Evolved Bmp6 enhancer alleles drive spatial shifts in gene expression during tooth development in sticklebacks.

Mutations in enhancers have been shown to often underlie natural variation but the evolved differences in enhancer activity can be difficult to identify in vivo. Threespine sticklebacks (Gasterosteus aculeatus) are a robust system for studying enhancer evolution due to abundant natural genetic variation, a diversity of evolved phenotypes between ancestral marine and derived freshwater forms, and the tractability of transgenic techniques. Previous work identified a series of polymorphisms within an intronic enhancer of the Bone morphogenetic protein 6 (Bmp6) gene that are associated with evolved tooth gain, a derived increase in freshwater tooth number that arises late in development. Here, we use a bicistronic reporter construct containing a genetic insulator and a pair of reciprocal two-color transgenic reporter lines to compare enhancer activity of marine and freshwater alleles of this enhancer. In older fish, the two alleles drive partially overlapping expression in both mesenchyme and epithelium of developing teeth, but the freshwater enhancer drives a reduced mesenchymal domain and a larger epithelial domain relative to the marine enhancer. In younger fish, these spatial shifts in enhancer activity are less pronounced. Comparing Bmp6 expression by in situ hybridization in developing teeth of marine and freshwater fish reveals similar evolved spatial shifts in gene expression. Together, these data support a model in which the polymorphisms within this enhancer underlie evolved tooth gain by shifting the spatial expression of Bmp6 during tooth development, and provide a general strategy to identify spatial differences in enhancer activity in vivo.

The Role of GH/IGF Axis in Dento-Alveolar Complex from Development to Aging and Therapeutics: A Narrative Review.

The GH/IGF axis is a major regulator of bone formation and resorption and is essential to the achievement of normal skeleton growth and homeostasis. Beyond its key role in bone physiology, the GH/IGF axis has also major pleiotropic endocrine and autocrine/paracrine effects on mineralized tissues throughout life. This article aims to review the literature on GH, IGFs, IGF binding proteins, and their respective receptors in dental tissues, both epithelium (enamel) and mesenchyme (dentin, pulp, and tooth-supporting periodontium). The present review re-examines and refines the expression of the elements of the GH/IGF axis in oral tissues and their in vivo and in vitro mechanisms of action in different mineralizing cell types of the dento-alveolar complex including ameloblasts, odontoblasts, pulp cells, cementoblasts, periodontal ligament cells, and jaw osteoblasts focusing on cell-specific activities. Together, these data emphasize the determinant role of the GH/IGF axis in physiological and pathological development, morphometry, and aging of the teeth, the periodontium, and oral bones in humans, rodents, and other vertebrates. These advancements in oral biology have elicited an enormous interest among investigators to translate the fundamental discoveries on the GH/IGF axis into innovative strategies for targeted oral tissue therapies with local treatments, associated or not with materials, for orthodontics and the repair and regeneration of the dento-alveolar complex and oral bones.

A show of Hands: Novel and conserved expression patterns of teleost hand paralogs during craniofacial, heart, fin, peripheral nervous system and gut development.

BACKGROUND: Hand genes are required for the development of the vertebrate jaw, heart, peripheral nervous system, limb, gut, placenta, and decidua. Two Hand paralogues, Hand1 and Hand2, are present in most vertebrates, where they mediate different functions yet overlap in expression. In ray-finned fishes, Hand gene expression and function is only known for the zebrafish, which represents the rare condition of having a single Hand gene, hand2. Here we describe the developmental expression of hand1 and hand2 in the cichlid Copadichromis azureus. RESULTS: hand1 and hand2 are expressed in the cichlid heart, paired fins, pharyngeal arches, peripheral nervous system, gut, and lateral plate mesoderm with different degrees of overlap. CONCLUSIONS: Hand gene expression in the gut, peripheral nervous system, and pharyngeal arches may have already been fixed in the lobe- and ray-finned fish common ancestor. In other embryonic regions, such as paired appendages, hand2 expression was fixed, while hand1 expression diverged in lobe- and ray-finned fish lineages. In the lateral plate mesoderm and arch associated catecholaminergic cells, hand1 and hand2 swapped expression between divergent lineages. Distinct expression of cichlid hand1 and hand2 in the epicardium and myocardium of the developing heart may represent the ancestral pattern for bony fishes.

