Localized sources of morphogens, called signalling centres, play a fundamental role in coordinating tissue growth and cell fate specification during organogenesis. However, how these signalling centres are established in tissues during embryonic development is still unclear. Here we show that the main signalling centre orchestrating development of rodent incisors, the enamel knot (EK), is specified by a cell proliferation-driven buildup in compressive stresses (mechanical pressure) in the tissue. Direct mechanical measurements indicate that the stresses generated by cell proliferation are resisted by the surrounding tissue, creating a circular pattern of mechanical anisotropy with a region of high compressive stress at its centre that becomes the EK. Pharmacological inhibition of proliferation reduces stresses and suppresses EK formation, and application of external pressure in proliferation-inhibited conditions rescues the formation of the EK. Mechanical information is relayed intracellularly through YAP protein localization, which is cytoplasmic in the region of compressive stress that establishes the EK and nuclear in the stretched anisotropic cells that resist the pressure buildup around the EK. Together, our data identify a new role for proliferation-driven mechanical compression in the specification of a model signalling centre during mammalian organ development.

Rebuilding mineralized tissues in skeletal and dental systems remains costly and challenging. Despite numerous demands and heavy clinical burden over the world, sources of autografts, allografts, and xenografts are far limited, along with massive risks including viral infections, ethic crisis, and so on. Per such dilemma, artificial scaffolds have emerged to provide efficient alternatives. To date, cell-free biomimetic mineralization (BM) and cell-dependent scaffolds have both demonstrated promising capabilities of regenerating mineralized tissues. However, BM and cell-dependent scaffolds have distinctive mechanisms for mineral genesis, which makes them methodically, synthetically, and functionally disparate. Herein, these two strategies in regenerative dentistry and orthopedics are systematically summarized at the level of mechanisms. For BM, methodological and theoretical advances are focused upon; and meanwhile, for cell-dependent scaffolds, it is demonstrated how scaffolds orchestrate osteogenic cell fate. The summary of the experimental advances and clinical progress will endow researchers with mechanistic understandings of artificial scaffolds in rebuilding hard tissues, by which better clinical choices and research directions may be approached.

Three-dimensional organogenesis in vivo is principally regulated by the spatiotemporal developmental process that relies on the cellular behavior such as cell growth, migration, differentiation, and cell-to-cell interaction. Organ development and morphogenesis have been elucidated to be regulated by the proper transient expression of various signaling molecules including cytokines, extracellular matrix, and adhesion molecules based on the epithelial and mesenchymal interactions. Current bioengineering technology for regenerating three-dimensional organ has progressed to the replication of organogenesis, thereby enabling the development of fully functional bioengineered organs using bioengineered organ germs that are generated from immature stem cells via tissue engineering technology in vitro.To achieve precise replication of organogenesis, we have developed a novel three-dimensional cell manipulation method designated the organ germ method, and enabled the generation of a structurally correct and fully functional bioengineered tooth in vivo. This method is also expected to be utilized for analyzing gene and protein functions during organogenesis. Here, we describe protocols for the tooth germ reconstitution by using the organ germ method and for the functional analysis of tooth development in vitro and in vivo.

Mineralized tissues, such as bones or teeth, are essential structures of all vertebrates. They enable rapid movement, protection, and food processing, in addition to providing physiological functions. Although the development, regeneration, and pathogenesis of teeth and bones have been intensely studied, there is currently no tool to accurately follow the dynamics of growth and healing of these vital tissues in space and time. Here, we present the BEE-ST (Bones and tEEth Spatio-Temporal growth monitoring) approach, which allows precise quantification of development, regeneration, remodeling, and healing in any type of calcified tissue across different species. Using mouse teeth as model the turnover rate of continuously growing incisors was quantified, and role of hard/soft diet on molar root growth was shown. Furthermore, the dynamics of bones and teeth growth in lizards, frogs, birds, and zebrafish was uncovered. This approach represents an effective, highly reproducible, and versatile tool that opens up diverse possibilities in developmental biology, bone and tooth healing, tissue engineering, and disease modeling.

The tooth provides an excellent system for deciphering the molecular mechanisms of organogenesis, and has thus been of longstanding interest to developmental and stem cell biologists studying embryonic morphogenesis and adult tissue renewal. In recent years, analyses of molecular signaling networks, together with new insights into cellular heterogeneity, have greatly improved our knowledge of the dynamic epithelial-mesenchymal interactions that take place during tooth development and homeostasis. Here, we review recent progress in the field of mammalian tooth morphogenesis and also discuss the mechanisms regulating stem cell-based dental tissue homeostasis, regeneration and repair. These exciting findings help to lay a foundation that will ultimately enable the application of fundamental research discoveries toward therapies to improve oral health.

