Disruption of kif3a results in defective osteoblastic differentiation in dental mesenchymal stem/precursor cells via the Wnt signaling pathway.

The anterograde intraflagellar transport motor protein, kif3a, regulates the integrity of primary cilia and various cellular functions, however, the role of kif3a in dental mesenchymal stem/precursor cell differentiation remains to be fully elucidated. In the present study, the expression of kif3a was knocked down in human dental follicle cells (hDFCs) and human dental pulp cells (hDPCs) using short hairpin RNA. The results of subsequent immunofluorescence revealed that knocking down kif3a resulted in the loss of primary cilia, which led to impairment of substantial mineralization and expression of the differentiation­associated markers, including alkaline phosphatase, Runt­related transcription factor 2, dentin matrix protein 1 and dentin sialophosphoprotein in the hDFCs and hDPCs. The results of reverse transcription­quantitative polymerase chain reaction and western blot analyses showed that the expression levels of Wnt3a­mediated active ß­catenin and lymphoid enhancer­binding factor 1 were attenuated, whereas the expression of phosphorylated glycogen synthase kinase 3ß was enhanced, in the kif3a­knockdown cells. In addition, exogenous Wnt3a partially rescued osteoblastic differentiation in the hDFCs and hDPCs. These results demonstrated that inhibition of kif3a in the hDFCs and hDPCs disrupted primary cilia formation and/or function, and indicated that kif3a is important in the differentiation of hDFCs and hDPCs through the Wnt pathway. These findings not only enhance current understanding of tooth development and diseases of tooth mineralization, but also indicate possible strategies to regulate mineralization during tooth repair and regeneration.

Tumorigenicity analysis of heterogeneous dental stem cells and its self-modification for chromosome instability.

Heterogeneity demonstrates that stem cells are constituted by several sub-clones in various differentiation states. The heterogeneous state is maintained by cross-talk among sub-clones, thereby ensuring stem cell adaption. In this study, we investigated the roles of heterogeneity on genetic stability. Three sub-clones (DF2, DF8 and DF18) were isolated from heterogeneous dental stem cells (DSCs), and were proved to be chromosome instability (CIN) after long term expansion. Cell apoptosis were not detected in sub-clones, which exhibited strong tumorigenesis tendency, coupled with weak expression of p53 and aberrant ultra-structure. However, 3 sub-clones did not overexpress tumor related markers or induce tumorigenesis in vivo. The mixed-culture study suggested that 3-clone-mixed culturing cells (DF1) presented apparent decrease in the ratio of aneuploidy. The screening experiment further proved that 3 sub-clones functioned separately in this modification procedure but only mixed culturing all 3 sub-clones, simulated heterogeneous microenvironment, could achieve complete modification. Additionally, osteogenesis capability of 3 sub-clones was partially influenced by CIN while DSCs still kept stronger osteogenesis than sub-clones. These results suggested aberrant sub-clones isolated from heterogeneous DSCs were not tumorigenesis and could modify CIN by cross-talk among themselves, indicating that the heterogeneity played a key role in maintaining genetic stability and differentiation capability in dental stem cells.

Comparison of human dental follicle cells and human periodontal ligament cells for dentin tissue regeneration.

AIM: To compare the odontogenic potential of human dental follicle cells (DFCs) and periodontal ligament cells (PDLCs). MATERIALS & METHODS: In vitro and in vivo characterization studies of DFCs and PDLCs were performed comparatively. DFCs and PDLCs were subcutaneously implanted into the dorsum of mice for 8 weeks after combined with treated dentin matrix scaffolds respectively. RESULTS: Proteomic analysis identified 32 differentially expressed proteins in DFCs and PDLCs. Examination of the harvested grafts showed PDLCs could form the dentin-like tissues as DFCs did. However, the structure of dentin tissues generated by DFCs was more complete. CONCLUSION: PDLCs could contribute to regenerate dentin-like tissues in the inductive microenvironment of treated dentin matrix. DFCs presented more remarkable dentinogenic capability than PDLCs did.

Comparison of the Odontogenic Differentiation Potential of Dental Follicle, Dental Papilla, and Cranial Neural Crest Cells.