Irrompimento do primeiro molar permanente em crianças da zona urbana e rural do município de Santa Helena - Paraná / Irrompement of the first permanent molar in children from the urban and rural area of the city of Santa Helena - Paraná

O objetivo deste trabalho foi comparar a cronologia de erupção do primeiro molar permanente em crianças de ambos os sexos, residentes na zona urbana e rural do munícipio de Santa Helena - PR, Brasil. Foi realizado um estudo transversal com 154 crianças da zona rural e 300 crianças da área urbana de 04 a 07 anos (48 a 84 meses). Os primeiros molares avaliados foram considerados irrompidos quando qualquer porção de sua coroa estivesse clinicamente visível. A média de idade para erupção do primeiro molar permanente se mostrou de 72 a 83 meses. Destes, o grupo da zona rural apresentou uma média para idade de erupção mais precoce. Contudo, verificou-se um resultado considerável em crianças na faixa de 48 a 59 meses (4 anos), mostrando mais uma vez a erupção precoce nas crianças da zona rural. Este dente irrompeu primeiro na mandíbula, irrompendo primeiro nas meninas do que nos meninos, e o dente 46 foi o que mais se mostrou presente. A média de idade para erupção do primeiro molar permanente correspondeu àquela descrita pela literatura aos seis anos, mas não correspondeu ao atraso na erupção das crianças residentes em zona rural. Bem como este dente irrompeu primeiro na mandíbula... (AU)

The objective of this study was to compare the chronology of eruption of the first permanent molar in children of both sexes, living in the urban and rural areas of the city of Santa Helena-PR, Brazil. A cross-sectional study was carried out with 154 children from the rural area and 300 children from the urban area from 4 to 7 years old (48 to 84 months). The first molars evaluated were considered erupted when any portion of their crown was clinically visible. The average age for eruption of the first permanent molar was 72 to 83 months. Of these, the rural group had an earlier average age for eruption than the urban group. However, a considerable result was found in children 48-59 months showing once again the early eruption in rural children. This tooth erupted first in the jaw, erupting first in girls rather than boys, and tooth 46 was most present. The mean age of eruption of the first permanent molar corresponded to that described in the literature at age six, but did not correspond to the delayed eruption of children living in rural areas. Just like this tooth erupted in the jaw first... (AU)

Ultrastructural studies of developing egg tooth in grass snake Natrix natrix (Squamata, Serpentes) embryos, supported by X-ray microtomography analysis.

The egg tooth development is similar to the development of all the other vertebrate teeth except earliest developmental stages because the egg tooth develops directly from the oral epithelium instead of the dental lamina similarly to null generation teeth. The developing egg tooth of Natrix natrix changes its curvature differently than the egg tooth of the other investigated unidentates due to the presence of the rostral groove. The developing grass snake egg tooth comprises dental pulp and the enamel organ. The fully differentiated enamel organ consists of outer enamel epithelium, stellate reticulum, and ameloblasts in its inner layer. The enamel organ directly in contact with the oral cavity is covered with periderm instead of outer enamel epithelium. Stellate reticulum cells in the grass snake egg tooth share intercellular spaces with the basal part of ameloblasts and are responsible for their nutrition. Ameloblasts during egg tooth differentiation pass through the following stages: presecretory, secretory, and mature. The ameloblasts from the grass snake egg tooth show the same cellular changes as reported during mammalian amelogenesis but are devoid of Tomes' processes. Odontoblasts of the developing grass snake egg tooth pass through the following classes: pre-odontoblasts, secretory odontoblasts, and ageing odontoblasts. They have highly differentiated secretory apparatus and in the course of their activity accumulate lipofuscin. Grass snake odontoblasts possess processes which are poor in organelles. In developing egg tooth cilia have been identified in odontoblasts, ameloblasts and cells of the stellate reticulum. Dental pulp cells remodel collagen matrix during growth of the grass snake egg tooth. They degenerate in a way previously not described in other teeth.

Tooth Repair and Regeneration: Potential of Dental Stem Cells.

Tooth defects are an extremely common health condition that affects millions of individuals. Currently used dental repair treatments include fillings for caries, endodontic treatment for pulp necrosis, and dental implants to replace missing teeth, all of which rely on the use of synthetic materials. By contrast, the fields of tissue engineering and regenerative medicine and dentistry (TERMD) use biologically based therapeutic strategies for vital tissue regeneration, and thus have the potential to regenerate living tissues. Methods to create bioengineered replacement teeth benefit from a detailed understanding of the molecular signaling networks regulating natural tooth development. We discuss how key signaling pathways regulating natural tooth development are being exploited for applications in TERMD approaches for vital tooth regeneration.

