Tooth Tissue Engineering Research Articles

Identification and Characterization of Key Genes Associated with Amelogenesis

Abstract Objectives The identification of key genes associated with amelogenesis would be helpful in finding solutions to genetic disorders in oral biology. The study aimed to use in silico analysis to identify the key genes involved in tooth development associated with preameloblasts (PABs) and secretory ameloblasts (SABs). Material and Methods The data was subjected to quality analysis and uniform manifold approximation and projection analysis. To examine the distribution of the genes and identify important upregulated loci, a p-value histogram, a quantile plot, a mean difference and mean-variance plot, and a volcano plot were generated. Finally, protein-protein interaction and gene enrichment analyses were performed to determine the ontology, relevant biological processes, and molecular functions of selected genes. Results A total of 157 genes were found to be significant in the PAB versus SAB comparison. HIST1H31 revealed strong interaction with HIST1H2BM, and EXO1, ASPM, SPC25, and TTK showed strong interactions with one other. The STRING database revealed that NCAPG, CENPU, NUSAP1, HIST1H2BM, and HIST1H31 are involved in biological processes. NCAPG, CENPU, SPC25, ETV5, TTK, ETV1, FAM9A, NUSAP1, HIST1H2BM, and HIST1H31 are involved in cellular components. Conclusion The TTK, NUSAP1, CENPU, NCAPG, FAM9A, ASPM, SPC25, and HIST1H31 genes demonstrate functions in cell division. These genes might play a role in ameloblast development. These results will be useful in developing new methods to stimulate ameloblast development, which is essential for tooth regeneration and tissue engineering. However, more research is required to validate the functions of these genes and the genes with which they interact. A wide variety of genetic, epigenetic, and exogenous signaling factors regulate these genes and pathways throughout development and differentiation, cell fate, and behavior.

Enhancing clinical safety in bioengineered-root regeneration: The use of animal component-free medium

BackgroundMost studies used animal serum-containing medium for bioengineered-root regeneration, but ethical and safety issues raised by animal serum are a potentially significant risk for clinical use. Thus, this study aimed to find a safer method for bioengineered-root regeneration. MethodsThe biological properties of human dental pulp stem cells (hDPSCs) cultured in animal component-free (ACF) medium or serum-containing medium (5%, 10% serum-containing medium, SCM) were compared in vitro. hDPSCs were cultured in a three-dimensional (3D) environment with human-treated dentin matrix (hTDM). The capacity for odontogenesis was compared using quantitative real-time PCR (qPCR) and Western blot. Subsequently, the hDPSCs/hTDM complexes were transplanted into nude mice subcutaneously. Histological staining was then used to verify the regeneration effect in vivo. ResultsACF medium promoted the migration of hDPSCs, but slightly inhibited the proliferation of hDPSCs in the first three days of culture compared to SCM. However, it had no significant effect on cell aging and apoptosis. After 7 days of 3D culture in ACF medium with hTDM, qPCR showed that DMP1, DSPP, OCN, RUNX2, and β-tubulin III were highly expressed in hDPSCs. In addition, 3D cultured hDPSCs/hTDM complexes in ACF medium regenerated dentin, pulp, and periodontal ligament-like tissues similar to SCM groups in vivo. ConclusionACF medium was proved to be an alternative medium for bioengineered-root regeneration. The strategy of using ACF medium to regenerate bioengineered-root can improve clinical safety for tooth tissue engineering.

Composite or Modified Hydroxyapatite Microspheres as Drug Delivery Carrier for Bone and Tooth Tissue Engineering.

Since hydroxyapatite (HAp) is an important constituent of bone and teeth, it has excellent biocompatibility and bioactivity, good osteoconductive effects and the ability to induce bone formation as a material for bone or tooth repair and replacement. At present, widely used HAp microspheres have some characteristics, such as large specific surface area, light mass, good injection properties, good fluidity, and low aggregation ability, but they are difficult to really meet the biological and clinical needs due to their own mechanical property defects, such as low strength, brittleness, and poor plasticity. Based on the current research status of HAp microspheres, we summarize the research progress of various types of composite microspheres, including inorganic materials, natural polymer materials and synthetic polymer materials, and further analyze the advantages of HAp composite microspheres loaded with drug molecules, proteins and bioactive factors, so as to explore the development prospect of HAp composite microspheres as scaffolds for constructing sustained release systems. It provides a theoretical basis and research direction to prepare HAp composite micro-spheres with superior comprehensive properties so that they can be better applied in bone tissue regeneration and tooth regeneration engineering.