INTRODUCTION: During tooth development, cells originating from the neural crest serve as precursors to the cells in the dental follicle and dental papilla. Therefore, the current study aimed to understand the associations of cranial neural crest cells (CNCCs), dental follicle cells (DFCs), and dental papilla cells (DPCs) by performing a parallel comparison to evaluate their odontogenic differentiation capacities. METHODS: In this study, we harvested the 3 cells from C57/green fluorescent protein-positive mice or embryos and compared the cell morphology, surface antigens, microstructures, and gene and protein expression. Under the odontogenic microenvironments provided by treated dentin matrix, the odontogenic differentiations of the 3 cells were further compared in vitro and in vivo. RESULTS: The gene levels of DFCs in neurofilament, tubulin, and nestin were close to the DPCs, and in alkaline phosphatase, osteopontin, dentin matrix protein 1, and dentin sialophosphoprotein were the lowest in the 3 cells. However, Western blot results showed that DFCs possessed more similar protein profiles to CNCCs than DPCs, including collagen 1, transforming growth factor beta 1, osteopontin, neurofilament, and dentin matrix protein 1. Meanwhile, DFCs as 1 source of dental stem cells possessed high potency in odontogenic differentiation in vitro. Moreover, similar dentinlike tissues were observed in all 3 groups in vivo. CONCLUSIONS: CNCCs, DFCs, and DPCs possessed different biological characteristics in odontogenic differentiation.

Combination of aligned PLGA/Gelatin electrospun sheets, native dental pulp extracellular matrix and treated dentin matrix as substrates for tooth root regeneration.

In tissue engineering, scaffold materials provide effective structural support to promote the repair of damaged tissues or organs through simulating the extracellular matrix (ECM) microenvironments for stem cells. This study hypothesized that simulating the ECM microenvironments of periodontium and dental pulp/dentin complexes would contribute to the regeneration of tooth root. Here, aligned PLGA/Gelatin electrospun sheet (APES), treated dentin matrix (TDM) and native dental pulp extracellular matrix (DPEM) were fabricated and combined into APES/TDM and DPEM/TDM for periodontium and dental pulp regeneration, respectively. This study firstly examined the physicochemical properties and biocompatibilities of both APES and DPEM in vitro, and further investigated the degradation of APES and revascularization of DPEM in vivo. Then, the potency of APES/TDM and DPEM/TDM in odontogenic induction was evaluated via co-culture with dental stem cells. Finally, we verified the periodontium and dental pulp/dentin complex regeneration in the jaw of miniature swine. Results showed that APES possessed aligned fiber orientation which guided cell proliferation while DPEM preserved the intrinsic fiber structure and ECM proteins. Importantly, both APES/TDM and DPEM/TDM facilitated the odontogenic differentiation of dental stem cells in vitro. Seeded with stem cells, the sandwich composites (APES/TDM/DPEM) generated tooth root-like tissues after being transplanted in porcine jaws for 12 w. In dental pulp/dentin complex-like tissues, columnar odontoblasts-like layer arranged along the interface between newly-formed predentin matrix and dental pulp-like tissues in which blood vessels could be found; in periodontium complex-like tissues, cellular cementum and periodontal ligament (PDL)-like tissues were generated on the TDM surface. Thus, above results suggest that APES and DPEM exhibiting appropriate physicochemical properties and well biocompatibilities, in accompany with TDM, could make up an ECM microenvironment for tooth root regeneration, which also offers a strategy for complex tissue or organ regeneration.

A therapeutic strategy for spinal cord defect: human dental follicle cells combined with aligned PCL/PLGA electrospun material.

Stem cell implantation has been utilized for the repair of spinal cord injury; however, it shows unsatisfactory performance in repairing large scale lesion of an organ. We hypothesized that dental follicle cells (DFCs), which possess multipotential capability, could reconstruct spinal cord defect (SCD) in combination with biomaterials. In the present study, mesenchymal and neurogenic lineage characteristics of human DFCs (hDFCs) were identified. Aligned electrospun PCL/PLGA material (AEM) was fabricated and it would not lead to cytotoxic reaction; furthermore, hDFCs could stretch along the oriented fibers and proliferate efficiently on AEM. Subsequently, hDFCs seeded AEM was transplanted to restore the defect in rat spinal cord. Functional observation was performed but results showed no statistical significance. The following histologic analyses proved that AEM allowed nerve fibers to pass through, and implanted hDFCs could express oligodendrogenic lineage maker Olig2 in vivo which was able to contribute to remyelination. Therefore, we concluded that hDFCs can be a candidate resource in neural regeneration. Aligned electrospun fibers can support spinal cord structure and induce cell/tissue polarity. This strategy can be considered as alternative proposals for the SCD regeneration studies.