Miniaturization: How many cells are needed to build a tooth?

BACKGROUND: Organs that develop early in life, and are replaced by a larger version as the animal grows, often represent a miniature version of the adult organ. Teeth constituting the first functional dentition in small-sized teleost fish, such as medaka (Oryzias latipes), are examples of such miniature organs. With a dentin cone as small as the size of one human cell, or even smaller, these teeth raise the question how many dentin-producing cells (odontoblasts) are required to build such a tooth, and whether this number can be as little as one. RESULTS: Based on detailed observations with transmission electron microscopy (TEM) and TEM-based 3D-reconstructions, we show that only one mesenchymal cell qualifies as a true odontoblast. A second mesenchymal cell potentially participates in dentin formation, but only at a late stage of tooth development. Moreover, the fate of these cells appears to be specified very early during tooth development. CONCLUSIONS: Our observations indicate that in this system, one single odontoblast fulfills roles normally exerted by a large and communicating cell population. First-generation teeth in medaka thus provide an exciting model to study integration of multiple functions into a single cell.

Evolutionary developmental genetics of teeth and odontodes in jawed vertebrates: a perspective from the study of elasmobranchs.

Most extant vertebrates display a high variety of tooth and tooth-like organs (odontodes) that vary in shape, position over the body and nature of composing tissues. The development of these structures is known to involve similar genetic cascades and teeth and odontodes are believed to share a common evolutionary history. Gene expression patterns have previously been compared between mammalian and teleost tooth development but we highlight how the comparative framework was not always properly defined to deal with different tooth types or tooth developmental stages. Larger-scale comparative analyses also included cartilaginous fishes: sharks display oral teeth and dermal scales for which the gene expression during development started to be investigated in the small-spotted catshark Scyliorhinus canicula during the past decade. We report several descriptive approaches to analyse the embryonic tooth and caudal scale gene expressions in S. canicula. We compare these expressions wih the ones reported in mouse molars and teleost oral and pharyngeal teeth and highlight contributions and biases that arise from these interspecific comparisons. We finally discuss the evolutionary processes that can explain the observed intra and interspecific similarities and divergences in the genetic cascades involved in tooth and odontode development in jawed vertebrates.

Exploiting teeth as a model to study basic features of signaling pathways.

Teeth constitute a classical model for the study of signaling pathways and their roles in mediating interactions between cells and tissues in organ development, homeostasis and regeneration. Rodent teeth are mostly used as experimental models. Rodent molars have proved fundamental in the study of epithelial-mesenchymal interactions and embryonic organ morphogenesis, as well as to faithfully model human diseases affecting dental tissues. The continuously growing rodent incisor is an excellent tool for the investigation of the mechanisms regulating stem cells dynamics in homeostasis and regeneration. In this review, we discuss the use of teeth as a model to investigate signaling pathways, providing an overview of the many unique experimental approaches offered by this organ. We discuss how complex networks of signaling pathways modulate the various aspects of tooth biology, and the models used to obtain this knowledge. Finally, we introduce new experimental approaches that allow the study of more complex interactions, such as the crosstalk between dental tissues, innervation and vascularization.

Developmental Roles of FUSE Binding Protein 1 (Fubp1) in Tooth Morphogenesis.

FUSE binding protein 1 (Fubp1), a regulator of the c-Myc transcription factor and a DNA/RNA-binding protein, plays important roles in the regulation of gene transcription and cellular physiology. In this study, to reveal the precise developmental function of Fubp1, we examined the detailed expression pattern and developmental function of Fubp1 during tooth morphogenesis by RT-qPCR, in situ hybridization, and knock-down study using in vitro organ cultivation methods. In embryogenesis, Fubp1 is obviously expressed in the enamel organ and condensed mesenchyme, known to be important for proper tooth formation. Knocking down Fubp1 at E14 for two days, showed the altered expression patterns of tooth development related signalling molecules, including Bmps and Fgf4. In addition, transient knock-down of Fubp1 at E14 revealed changes in the localization patterns of c-Myc and cell proliferation in epithelium and mesenchyme, related with altered tooth morphogenesis. These results also showed the decreased amelogenin and dentin sialophosphoprotein expressions and disrupted enamel rod and interrod formation in one- and three-week renal transplanted teeth respectively. Thus, our results suggested that Fubp1 plays a modulating role during dentinogenesis and amelogenesis by regulating the expression pattern of signalling molecules to achieve the proper structural formation of hard tissue matrices and crown morphogenesis in mice molar development.