Spatiotemporal monitoring of hard tissue development reveals unknown features of tooth and bone development.

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.

Dental Biomaterial Scaffolds in Tooth Tissue Engineering: a Review

Dental Biomaterial Scaffolds in Tooth Tissue Engineering: a Review

Dentin regeneration based on tooth tissue engineering: A review.

Missing or damaged teeth due to caries, genetic disorders, oral cancer, or infection may contribute to physical and mental impairment that reduces the quality of life. Despite major progress in dental tissue repair and those replacing missing teeth with prostheses, clinical treatments are not yet entirely satisfactory, as they do not regenerate tissues with natural teeth features. Therefore, much of the focus has centered on tissue engineering (TE) based on dental stem/progenitor cells to create bioengineered dental tissues. Many in vitro and in vivo studies have shown the use of cells in regenerating sections of a tooth or a whole tooth. Tooth tissue engineering (TTE), as a promising method for dental tissue regeneration, can form durable biological substitutes for soft and mineralized dental tissues. The cell-based TE approach, which directly seeds cells and bioactive components onto the biodegradable scaffolds, is currently the most potential method. Three essential components of this strategy are cells, scaffolds, and growth factors (GFs). This study investigates dentin regeneration after an injury such as caries using TE and stem/progenitor cell-based strategies. We begin by discussing about the biological structure of a dentin and dentinogenesis. The engineering of teeth requires knowledge of the processes that underlie the growth of an organ or tissue. Then, the three fundamental requirements for dentin regeneration, namely cell sources, GFs, and scaffolds are covered in the current study, which may ultimately lead to new insights in this field.

Recent Advances in Development of Natural Cellulosic Non-Woven Scaffolds for Tissue Engineering.

In recent years, tissue engineering researchers have exploited a variety of biomaterials that can potentially mimic the extracellular matrix (ECM) for tissue regeneration. Natural cellulose, mainly obtained from bacterial (BC) and plant-based (PC) sources, can serve as a high-potential scaffold material for different regenerative purposes. Natural cellulose has drawn the attention of researchers due to its advantages over synthetic cellulose including its availability, cost effectiveness, perfusability, biocompatibility, negligible toxicity, mild immune response, and imitation of native tissues. In this article, we review recent in vivo and in vitro studies which aimed to assess the potential of natural cellulose for the purpose of soft (skin, heart, vein, nerve, etc.) and hard (bone and tooth) tissue engineering. Based on the current research progress report, it is sensible to conclude that this emerging field of study is yet to satisfy the clinical translation criteria, though reaching that level of application does not seem far-fetched.

BMP9-initiated osteogenic/odontogenic differentiation of mouse tooth germ mesenchymal cells (TGMCS) requires Wnt/β-catenin signalling activity.

Teeth arise from the tooth germ through sequential and reciprocal interactions between immature epithelium and mesenchyme during development. However, the detailed mechanism underlying tooth development from tooth germ mesenchymal cells (TGMCs) remains to be fully understood. Here, we investigate the role of Wnt/β‐catenin signalling in BMP9‐induced osteogenic/odontogenic differentiation of TGMCs. We first established the reversibly immortalized TGMCs (iTGMCs) derived from young mouse mandibular molar tooth germs using a retroviral vector expressing SV40 T antigen flanked with the FRT sites. We demonstrated that BMP9 effectively induced expression of osteogenic markers alkaline phosphatase, collagen A1 and osteocalcin in iTGMCs, as well as in vitro matrix mineralization, which could be remarkably blunted by knocking down β‐catenin expression. In vivo implantation assay revealed that while BMP9‐stimulated iTGMCs induced robust formation of ectopic bone, knocking down β‐catenin expression in iTGMCs remarkably diminished BMP9‐initiated osteogenic/odontogenic differentiation potential of these cells. Taken together, these discoveries strongly demonstrate that reversibly immortalized iTGMCs retained osteogenic/odontogenic ability upon BMP9 stimulation, but this process required the participation of canonical Wnt signalling both in vitro and in vivo. Therefore, BMP9 has a potential to be applied as an efficacious bio‐factor in osteo/odontogenic regeneration and tooth engineering. Furthermore, the iTGMCs may serve as an important resource for translational studies in tooth tissue engineering.

