Osteodentin is structurally different from orthodentin, and its appearance is reminiscent of mammalian osteonal bone, an observation which led to its name (Ørvig, 1976). In particular, two-dimensional (2D) sections of osteodentin observed by light- and electron microscopy reveal multiple units comprising a central canal of a diameter 1–2 orders of magnitude larger than orthodentinal tubules, surrounded by several concentric, lamellar-like layers (Ørvig, 1976). In some cases, both orthodentin and osteodentin are present in the same tooth (Li et al., 2022).

In a previous study we proposed that osteodentin may in fact be structurally closer to anosteocytic bone than to true dentin (Thangadurai et al., 2021). In that study we described the macro-and micro-structure of wolfish osteodentin, as well as its mechanical properties and composition. The current study set out to examine further the hypothesis that osteodentin is a bony tissue, by exploring the structure of osteodentin also at the nanometer length scale. Here we present the results of a high-resolution, three-dimensional (3D) study of the nano-structure of osteodentin in the teeth of the Atlantic wolfish, Anarhichas lupus. Such characterization is of evolutionary significance, and can provide further insights into the co-appearance of two similar but different mineralized tissues, dentin and bone, close to 500 million years ago.