Signaling Modulations of miR-206-3p in Tooth Morphogenesis.

MicroRNAs (miRNAs) are a class of naturally occurring small non-coding RNAs that post-transcriptionally regulate gene expression in organisms. Most mammalian miRNAs influence biological processes, including developmental changes, tissue morphogenesis and the maintenance of tissue identity, cell growth, differentiation, apoptosis, and metabolism. The miR-206-3p has been correlated with cancer; however, developmental roles of this miRNA are unclear. In this study, we examined the expression pattern and evaluated the developmental regulation of miR-206-3p during tooth morphogenesis using ex-vivo culture method. The expression pattern of miR-206-3p was examined in the epithelium and mesenchyme of developing tooth germ with stage-specific manners. Perturbation of the expression of miR-206-3p clearly altered expression patterns of dental-development-related signaling molecules, including Axin2, Bmp2, Fgf4, Lef1 and Shh. The gene expression complemented with change in cellular events including, apoptosis and proliferation which caused altered crown and pulp morphogenesis in renal-capsule-calcified teeth. Especially, mislocalization of ß-Catenin and SMAD1/5/8 were observed alongside dramatic alterations in the expression patterns of Fgf4 and Shh. Overall, our data suggest that the miR-206-3p regulate the cellular physiology during tooth morphogenesis through modulation of the Wnt, Bmp, Fgf, and Shh signaling pathways to form proper tooth pulp and crown.

Retinoic Acid Signaling and the Zebrafish Dentition During Development and Evolution.

Explaining how the extensive diversity in form of vertebrate teeth arose in evolution and the mechanisms by which teeth are made during embryogenesis are intertwined questions that can merit from a better understanding of the roles of retinoic acid (RA) in tooth development. Pioneering studies in rodents showed that dietary vitamin A (VA), and eventually RA (one of the major active metabolites of VA), are required for proper tooth formation and that dentin-forming odontoblast cells seem to be especially sensitive to changes in RA levels. Later, rodent studies further indicated that RA signaling interactions with other cell-signaling pathways are an important part of RA's actions in odontogenesis. Recent investigations employing zebrafish and other teleost fish continued this work in an evolutionary context, and specifically demonstrated that RA is required for the initiation of tooth development. RA is also sufficient in certain circumstances to induce de novo tooth formation. Both effects appear to involve cranial-neural crest cells, again suggesting that RA signaling has a particular influence on odontoblast development. These teleost studies have also highlighted both evolutionary conservation and change in how RA is employed during odontogenesis in different vertebrate lineages, and thus raises the possibility that developmental changes to RA signaling has led to some of the diversity of form seen across vertebrate dentitions. Future progress in this area will come at least in part from expanding the species examined to get a better picture of how often RA signaling has changed in evolution and how this relates to the evolution of dental form.

The spatiotemporal expression pattern of Syndecans in murine embryonic teeth.

The hierarchical interactions between the dental epithelium and dental mesenchyme represent a common paradigm for organogenesis. During tooth development, various morphogens interact with extracellular components in the extracellular matrix and on the cell surfaces to transmit regulatory signaling into cells. We recently found pivotal roles of FAM20B-catalyzed proteoglycans in the control of murine tooth number at embryonic stages. However, the expression pattern of proteoglycans in embryonic teeth has not been well understood. We extracted total RNA from E14.5 murine tooth germs for semi-quantitative RT-PCR analysis of 29 proteoglycans, and identified 23 of them in the embryonic teeth. As a major subfamily of FAM20B-catalyzed proteoglycans, Syndecans are important candidates being potentially involved in the tooth development of mice. We examined the expression pattern of Syndecans in embryonic teeth using in situ hybridization (ISH) and immunohistochemistry (IHC) approaches. Syndecan-1 is mainly present in the dental mesenchyme at early embryonic stages. Subsequently, its expression expands to both dental epithelium and dental mesenchyme. Syndecan-2 is strongly expressed in the dental mesenchyme at early embryonic stages, then shifts to the stratum intermedium and inner dental epithelium at cap stages. Syndecan-3 shows a gradually increased expression that initially in the dental epithelium of both incisors and molars and then in the inner dental epithelium and stratum intermedium in molars alone. Syndecan-4 is localized in the dental epithelium in incisors and the dental follicle mesenchyme in molars at early cap stage. The spatiotemporal expression pattern of Syndecans in murine embryonic teeth suggest potential roles of these proteoglycans in murine tooth morphogenesis.

