Pierfrancesco Pagella

Roles of innervation in developing and regenerating orofacial tissues

The head is innervated by 12 cranial nerves (I–XII) that regulate its sensory and motor functions. Cranial nerves are composed of sensory, motor, or mixed neuronal populations. Sensory neurons perceive generally somatic sensations such as pressure, pain, and temperature. These neurons are also involved in smell, vision, taste, and hearing. Motor neurons ensure the motility of all muscles and glands. Innervation plays an essential role in the development of the various orofacial structures during embryogenesis. Hypoplastic cranial nerves often lead to abnormal development of their target organs and tissues. For example, Möbius syndrome is a congenital disease characterized by defective innervation (i.e., abducens (VI) and facial (VII) nerves), deafness, tooth anomalies, and cleft palate. Hence, it is obvious that the peripheral nervous system is needed for both development and function of orofacial structures. Nerves have a limited capacity to regenerate. However, neural stem cells, which could be used as sources for neural tissue maintenance and repair, have been found in adult neuronal tissues. Similarly, various adult stem cell populations have been isolated from almost all organs of the human body. Stem cells are tightly regulated by their microenvironment, the stem cell niche. Deregulation of adult stem cell behavior results in the development of pathologies such as tumor formation or early tissue senescence. It is thus essential to understand the factors that regulate the functions and maintenance of stem cells. Yet, the potential importance of innervation in the regulation of stem cells and/or their niches in most organs and tissues is largely unexplored. This review focuses on the potential role of innervation in the development and homeostasis of orofacial structures and discusses its possible association with stem cell populations during tissue repair.

Microfluidics co-culture systems for studying tooth innervation

Innervation plays a key role in the development and homeostasis of organs and tissues of the orofacial complex. Among these structures, teeth are peculiar organs as they are not innervated until later stages of development. Furthermore, the implication of neurons in tooth initiation, morphogenesis and differentiation is still controversial. Co-cultures constitute a valuable method to investigate and manipulate the interactions of nerve fibers with their target organs in a controlled and isolated environment. Conventional co-cultures between neurons and their target tissues have already been performed, but these cultures do not offer optimal conditions that are closely mimicking the in vivo situation. Indeed, specific cell populations require different culture media in order to preserve their physiological properties. In this study we evaluate the usefulness of a microfluidics system for co-culturing mouse trigeminal ganglia and developing teeth. This device allows the application of specific media for the appropriate development of both neuronal and dental tissues. The results show that mouse trigeminal ganglia and teeth survive for long culture periods in this microfluidics system, and that teeth maintain the attractive or repulsive effect on trigeminal neurites that has been observed in vivo. Neurites are repealed when co-cultured with embryonic tooth germs, while postnatal teeth exert an attractive effect to trigeminal ganglia-derived neurons. In conclusion, microfluidics system devices provide a valuable tool for studying the behavior of neurons during the development of orofacial tissues and organs, faithfully imitating the in vivo situation.

Innovative Dental Stem Cell-Based Research Approaches: The Future of Dentistry

Over the past decade, the dental field has benefited from recent findings in stem cell biology and tissue engineering that led to the elaboration of novel ideas and concepts for the regeneration of dental tissues or entire new teeth. In particular, stem cell-based regenerative approaches are extremely promising since they aim at the full restoration of lost or damaged tissues, ensuring thus their functionality. These therapeutic approaches are already applied with success in clinics for the regeneration of other organs and consist of manipulation of stem cells and their administration to patients. Stem cells have the potential to self-renew and to give rise to a variety of cell types that ensure tissue repair and regeneration throughout life. During the last decades, several adult stem cell populations have been isolated from dental and periodontal tissues, characterized, and tested for their potential applications in regenerative dentistry. Here we briefly present the various stem cell-based treatment approaches and strategies that could be translated in dental practice and revolutionize dentistry.

