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Different stem cell–based strategies, especially induced pluripotent stem cells (iPSCs), have been exploited to regenerate teeth or restore biological and physiological functions after tooth loss. Further research is needed to establish an optimized protocol to effectively differentiate human iPSCs (hiPSCs) into dental epithelial cells (DECs). In this study, various factors were precisely modulated to facilitate differentiation of hiPSCs into DECs, which are essential for the regeneration of functional teeth. Embryoid bodies (EBs) were formed from hiPSCs as embryo-like aggregates, retinoic acid (RA) was used as an early ectodermal inducer, and bone morphogenic protein 4 (BMP4) activity was manipulated. The characteristics of DECs were enhanced and preserved after culture in keratinocyte serum-free medium (K-SFM). The yielded cell population exhibited noticeable DEC characteristics, consistent with the expression of epithelial cell and ameloblast markers. DECs demonstrated odontogenic abilities by exerting an inductive effect on human dental pulp stem cells (hDPSCs) and forming a tooth-like structure with the mouse tooth mesenchyme. Overall, our differentiation protocol provides a practical approach for applying hiPSCs for tooth regeneration. [ABSTRACT FROM AUTHOR]

The establishment of a method to derive dental epithelial cells seems to be an important challenge toward realizing the whole tooth regeneration. In order to obtain a source of dental epithelial-like cells, a new methodology has been previously developed by our research group. In the method, induced pluripotent stem cells are cultured in suspension in the presence of neurotrophin-4 to form embryoid bodies followed by further adherent culture of the embryoid bodies in DMEM basal nutrient medium. The present study was directed to improve the efficiency of dental epithelial-like cell production, by focusing on the optimization of initial cell number for the formation of embryoid bodies and the addition of epidermal growth factor as well as its timing. Our results demonstrated that an initial cell number of 1000 cells/drop gives the highest efficiency of dental epithelial-like cell production. It appears that, under this condition, medium deterioration is moderated, and that cell-cell interactions are optimized within embryoid bodies. On the other hand, epidermal growth factor serves to increase the abundance of dental epithelial-like cells when added to the medium together with neurotrophin-4 during embryoid body formation. The promotive effect of epidermal growth factor may involve the transactivation of TrkB, mediated by the effectors of epidermal growth factor receptor signaling. [ABSTRACT FROM AUTHOR]

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Tooth defect and tooth loss are common clinical diseases in stomatology. Compared with the traditional oral restoration treatment, tooth regeneration has unique advantages and is currently the focus of oral biomedical research. It is known that dozens of cytokines/growth factors and other bioactive factors are expressed in a spatial-temporal pattern during tooth development. On the other hand, the technology for spatial-temporal control of drug release has been intensively studied and well developed recently, making control release of these bioactive factors mimicking spatial-temporal pattern more feasible than ever for the purpose of tooth regeneration. This article reviews the research progress on the tooth development and discusses the future of tooth regeneration in the context of spatial-temporal release of developmental factors. [ABSTRACT FROM AUTHOR]

Previous studies have demonstrated that several types of human stem cells of non-dental origin can be induced to differentiate into enamel-secreting ameloblasts after recombined with mouse embryonic dental mesenchyme. However, the successful rate of ameloblastic differentiation is about rather low, which presents a major obstacle for future stem cell-based whole tooth bioengineering. Previous studies have shown that cultures at reduced temperature could improve the differentiation capability of stem cells in tissue engineering. In this study, we systematically investigated the effects of low temperature on the viability, proliferation and stemness of human keratinocytes stem cells (hKSCs) in cell culture and further examined ameloblastic differentiation of the hKSCs in human–mouse recombinant chimeric tooth germs. Our results demonstrated that low temperature indeed reduces growth rate and maintains healthy undifferentiated morphology of hKSCs without any effects on cell viability. Moreover, examination of stemness makers revealed improved stemness of hKSCs cultured at low temperature with increased expression of stemness markers K15, CD29 and p63 and decreased expression differentiation marker K10, as compared to those cultured at 37 °C. These low temperature treated hKSCs, when recombined with mouse embryonic dental mesenchyme, exhibited significantly increased rate (40%) of ameloblastic differentiation, as compared to that (17%) in tissue recombinants with those hKSCs treated at standard temperature. Our studies demonstrate that low temperature cell culture improves the stemness and plasticity of hKSCs, which in turn enhances ameloblastic differentiation capability of the stem cells in bioengineered teeth. [ABSTRACT FROM AUTHOR]