Oxygen plays a crucial role in human embryogenesis, homeostasis, and tissue regeneration. Emerging engineered regenerative solutions call for novel oxygen delivery systems. To become a reality, these systems must consider physiological processes, oxygen release mechanisms and the target application. In this review, we explore the biological relevance of oxygen at both a cellular and tissue level, and the importance of its controlled delivery via engineered biomaterials and devices. Recent advances and upcoming trends in the field are also discussed with a focus on tissue-engineered constructs that could meet metabolic demands to facilitate regeneration.

Cementum, a thin layer of mineralized tissue covering tooth root surface, is recognized as the golden standard in periodontal regeneration. However, current efforts mainly focus on alveolar bone regeneration rather than cementum regeneration, and rarely take (Pg), the keystone pathogen responsible for periodontal tissue destruction, into consideration. Though M2 macrophage-derived exosomes (M2-EXO) show promise in tissue regeneration, the exosome-producing M2 macrophages are induced by exogenous cytokines with transitory and unstable effects, restricting the regeneration potential of M2-EXO. Here, exosomes derived from genetically engineered M2-like macrophages are constructed by silencing of casein kinase 2 interacting protein-1 (Ckip-1), a versatile player involved in various biological processes. Ckip-1 silencing is proved to be an effective gene regulation strategy to obtain permanent M2-like macrophages with mineralization-promoting effect. Further, exosomes derived from Ckip-1-silenced macrophages (sh-Ckip-1-EXO) rescue Pg-suppressed cementoblast mineralization and cementogenesis. Mechanismly, sh-Ckip-1-EXO delivers targeting and silencing Ckip-1, a negative regulator also for cementum formation and cementoblast mineralization. More deeply, downregulation of Ckip-1 in cementoblasts by exosomal activates PGC-1α-dependent mitochondrial biogenesis. In all, this study provides a new strategy of genetically engineered M2-like macrophage-derived exosomes for cementum regeneration under Pg-dominated inflammation.

The remarkable evolution of teeth morphological complexity represents a giant leap for vertebrate. Despite its importance in life history, the understanding of spatiotemporal organization of teeth remains rudimentary. Herein, a high-resolution genome-wide molecular patterning of lineage allocation and cellular organization in tooth morphogenesis is described, constructed by integrating spatial transcriptome and single-cell RNA sequencing. Twelve spatial compartments and seventeen heterogeneous cell clusters linked to tooth morphogenic milestones are identified. Eighty-eight percent of total lineage species has already appeared in the initial tooth bud rather than the generally considered sequential emergence. A previously unrecognized sprouting-like patterning mode of the dental papilla is discovered, that the inner compartment can break through the outer shell compartment to build up the final papilla cusp. Meanwhile, the continuum differentiation hierarchies of enamel knots in time and space are revealed. Furthermore, the regulatory network directing tooth morphogenesis is established, whereby a series of mechanotransduction signals are spatiotemporally involved beyond the well-established classical odontogenesis signals. Finally, genes underlying tooth dysplasia are successfully tracked to highly specific time points and cell types. The results raise the idea that tooth morphogenesis is orchestrated by mechanical niches combined with biochemical signaling.

Tooth root development involves intricate spatiotemporal cellular dynamics and molecular regulation. The initiation of Hertwig's epithelial root sheath (HERS) induces odontoblast differentiation and the subsequent radicular dentin deposition. Precisely controlled signaling pathways modulate the behaviors of HERS and the fates of dental mesenchymal stem cells (DMSCs). Disruptions in these pathways lead to defects in root development, such as shortened roots and furcation abnormalities. Advances in dental stem cells, biomaterials, and bioprinting show immense promise for bioengineered tooth root regeneration. However, replicating the developmental intricacies of odontogenesis has not been resolved in clinical treatment and remains a major challenge in this field. Ongoing research focusing on the mechanisms of root development, advanced biomaterials, and manufacturing techniques will enable next-generation biological root regeneration that restores the physiological structure and function of the tooth root. This review summarizes recent discoveries in the underlying mechanisms governing root ontogeny and discusses some recent key findings in developing of new biologically based dental therapies.