A Biphasic Calcium Phosphate Cement Enhances Dentin Regeneration by Dental Pulp Stem Cells and Promotes Macrophages M2 Phenotype In Vitro.

Calcium phosphate cement (CPC) is promising for bone and dentin repair and regeneration. However, there has been no report of biphasic CPC for inducing dentin regeneration. The aim of this study was to develop a novel biphasic CPC containing β-tricalcium phosphate (β-TCP), and investigate its effects on odontogenic differentiation of human dental pulp stem cells (hDPSCs) and macrophage polarization. New biphasic CPC was formulated with different ratios of β-TCP to an equimolar mixture of tetracalcium phosphate and dicalcium phosphate anhydrous. Mechanical properties, biocompatibility, and odontogenic differentiation induction ability of the cements and the inflammatory reaction to the cements were examined. A series of CPC containing β-TCP were developed. CPC with 20% β-TCP exhibited homogeneity and injectability, an acceptable setting time, and a twofold increase in compressive strength. Significant increases in hDPSCs' alkaline phosphatase activity, mineral deposit, DMP1 and DSPP gene, and protein expressions were obtained for 20% TCP-CPC, compared with traditional CPC (p < 0.01). The addition of β-TCP did not promote macrophage polarization to the proinflammation phenotype. The addition of 10% and 20% β-TCP promoted macrophage polarization to the anti-inflammatory phenotype. In conclusion, a biphasic β-TCP-modified CPC was developed for the first time, demonstrating substantially increased dentin regeneration capability, while promoting macrophages to an anti-inflammation phenotype. The novel biphasic CPC is promising for tooth tissue engineering and dentin regeneration applications. Impact statement Dental pulp exposure from dental caries, wounds, or deep cavity preparation can cause pain and infection, which may lead to root canal treatment or tooth extraction. Maintaining pulp vitality is a major challenge in stomatology. We developed a new injectable β-tricalcium phosphate (β-TCP)-calcium phosphate cement (CPC) for the regeneration of dentin. Compared with traditional CPC, the new CPC +20%TCP possessed good cytocompatibility, acceptable injection force, higher compressive strength (increased by 63%), and greater odontogenic expression and mineral deposits (increased by more than twofolds), while avoiding any proinflammatory drawback. This new biphasic CPC is promising for dental pulp-capping, base, and liner applications to promote dentin regeneration, as well as potentially for bone tissue engineering applications, which warrant further studies.

Differentiation and Establishment of Dental Epithelial-Like Stem Cells Derived from Human ESCs and iPSCs.

Tooth development and regeneration occur through reciprocal interactions between epithelial and ectodermal mesenchymal stem cells. However, the current studies on tooth development are limited, since epithelial stem cells are relatively difficult to obtain and maintain. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) may be alternative options for epithelial cell sources. To differentiate hESCs/hiPSCs into dental epithelial-like stem cells, this study investigated the hypothesis that direct interactions between pluripotent stem cells, such as hESCs or hiPSCs, and Hertwig’s epithelial root sheath/epithelial rests of Malassez (HERS/ERM) cell line may induce epithelial differentiation. Epithelial-like stem cells derived from hES (EPI-ES) and hiPSC (EPI-iPSC) had morphological and immunophenotypic characteristics of HERS/ERM cells, as well as similar gene expression. To overcome a rare population and insufficient expansion of primary cells, EPI-iPSC was immortalized with the SV40 large T antigen. The immortalized EPI-iPSC cell line had a normal karyotype, and a short tandem repeat (STR) analysis verified that it was derived from hiPSCs. The EPI-iPSC cell line co-cultured with dental pulp stem cells displayed increased amelogenic and odontogenic gene expression, exhibited higher dentin sialoprotein (DSPP) protein expression, and promoted mineralized nodule formation. These results indicated that the direct co-culture of hESCs/hiPSCs with HERS/ERM successfully established dental epithelial-like stem cells. Moreover, this differentiation protocol could help with understanding the functional roles of cell-to-cell communication and tissue engineering of teeth.