Prenatal Development of the Humpback Whale: Growth Rate, Tooth Loss and Skull Shape Changes in an Evolutionary Framework.

Extant baleen whales (Mysticeti) share a distinct suite of extreme and unique adaptations to perform bulk filter feeding, such as a long, arched skull, and mandible and the complete loss of adult dentition in favor of baleen plates. However, mysticetes still develop tooth germs during ontogeny. In the fossil record, multiple groups document the transition from ancestral raptorial feeding to filter feeding. Fetal specimens give us an extraordinary opportunity to observe when and how this macroevolutionary transition occurs during gestation. We used iodine-enhanced and traditional CT scanning to visualize the internal anatomy of five fetuses of humpback whale representing the first two-thirds of gestation, and we combine these data with previously published reports to provide the first comprehensive qualitative description of the sequence of developmental changes that characterize the skull and dentition. We also use quantitative methods based on 3D landmarks to investigate the shape changes in the fetuses in relation to a juvenile cranial morphology. We found similarities in the ossification patterns of the humpback and other cetaceans (dolphins), but there appear to be major differences when comparing them to terrestrial artiodactyls. As for the tooth germs, this developmental sequence confirms that the tooth-to-baleen transition occurs in the last one-third of gestation. Analysis of cranial shape development revealed a progressive elongation of the rostrum and a resulting posterior movement of the nasals relative to the braincase. Future work will involve acquisition of data from other species to complete our documentation of the teeth-to-baleen transition. Anat Rec, 2018. © 2018 American Association for Anatomy.

Shark tooth regeneration reveals common stem cell characters in both human rested lamina and ameloblastoma.

The human dentition is a typical diphyodont mammalian system with tooth replacement of most positions. However, after dental replacement and sequential molar development, the dental lamina undergoes apoptosis and fragments, leaving scattered epithelial units (dental lamina rests; DLRs). DLRs in adult humans are considered inactive epithelia, thought to possess limited capacity for further regeneration. However, we show that these tissues contain a small proportion of proliferating cells (assessed by both Ki67 and PCNA) but also express a number of common dental stem cell markers (Sox2, Bmi1, ß-catenin and PH3) similar to that observed in many vertebrates that actively, and continuously regenerate their dentition. We compared these human tissues with the dental lamina of sharks that regenerate their dentition throughout life, providing evidence that human tissues have the capacity for further and undocumented regeneration. We also assessed cases of human ameloblastoma to characterise further the proliferative signature of dental lamina rests. Ameloblastomas are assumed to derive from aberrant lamina rests that undergo changes, which are not well understood, to form a benign tumour. We suggest that dental lamina rests can offer a potential source of important dental stem cells for future dental regenerative therapy. The combined developmental genetic data from the shark dental lamina and ameloblastoma may lead to the development of novel methods to utilise these rested populations of adult lamina stem cells for controlled tooth replacement in humans.

Advances of Wnt signalling pathway in dental development and potential clinical application.

Wnt signalling pathway is widely studied in many processes of biological development, like embryogenesis, tissue homeostasis and wound repair. It is universally known that Wnt signalling pathway plays an important role in tooth development. Here, we summarized the function of Wnt signalling pathway during tooth initiation, crown morphogenesis, root formation, and discussed the therapeutic potential of Wnt modulators.

Homeobox code model of heterodont tooth in mammals revised.

Heterodonty is one of the hallmarks of mammals. It has been suggested that, homeobox genes, differentially expressed in the ectomesenchyme of the jaw primordium along the distal-proximal axis, would determine the tooth classes (homeobox code model) based on mouse studies. Because mouse has highly specialized tooth pattern lacking canine and premolars (dental formula: 1003/1003, for upper and lower jaws, respectively), it is unclear if the suggested model could be applied for mammals with all tooth classes, including human. We thus compared the homeobox code gene expressions in various mammals, such as opossum (5134/4134), ferret (3131/3132), as well as mouse. We found that Msx1 and BarX1 expression domains in the jaw primordium of the opossum and ferret embryos show a large overlap, but such overlap is small in mouse. Detailed analyses of gene expressions and subsequent morphogenesis of tooth germ in the opossum indicated that the Msx1/BarX1 double-positive domain will correspond to the premolar region, and Alx3-negative/Msx1-positive/BarX1-negative domain will correspond to canine. This study therefore provides a significant update of the homeobox code model in the mammalian heterodonty. We also show that the modulation of FGF-mediated Msx1 activation contributes to the variation in the proximal Msx1 expression among species.