A cytoplasmic role of Wnt/β-catenin transcriptional cofactors Bcl9, Bcl9l, and Pygopus in tooth enamel formation

Transcriptional cofactors that participate in Wnt/β-catenin signaling act in the cytoplasm to promote proper development of tooth enamel. Cytoplasmic functions for transcriptional cofactors in teeth Bcl9, Bcl9l, and Pygo2 interact with transcription factors, such as the Wnt-regulated protein β-catenin, to regulate gene expression. Cantù et al. reveal that these proteins also have cytoplasmic functions during tooth development and are particularly important for the formation of enamel. Mice lacking both Pygo1 and Pygo2 or both Bcl9 and Bcl9l developed teeth, a process that requires Wnt/β-catenin transcriptional regulation, but the enamel was structurally disorganized and contained less iron than teeth from control mice. Bcl9, Bcl9l, and Pygo2 were present in the cytoplasm of ameloblasts, the cells that secrete enamel proteins, and colocalized in these cells with amelogenin, the main component of enamel. Bcl9 interacted with amelogenin and proteins involved in exocytosis and vesicular trafficking, suggesting that these proteins function in the trafficking or secretion of enamel proteins. These results demonstrate that Bcl9, Bcl9l, and Pygo2 have cytoplasmic functions distinct from their roles as transcriptional cofactors downstream of Wnt signaling. Wnt-stimulated β-catenin transcriptional regulation is necessary for the development of most organs, including teeth. Bcl9 and Bcl9l are tissue-specific transcriptional cofactors that cooperate with β-catenin. In the nucleus, Bcl9 and Bcl9l simultaneously bind β-catenin and the transcriptional activator Pygo2 to promote the transcription of a subset of Wnt target genes. We showed that Bcl9 and Bcl9l function in the cytoplasm during tooth enamel formation in a manner that is independent of Wnt-stimulated β-catenin–dependent transcription. Bcl9, Bcl9l, and Pygo2 localized mainly to the cytoplasm of the epithelial-derived ameloblasts, the cells responsible for enamel production. In ameloblasts, Bcl9 interacted with proteins involved in enamel formation and proteins involved in exocytosis and vesicular trafficking. Conditional deletion of both Bcl9 and Bcl9l or both Pygo1 and Pygo2 in mice produced teeth with defective enamel that was bright white and deficient in iron, which is reminiscent of human tooth enamel pathologies. Overall, our data revealed that these proteins, originally defined through their function as β-catenin transcriptional cofactors, function in odontogenesis through a previously uncharacterized cytoplasmic mechanism, revealing that they have roles beyond that of transcriptional cofactors.

Distribution of syndecan-1 protein in developing mouse teeth.

Syndecan-1 is a cell surface proteoglycan involved in the regulation of various biological processes such as proliferation, migration, condensation and differentiation of cells, intercellular communication, and morphogenesis. The extracellular domain of syndecan-1 can bind to extracellular matrix components and signaling molecules, while its intracellular domain interacts with cytoskeletal proteins, thus allowing the transfer of information about extracellular environment changes into the cell that consequently affect cellular behavior. Although previous studies have shown syndecan-1 expression during precise stages of tooth development, there is no equivalent study regrouping the expression patterns of syndecan-1 during all stages of odontogenesis. Here we examined the distribution of syndecan-1 protein in embryonic and post-natal developing mouse molars and incisors. Syndecan-1 distribution in mesenchymal tissues such as dental papilla and dental follicle was correlated with proliferating events and its expression was often linked to stem cell niche territories. Syndecan-1 was also expressed in mesenchymal cells that will differentiate into the dentin producing odontoblasts, but not in differentiated functional odontoblasts. In the epithelium, syndecan-1 was detected in all cell layers, by the exception of differentiated ameloblasts that form the enamel. Furthermore, syndecan-1 was expressed in osteoblast precursors and osteoclasts of the alveolar bone that surrounds the developing tooth germs. Taken together these results show the dynamic nature of syndecan-1 expression during odontogenesis and suggest its implication in various processes of tooth development and homeostasis.

Nerve growth factor signalling in pathology and regeneration of human teeth

Nerve growth factor (NGF) is a key regulator of the development and differentiation of neuronal and non-neuronal cells. In the present study we examined the distribution of NGF and its low and high-affinity receptors, p75NTR and TrkA respectively, in permanent human teeth under normal and pathological conditions. In intact functional teeth, NGF, p75NTR and TrkA are weakly expressed in dental pulp fibroblasts and odontoblasts that are responsible for dentine formation, while the NGF and p75NTR molecules are strongly expressed in nerve fibres innervating the dental pulp. In carious and injured teeth NGF and TrkA expression is upregulated in a selective manner in odontoblasts surrounding the injury sites, indicating a link between NGF signalling and dental tissue repair events. Accordingly, NGF and TrkA expression is strongly upregulated in cultured primary human dental mesenchymal cells during their differentiation into odontoblasts. Targeted release of NGF in cultured human tooth slices induced extensive axonal growth and migration of Schwann cells towards the NGF administration site. These results show that NGF signalling is strongly linked to pathological and regenerative processes in human teeth and suggest a potential role for this neurotrophic molecule in pulp regeneration.