In vitro evaluation of Zeolite-nHA blended PCL/PLA nanofibers for dental tissue engineering

This study aimed to investigate the potential of a composite poly(lactic acid)/polycaprolactone (PLA/PCL) blend electrospun nanofiber containing both nano-hydroxyapatite (nHA) and zeolite for promoting the proliferation of human dental pulp-derived stem cells (hDPSCs) with possible application in dental tissue engineering. For this purpose, nHA and Zeolite were synthesized via the hydrothermal method and their morphological features and crystals properties were studied by FE-SEM and X-ray diffraction, respectively. PCL-PLA/nHA/zeolite nanofibers were fabricated via electrospinning and characterized through FT-IR and water contact angle measurement methods. Then, the viability and adhesion of hDPSCs on the fibers were investigated by MTT assay and FE-SEM, respectively. The results showed that the viability of hDPSCs on the PCL-PLA/Zeolite scaffolds was significantly improved after 1, 7 and 14 days of culturing and maximum enhancing on the cell viability was detected after 3 days on PCL-PLA/nHA scaffolds (P ≤ 0.001). After 7 and 14 days of incubation, cell growth on the scaffolds containing both nHA and Zeolite was better than the nanofibers which were loaded with nHA alone. Briefly, these results revealed that Zeolite-loaded nanofiber is the most suitable scaffold for bone and tooth tissue engineering applications. More studies are required to investigate the efficiency of zeolite-based nanofibrous scaffolds for the development of artificial scaffolds for dental tissue regeneration.

Scaffolds in Dental Tissue Engineering: A Review

Context: The major aim of tissue engineering is inducing of the body’s mechanisms to regenerate damaged tissues to original condition and task. Scaffolds are 3D porous constructs which provide a cellular microenvironment needed for tissue engineering. Choosing a biomaterial with proper biological, physical, and mechanical properties is of great importance in tooth tissue engineering. Thus, the current study reviews the properties of different polymers and ceramics for use as scaffolds in tooth dental regeneration. Evidence Acquisition: The current study searched databases such as Elsevier, Wiley, Google Scholar, and PubMed in English from 1972 to 2018. After going through the required process, 29 articles were eventually confirmed and enrolled in this review paper. Conclusions: The results of this work confirmed that thanks to having the ability to provide suitable amounts of interconnected porosities, high ratio of surface area to volume, proper mechanical strength, and different geometries required for tissue engineering, polymers are a good option for regeneration of tooth tissues. Although bioceramics such as calcium phosphate and glasses have good biocompatibility and bioactivity, their poor mechanical properties and low degradation rate limit their extensive use in tooth tissue regeneration.

Human Freeze-dried Dentin Matrix as a Biologically Active Scaffold for Tooth Tissue Engineering

IntroductionDentin is a suitable scaffold for tooth tissue engineering and can provide the appropriate combinations and concentrations of bioactive proteins to accommodate odontogenic cells. Freeze drying is a possible strategy to treat dentin because it ensures long-term stability of biopharmaceutical products and can increase the stability of dentin for improved distribution and storage. We hypothesized that freeze-dried dentin matrix (FDDM) constitutes a novel bioinstructive scaffold for tooth tissue engineering. MethodsThis study used a modified technique to prepare an FDDM that preserved the mechanical and biological properties of dentin. The compression resistance and microhardness of FDDM were determined. Similarly, various biological characteristics, including cell morphology, cell proliferation, collagen secretion, alkaline phosphatase activity, and gene and protein expression, were investigated. To assess the inductive capacity of FDDM in vivo, FDDM and human dental pulp stem cell (DPSC) sheets were subcutaneously implanted in the dorsal pockets of nude mice. At 8 weeks postimplantation, the transplants were removed and histologically studied. ResultsThe results show that FDDM has mechanical and biological characteristics similar to those of dentin (P > .05). DPSCs cultured on FDDM and dentin showed superior attachment, growth, viability, and collagen secretion capacity but decreased mineral capability compared with DPSCs cultured with alpha minimum essential medium or hydroxyapatite (P < .05). The histologic results show that FDDM, which is similar to dentin, supported dentin/pulplike tissue regeneration in vivo as shown by the related expression levels of dentin markers, such as dentin sialoprotein and alkaline phosphatase. ConclusionsThese results suggest that FDDM constitutes a novel superior scaffold for tooth tissue engineering.