Correlations between morphology of cervical vertebrae and dental eruption.

The process of dental eruption is submitted to physiological and pathological variables. A series of discrepancies may occur, one of these being a disturbance between dental age and bone age. The assessment of bone age is best made with the cervical vertebral maturation (CVM) method, simplified by Baccetti et al. (2005). The sample studied consisted of 215 orthodontic patients. The dental age was assessed on the orthopantomograph radiographies and the bone age on the lateral cephalograms. For determining the bone age, CVM method was used. Considering dental age, most of the patients (50.2%) have a premature dental age compared to bone age, while patients with normal dental age (27.9%) and patients with late dental age (21.9%) have a lower frequency. The correlation between the dental age and the bone age of the patients shows that patients who have higher values of dental age also have higher values of bone age (p<0.001). The correlation between genders shows that female patients tend to have a higher average value of bone age in comparison to male patients (p<0.001). The authors conclude that assessing bone age based on the morphology of cervical vertebrae and correlating it with the dental age could be of great use in opting for a certain orthodontic treatment plan.

Mesenchymal Wnt/ß-catenin signaling induces Wnt and BMP antagonists in dental epithelium.

Previous studies indicated that the elevated mesenchymal Wnt/ß-catenin signaling deprived dental mesenchyme of odontogenic fate. By utilizing ex vivo or pharmacological approaches, Wnt/ß-catenin signaling in the developing dental mesenchyme was suggested to suppress the odontogenic fate by disrupting the balance between Axin2 and Runx2. In our study, the Osr2-creKI; Ctnnb1ex3f mouse was used to explore how mesenchymal Wnt/ß-catenin signaling suppressed the odontogenic fate in vivo. We found that all of the incisor and half of the molar germs of Osr2-creKI; Ctnnb1ex3fmice started to regress at E14.5 and almost disappeared at birth. The expression of Fgf3 and Msx1 was dramatically down-regulated in the E14.5 Osr2-creKI; Ctnnb1ex3f incisor and molar mesenchyme, while Runx2transcription was only diminished in incisor mesenchyme. Intriguingly, in the E14.5 Osr2-creKI; Ctnnb1ex3f incisor epithelium, the expression of Noggin was activated, while Shh was abrogated. Similarly, the Wnt and BMP antagonists, Ectodin and Noggin were also ectopically activated in the E14.5 Osr2-creKI; Ctnnb1ex3f molar epithelium. Recombination of E13.5 Osr2-creKI; Ctnnb1ex3f molar mesenchyme with E10.5 and E13.5 WT dental epithelia failed to develop tooth. Taken together, the mesenchymal Wnt/ß-catenin signaling resulted in the loss of odontogenic fate in vivo not only by directly suppressing odontogenic genes expression but also by inducing Wnt and BMP antagonists in dental epithelium.

Parenchymal and stromal tissue regeneration of tooth organ by pivotal signals reinstated in decellularized matrix.

Cells are transplanted to regenerate an organs' parenchyma, but how transplanted parenchymal cells induce stromal regeneration is elusive. Despite the common use of a decellularized matrix, little is known as to the pivotal signals that must be restored for tissue or organ regeneration. We report that Alx3, a developmentally important gene, orchestrated adult parenchymal and stromal regeneration by directly transactivating Wnt3a and vascular endothelial growth factor. In contrast to the modest parenchyma formed by native adult progenitors, Alx3-restored cells in decellularized scaffolds not only produced vascularized stroma that involved vascular endothelial growth factor signalling, but also parenchymal dentin via the Wnt/ß-catenin pathway. In an orthotopic large-animal model following parenchyma and stroma ablation, Wnt3a-recruited endogenous cells regenerated neurovascular stroma and differentiated into parenchymal odontoblast-like cells that extended the processes into newly formed dentin with a structure-mechanical equivalency to native dentin. Thus, the Alx3-Wnt3a axis enables postnatal progenitors with a modest innate regenerative capacity to regenerate adult tissues. Depleted signals in the decellularized matrix may be reinstated by a developmentally pivotal gene or corresponding protein.