Recent studies have successfully generated tooth-like structure by mimicking the reciprocal interaction between dental epithelial and mesenchymal cells in tooth organogenesis. However, clinical applications of these methods are limited primarily due to the lack of appropriate sources for dental epithelial cells. Induced pluripotent stem cells (iPSCs) are attractive as a source for dental epithelial cells due to their unique characteristics. In this study, we examined the effect of neurotrophin-4 (NT-4) on the differentiation of mouse iPSCs (miPSCs) into dental epithelial cells. Our results showed that the addition of NT-4 during the formation of embryoid body significantly triggered the upregulation of epithelial markers such as p63 and CK14, suggesting that NT-4 provides an inductive condition for the differentiation of miPSCs into epithelial cells. Expansion of the NT-4-treated cells under serum-free culture conditions improves the formation of cells with cobblestone-like morphology and significantly downregulated the expression of pluripotent and ectodermal markers. Phenotypic analysis revealed that a dental epithelial surface marker, CD49f, was highly expressed on these cells. Formation of miPSCs-derived dental epithelial-like cells was further confirmed by the expression of ameloblast-specific markers. These results suggest that the addition of NT-4 during the formation of embryoid body together with the serum-free culture condition promoted the differentiation of miPSCs into dental epithelial-like cells. [ABSTRACT FROM AUTHOR]

Over the last several decades, a wealth of information has become available regarding various sources of stem cells and their potential use for regenerative purposes. Given the intense debate regarding embryonic stem cells, much of the focus has centered around application of adult stem cells for regenerative engineering along with other relevant aspects including use of growth factors and scaffolding materials. The more recent discovery of tooth-derived stem cells has sparked much interest in their application to regenerative dentistry to treat and alleviate the most prevalent oral diseases—i.e., dental caries and periodontal diseases. Also exciting is the advent of induced pluripotent stem cells, which provides the means of using patient-derived somatic cells for their creation, and their eventual application for generation of the dental complex. Thus, evolving developments in the field of regenerative dentistry indicate the prospect of constructing "custom-made" tooth and supporting structures thereby fostering the realization of "personalized dentistry." On the other hand, others have explored the possibility of augmenting endogenous regenerative capacity through utilization of small molecules to regulate molecular signaling mechanisms that mediate regeneration of tooth structure. This review is focused on these aspects of regenerative dentistry in view of their relevance to personalized dentistry. [ABSTRACT FROM AUTHOR]

The stem-cell-based tissue-engineering approaches are widely applied in establishing functional organs and tissues for regenerative medicine. Successful generation of induced pluripotent stem cells (iPS cells) and rapid progress of related technical platform provide great promise in the development of regenerative medicine, including organ regeneration. We have previously reported that iPS cells could be an appealing stem cells source contributing to tooth regeneration. In the present paper, we mainly review the application of iPS technology in dental bioengineering and discuss the challenges for iPS cells in the whole tooth regeneration. [ABSTRACT FROM AUTHOR]

Oral and general health is compromised by irreversible dental problems, including dental caries, periodontal disease and tooth injury. Regenerative therapy for tooth tissue repair and whole-tooth replacement is currently considered a novel therapeutic concept with the potential for the full recovery of tooth function. Several types of stem cells and cell-activating cytokines have been identified in oral tissues. These cells are thought to be candidate cell sources for tooth tissue regenerative therapies because they have the ability to differentiate into tooth tissues in vitro and in vivo. Whole-tooth replacement therapy is regarded as an important model for the development of an organ regenerative concept. A novel three-dimensional cell-manipulation method, designated the organ germ method, has been developed to recapitulate organogenesis. This method involves compartmentalisation of epithelial and mesenchymal cells at a high cell density to mimic multicellular assembly conditions and epithelial-mesenchymal interactions. A bioengineered tooth germ can generate a structurally correct tooth in vitro and erupt successfully with the correct tooth structure when transplanted into the oral cavity. We have ectopically generated a bioengineered tooth unit composed of a mature tooth, periodontal ligament and alveolar bone, and that tooth unit was successfully engrafted into an adult jawbone through bone integration. Such bioengineered teeth were able to perform normal physiological tooth functions, such as developing a masticatory potential in response to mechanical stress and a perceptive potential for noxious stimuli. In this review, we describe recent findings and technologies underpinning tooth regenerative therapy. [ABSTRACT FROM AUTHOR]

Continuous replacement of teeth throughout the lifespan of an individual is possibly basal for most of the vertebrates including fish and reptiles; however, mammals generally have a limited capacity of tooth renewal. The ability to induce cellular differentiation in adults to replace lost or damaged cells in mammals, or to tissue-engineer organs in vitro, has hence become one of the major goals of regenerative medicine. In this article, we will revisit some of the important signals and tissue interactions that regulate mammalian tooth development, and will offer a synopsis of the latest progress in tooth regeneration and repair via molecular and engineering approaches. It is hoped that this article will not only offer an overview of recent technologies in tooth regeneration and repair but will also stimulate more interdisciplinary research in this field to turn the pursuit of tooth regeneration and repair into practical reality. [ABSTRACT FROM AUTHOR]