Development of immortalized Hertwig\u2019s epithelial root sheath cell lines for cementum and dentin regeneration

BackgroundHertwig’s epithelial root sheath (HERS) is important in guiding tooth root formation by differentiating into cementoblasts through epithelial–mesenchymal transition (EMT) and inducing odontoblastic differentiation of dental papilla through epithelial–mesenchymal interaction (EMI) during the tooth root development. Thus, HERS cells are critical for cementum and dentin formation and might be a potential cell source to achieve tooth root regeneration. However, limited availability and lifespan of primary HERS cells may represent an obstacle for biological investigation and therapeutic use of tooth tissue engineering. Therefore, we constructed, characterized, and tested the functionality of immortalized cell lines in order to produce a more readily available alternative to HERS cells.MethodsPrimary HERS cells were immortalized via infection with lentivirus vector containing the gene encoding simian virus 40 Large T Antigen (SV40LT). Immortalized HERS cell subclones were isolated using a limiting dilution method, and subclones named HERS-H1 and HERS-C2 cells were isolated. The characteristics of HERS-H1 and HERS-C2 cells, including cell proliferation, ability of epithelial–mesenchymal transformation and epithelial–mesenchymal interaction, were determined by CCK-8 assay, immunofluorescence staining, and real-time PCR. The cell differentiation into cementoblast-like cells or periodontal fibroblast-like cells was confirmed in vivo. And the inductive influence of the cell lines on dental papilla cells (DPCs) was also confirmed in vivo.ResultsHERS-H1 and HERS-C2 cells share some common features with primary HERS cells such as epithelial-like morphology, positive expression of CK14, E-Cadherin, and Vimentin, and undergoing EMT in response to TGF-beta. HERS-C2 cells showed the EMT characteristics and could differentiate into cementum-forming cells in vitro and generate cementum-like tissue in vivo. HERS-H1 could induce the differentiation of DPCs into odontoblasts in vitro and generation of dentin-like tissue in vivo.ConclusionsWe successfully isolated and characterized novel cell lines representing two key features of HERS cells during the tooth root development and which were useful substitutes for primary HERS cells, thereby providing a biologically relevant, unlimited cell source for studies on cell biology, developmental biology, and tooth root regeneration.

Bioprinting of three-dimensional dentin–pulp complex with local differentiation of human dental pulp stem cells

Numerous approaches have been introduced to regenerate artificial dental tissues. However, conventional approaches are limited when producing a construct with three-dimensional patient-specific shapes and compositions of heterogeneous dental tissue. In this research, bioprinting technology was applied to produce a three-dimensional dentin–pulp complex with patient-specific shapes by inducing localized differentiation of human dental pulp stem cells within a single structure. A fibrin-based bio-ink was designed for bioprinting with the human dental pulp stem cells. The effects of fibrinogen concentration within the bio-ink were investigated in terms of printability, human dental pulp stem cell compatibility, and differentiation. The results show that micro-patterns with human dental pulp stem cells could be achieved with more than 88% viability. Its odontogenic differentiation was also regulated according to the fibrinogen concentration. Based on these results, a dentin–pulp complex having patient-specific shape was produced by co-printing the human dental pulp stem cell–laden bio-inks with polycaprolactone, which is a bio-thermoplastic used for producing the overall shape. After culturing with differentiation medium for 15 days, localized differentiation of human dental pulp stem cells in the outer region of the three-dimensional cellular construct was successfully achieved with localized mineralization. This result demonstrates the possibility to produce patient-specific composite tissues for tooth tissue engineering using three-dimensional bioprinting technology.