Expression of Nerve Growth Factor (NGF), TrkA, and p75NTR in Developing Human Fetal Teeth

Nerve growth factor (NGF) is important for the development and the differentiation of neuronal and non-neuronal cells. NGF binds to specific low- and high-affinity cell surface receptors, respectively, p75NTR and TrkA. In the present study, we examined by immunohistochemistry the expression patterns of the NGF, p75NTR, and TrkA proteins during human fetal tooth development, in order to better understand the mode of NGF signaling action in dental tissues. The results obtained show that these molecules are expressed in a wide range of dental cells of both epithelial and mesenchymal origin during early stages of odontogenesis, as well as in nerve fibers that surround the developing tooth germs. At more advanced developmental stages, NGF and TrkA are localized in differentiated cells with secretory capacities such as preameloblasts/ameloblasts secreting enamel matrix and odontoblasts secreting dentine matrix. In contrast, p75NTR expression is absent from these secretory cells and restricted in proliferating cells of the dental epithelium. The temporospatial distribution of NGF and p75NTR in fetal human teeth is similar, but not identical, with that observed previously in the developing rodent teeth, thus indicating that the genetic information is well-conserved during evolution. The expression patterns of NGF, p75NTR, and TrkA during odontogenesis suggest regulatory roles for NGF signaling in proliferation and differentiation of epithelial and mesenchymal cells, as well as in attraction and sprouting of nerve fibers within dental tissues.

Monitoring Notch Signaling-Associated Activation of Stem Cell Niches within Injured Dental Pulp

Dental pulp stem/progenitor cells guarantee tooth homeostasis, repair and regeneration throughout life. The decision between renewal and differentiation of these cells is influenced by physical and molecular interactions with stromal cells and extracellular matrix molecules forming the specialized microenvironment of dental pulp stem cell niches. Here we study the activation of putative pulp niches after tooth injury through the upregulation of Notch signaling pathway. Notch1, Notch2, and Notch3 molecules were used as markers of dental pulp stem/progenitor cells. Upon dental injury, Notch1 and Notch3 are detected in cells related to vascular structures suggesting a role of these proteins in the activation of specific pulpal perivascular niches. In contrast, a population of Notch2-positive cells that are actively proliferative is observed in the apical part of the pulp. Kinetics of these cells is followed up with a lipophilic DiI labeling, showing that apical pulp cells migrate toward the injury site where dynamic regenerative/repair events occur. The knowledge of the activation and regulation of dental pulp stem/progenitor cells within their niches in pathologic conditions may be helpful for the realization of innovative dental treatments in the near future.

Linking dental pathologies and cancer via Wnt signalling

Great efforts have been made over the past decades to discover new therapeutic targets for a big variety of human pathologies. Most of the studies dealing with severe pathological conditions such as cancers and tissue malformations are focused on the role of either widely recognized master controlling genes such as ras and myc or pivotal components of key signalling pathways, among which Wnt and Notch [1, 2]. However, these genes and molecules are fundamental for proper embryogenesis as well as for tissue and organ homeostasis and regeneration, where they regulate cell proliferation, migration, differentiation and apoptosis. The precise timing and localization of their activation are important to ensure the appropriate cellular functions in physiological conditions. Thus, their indiscriminate targeting is not desirable, due to both the high risk of severe side effects and the certainty of broad phenotypic consequences. On the contrary, optimal therapeutic targets should be selected based on their tissue, time and pathology specific roles. Here, we suggest a paradigmatic example of such target molecules that could be represented by the Wnt/β-catenin signalling components Bcl9 and Bcl9l.

Modern trends in dental medicine: An update for internists

Traumatic injuries, genetic diseases, and external harmful agents such as bacteria and acids often compromise tooth integrity. There is an unmet medical need to develop alternative, innovative dental treatments that complement traditional restorative and surgery techniques. Stem cells have transformed the medical field in recent years. The combination of stem cells with bioactive scaffolds and nanostructured materials turns out to be increasingly beneficial in regenerative dental medicine. Stem cell-based regenerative approaches for the formation of dental tissues will significantly improve treatments and will have a major impact in dental practice. To date there is no established and reliable stem cell-based treatment translated into the dental clinics, however, the advances and improved technological knowledge are promising for successful dental therapies in the near future. Here, we review some of the contemporary challenges in dental medicine and describe the benefits and future possibilities of certain novel approaches in the emerging field of regenerative dentistry.