Pulp regeneration using human dental pulp stem cells (hDPSCs) maintains tooth vitality compared with conventional root canal therapy. Our previous study demonstrated that preameloblast-conditioned medium (PA-CM) from murine apical bud cells induces the odontogenic differentiation of hDPSCs and promoted dentin formation in mouse subcutaneous tissue. The purpose of the present study is to evaluate the effects of PA-CM with human whole pulp cells on pulp regeneration in an empty root canal space. Human pulp cells were seeded in the pulp cavities of 5 mm-thick human tooth segments with or without PA-CM treatment, and then transplanted subcutaneously into immunocompromised mice. In the pulp cell-only group, skeletal muscle with pulp-like tissue was generated in the pulp cavity. A reparative dentin-like structure with entrapped cells lined the existing dentin wall. However, in the PA-CM-treated group, only pulp-like tissue was regenerated without muscle or a reparative dentin-like structure. Moreover, human odontoblast-like cells exhibited palisade arrangement around the pulp, and typical odontoblast processes elongated into dentinal tubules. The results suggest that PA-CM can induce pulp regeneration of human pulp cells with physiological structures in an empty root canal space. [ABSTRACT FROM AUTHOR]

Multipotent mesenchymal stem cells from bone marrow are expected to be a somatic stem cell source for the development of new cell-based therapy in regenerative medicine. However, dental clinicians are unlikely to carry out autologous cell/tissue collection from patients (i.e., marrow aspiration) as a routine procedure in their clinics; hence, the utilization of bone marrow stem cells seems impractical in the dental field. Dental tissues harvested from extracted human teeth are well known to contain highly proliferative and multipotent stem cell compartments and are considered to be an alternative autologous cell source in cell-based medicine. This article provides a short overview of the ongoing studies for the potential application of dental stem cells and suggests the utilization of 2 concepts in future regenerative medicine: (1) dental stem cell-based therapy for hepatic and other systemic diseases and (2) tooth replacement therapy using the bioengineered human whole tooth, called the 'test-tube dental implant.' Regenerative therapies will bring new insights and benefits to the fields of clinical medicine and dentistry. [ABSTRACT FROM AUTHOR]

The article discusses research into the regeneration of lost teeth with the use of dental pulp stem cells (DPSCs). An overview is presented of the potential location of and isolation methods for DPSCs. It is theorized that DPSCs have the capacity of differentiation into odontoblast tooth components such as dentin, pulp, and enamel that can be reconstructed into a biological tooth (bio-tooth).

The present study attempted to examine whether clonal cell lines of the oral epithelium can differentiate into ameloblasts and regenerate tooth when combined with dental germ mesenchyme. Clonal cell lines with a distinct morphology were established from the oral epithelium of p53-deficient fetal mice at embryonic day 18 (E18). The strain of mouse is shown to be a useful source for establishing clonal and immortalized cell lines from various tissues and at various stages of development. Tooth morphogenesis is almost completed and the oral epithelium is segregated from the dental epithelium at E18. In RT-PCR analysis of cell lines, mucosal epithelial markers (cytokeratin 14) were detected, but ameloblast markers such as amelogenin and ameloblastin were not detected when cells were cultured on plastic dish. They formed stratified epithelia and expressed a specific differentiation marker (CK13) in the upper layer when cultured on feeder layer or on collagen gel for 1–3 wk, demonstrating that they are of oral mucosa origin. Next, bioengineered tooth germs were prepared with cell lines and fetal molar mesenchymal tissues and implanted under kidney capsule for 2–3 wk. Five among six cell lines regenerated calcified structures as seen in natural tooth. Our results indicate that some oral epithelial cells at E18 possess the capability to differentiate into ameloblasts. Furthermore, cell lines established in the present study are useful models to study processes in tooth organogenesis and tooth regeneration. [ABSTRACT FROM AUTHOR]

Humans are genetically programmed to replace their teeth once during childhood. Therefore, when adult teeth are lost or damaged, they cannot be regenerated or regrown. However, with the advancement of stem cell biology and tissue engineering, regenerating the whole tooth has become a realistic and attractive option to replace a lost or damaged tooth, and therefore has strongly attracted attention in the field of dental research. During the past several years, significant progress has been made in this research endeavor, providing greater understanding of the production of an entire biological tooth by tissue engineering using stem cells. There are several ways to reproduce an entire biological tooth. Approaches are categorized according to the cell sources that have the potential to produce teeth. One source is the embryonic tooth bud, and the other is the postnatal tooth bud. The results from embryonic and postnatal tooth buds differ considerably. In particular, the potential to regulate the shape of the tooth crown from embryonic tooth bud is higher than from postnatal tooth bud. This article describes the achievements to date in production of biological teeth, mostly from our laboratory. In particular, we describe the potential to produce teeth from embryonic and postnatal tooth buds. [ABSTRACT FROM AUTHOR]