Long non-coding RNA H19 promotes the osteogenic differentiation of rat ectomesenchymal stem cells via Wnt/β-catenin signaling pathway.

To investigate the molecular mechanism of long noncoding RNA (lncRNA) H19 that promotes osteogenic differentiation in rat ectomesenchymal stem cells (EMSCs). EMSCs were isolated from rat fetal facial processes by flow cytometry. Osteogenic markers CD29, CD90, CD44, CD57, Nestin and sox10 were detected by fluorescent immunoassay. β-catenin and Wnt pathway target genes were detected by Real-time polymerase chain reaction (RT-PCR) and Western blot after the construction of transient interference H19, a stable expression of H19 EMSCs cell line and the induction of osteogenic differentiation of EMSCs cells. EMSCs of H19 overexpression were treated with Wnt/beta-catenin signaling pathway inhibitor Wnt-C59, and the expressions of beta-catenin and osteogenic markers were detected by RT-PCR and Western blot. Furthermore, the mechanism of H19 regulating Wnt/beta-catenin signaling pathway was explored by transfecting miR-22 and miR-141 mimics and luciferase reporter assays. EMSCs were successfully isolated and identified, osteogenic markers CD29, CD90, CD44, CD57, Nestin and sox10 were significantly overexpressed. Osteogenesis-induced solution significantly increased the expression of H19 and osteogenic markers ALP, Runx2, BMP and OCN in EMSCs (p<0.05). Interference with H19 significantly inhibited the expressions of osteogenic markers, beta-catenin and target genes of Wnt/beta-catenin signaling pathway (p<0.05), while upregulation of H19 significantly promoted the expressions of these markers and genes in EMSCs (p<0.05). Wnt-C59 inhibitors treatment inhibited the Wnt/beta-catenin signaling pathway and osteogenic differentiation in EMSCs with H19 overexpression (p<0.05). Furthermore, H19 could block the inhibitory effect of miR-22 and miR-141 on β-catenin and activate the Wnt/beta-catenin signaling pathway after transfecting miR-22 mimics and miR-141 mimics in EMSCs (p<0.05). LncRNA H19 can promote the osteogenic differentiation of rat EMSCs by activating Wnt/beta-catenin signal, providing a theoretical basis for the application of EMSCs in tooth tissue engineering regeneration and repair.

Stathmin inhibits proliferation and differentiation of dental pulp stem cells via sonic hedgehog/Gli.

The mineralization of dental pulp stem cells is an important factor in the tissue engineering of teeth, but the mechanism is not yet obvious. This study aimed to identify the effect of Stathmin on the proliferation and osteogenic/odontoblastic differentiation of human dental pulp stem cells (hDPSCs) and to explore whether the Shh signalling pathway was involved in this regulation. First, Stathmin was expressed in the cytoplasm and on the cell membranes of hDPSCs by cell immunofluorescence. Then, by constructing a lentiviral vector, the expression of Stathmin in hDPSCs was inhibited. Treatment with Stathmin shRNA (shRNA‐Stathmin group) inhibited the ability of hDPSCs to proliferate, as demonstrated by a CCK8 assay and flow cytometry analysis, and suppressed the osteogenic/odontoblastic differentiation ability, as demonstrated by alizarin red S staining and osteogenic/odontoblastic differentiation‐related gene (ALP, BSP, OCN, DSPP) activity, compared to that of hDPSCs from the control shRNA group. Molecular analyses showed that the Shh/GLI1 signalling pathway was inhibited when Stathmin was silenced, and purmorphamine, the Shh signalling pathway activator, was added to hDPSCs in the shRNA‐Stathmin group, real‐time PCR and Western blotting confirmed that expression of Shh and its downstream signalling molecules PTCH1, SMO and GLI1 increased significantly. After activating the Shh signalling pathway, the proliferation of hDPSCs increased markedly, as demonstrated by a CCK8 assay and flow cytometry analysis; osteogenic/odontoblastic differentiation‐related gene (ALP, BSP, OCN, DSPP) expression also increased significantly. Collectively, these findings firstly revealed that Stathmin‐Shh/GLI1 signalling pathway plays a positive role in hDPSC proliferation and osteogenic/odontoblastic differentiation.

Induction of Rhesus Keratinocytes into Functional Ameloblasts by Mouse Embryonic Dental Mesenchyme.

Fast progresses in stem cell-based tooth tissue engineering have been achieved in recent years in several animal models including the mouse, rat, dog, and pig. Moreover, various postnatal mesenchymal stem cells of dental origin have been isolated and shown capable of differentiating into odontoblasts and generating dentin. Meanwhile, human keratinocyte stem/progenitor cells, gingival epithelial cells, and even iPSC-derived epithelium have been demonstrated to be able to differentiate into functional ameloblasts. Translational medicine studies in the nonhuman primate are irreplaceable steps towards clinical application of stem cell-based tissue engineering therapy. In the present study, we first examined the epithelial stem cell markers in the rhesus skin using immunostaining. Keratinocyte stem cells were then isolated from rhesus epidermis, cultured in vitro, and characterized by epithelial stem cell markers. Epithelial sheets of these cultured keratinocytes, which were recombined with E13.5 mouse dental mesenchyme that possesses odontogenic potential in the presence of exogenous FGF8, were induced to differentiate into enamel-secreting ameloblasts. Our results demonstrate that in the presence of appropriate odontogenic signals, rhesus keratinocytes can be induced to gain odontogenic competence and are capable of participating in odontogenesis, indicating that rhesus keratinocytes are an ideal epithelial cell source for further translational medicine study of tooth tissue engineering in nonhuman primates.

Bone marrow mesenchymal stem cells combine with Treated dentin matrix to build biological root

Treated dentin matrix (TDM) as a kind of scaffolding material has been proved odontogenic induction ability on dental-derived stem cells. Given the limited resources of dental stem cells, it is necessary to seek new seed cell which easily obtained. Jaw bone marrow mesenchymal stem cell (JBMMSC) as non-dental-derived stem cell relates to the development of teeth and jaws which suggest us JBMMSCs could act as a new seed cell for tooth tissue engineering. To assess the odontogenic and osteogenic potential of JBMMSCs, cells were induced by TDM extraction in vitro and combined with TDM in vivo. Results were analyzed by PCR, Western Blotting and histology. PCR and Western Blotting showed odontogenic and osteogenic makers were significantly enhanced in varying degrees after induced by TDM extraction in vitro. In vivo, JBMMSCs expressed both odontogenic and osteogenic-related protein, and the latter showed stronger positive expression. Furthermore, histological examination of the harvested grafts was observed the formation of bone-like tissue. Therefore, osteogenic differentiation ability of JBMMSCs were enhanced significantly after being inducted by TDM which illustrates that non-odontogenic derived stem cells are still promising seed cells in tooth root tissue engineering.

Advances and perspectives in tooth tissue engineering.

Bio-engineered teeth that can grow and remodel in a manner similar to that of natural teeth have the potential to serve as permanent replacements to the currently used prosthetic teeth, such as dental implants. A major challenge in designing functional bio-engineered teeth is to mimic both the structural and anisotropic mechanical characteristics of the native tooth. Therefore, the field of dental and whole tooth regeneration has advanced towards the molecular and nanoscale design of bio-active, biomimetic systems, using biomaterials, drug delivery systems and stem cells. The focus of this review is to discuss recent advances in tooth tissue engineering, using biomimetic scaffolds that provide proper architectural cues, exhibit the capacity to support dental stem cell proliferation and differentiation and sequester and release bio-active agents, such as growth factors and nucleic acids, in a spatiotemporal controlled manner. Although many in vitro and in vivo studies on tooth regeneration appear promising, before tooth tissue engineering becomes a reality for humans, additional research is needed to perfect methods that use adult human dental stem cells, as opposed to embryonic dental stem cells, and to devise the means to generate bio-engineered teeth of predetermined size and shape. Copyright © 2016 John Wiley & Sons, Ltd.