Expression and roles of syndecan-4 in dental epithelial cell differentiation.

Syndecan-4 (SDC4), a transmembrane heparan sulfate proteoglycan, acts as a signal transducer. It affects the growth and differentiation of a number of tissues and organs. However, the specific mechanisms through which SDC4 regulates the differentiation of dental epithelial cells (amelogenesis) and tooth development remains largely unknown. In the present study, to identify the SDC4-regulated processes in dental epithelial cells, the SDC4 expression pattern was examined in mouse molar and postnatal incisor tooth germs during the late bell stage of development. Small interfering RNA (siRNA) was designed for this study and used to downregulate SDC4 expression in the rat dental epithelial cell line, HAT-7. The results revealed that SDC4 was mainly present in the oral epithelium, the dental epithelial cells of enamel organs in the molars and the cervical loops in the incisors. When the inner enamel epithelial cells gave rise to ameloblasts, however, the loss of SDC4 expression was evident. SDC4 was also expressed in stratum intermedium (SI) cells in the incisors and in dental mesenchymal cells adjacent to the cervical loops in molars (E18) and postnatal incisors. Fibroblast growth factor 10 (FGF10) promoted proliferation and slightly decreased cell differentiation. The knockdown of SDC4 using specific siRNA led to a decrease in cell proliferation and a highly significant increase in amelogenin, ameloblastin, kallikrein 4 and matrix metalloproteinase 20 expression, molecules that are known to participate in the formation of enamel. These effects were attenuated by FGF10, which upregulated SDC4 expression. Taken together, these results suggest that SDC4 participates in amelogenesis, and FGF10 may modulate dental epithelial cell behaviors through the regulation of SDC4 expression.

Expression of Nfic during root formation in first mandibular molar of rat.

The transcription factor Nfic is a key regulator during tooth development. Nfic deficient mice exhibit short and abnormal molar roots and severely deformed incisors. Dental epithelial cells, known as Hertwig's epithelial root sheath (HERS), participate in root formation. However, whether Nfic is involved in HERS-mesenchyme interaction remains unclear. In this study, the detail temporal and spatial expression pattern of Nfic during rat molar development was examined using immunohistochemistry and in situ hybridization. Nfic was detected in ameloblasts, dental follicle cells (DFCs) and dental papilla cells (DPCs), especially the DPCs close to dentin, from postnatal day 5 to day 16. Nfic expression in DPCs, DFCs and HERS cells was also examined by western blot and RT-PCR. Nfic was detected in DPCs and DFCs, but not in HERS cells. Co-culture experiment further indicated that Nfic mRNA expression in DPCs was elevated by the presence of HERS cells. Our results revealed that Nfic could be a marker gene for root odontoblasts differentiation initiation and its expression might be regulated through epithelial-mesenchymal interaction.

The potential of dental stem cells differentiating into neurogenic cell lineage after cultivation in different modes in vitro.

Trauma or degenerative diseases of the central nervous system (CNS) cause the loss of neurons or glial cells. Stem cell transplantation has become a vital strategy for CNS regeneration. It is necessary to effectively induce nonneurogenic stem cells to differentiate into neurogenic cell lineages because of the limited source of neurogenic stem cells, relatively difficult cultivation, and ethical issues. Previous studies have found that dental stem cells can be used for transplantation therapy. The aim of this study was to explore a better inductive mode and time point for dental stem cells to differentiate into neural-like cells and evaluate a better candidate cell. In this study, dental follicle stem cells (DFSCs), dental papilla stem cells (DPSCs), and stem cells from apical papilla (SCAPs) were cultivated in five different modes. The proliferation ability, morphology, and expression of neural marker genes were analyzed. Results showed that DFSCs showed a higher proliferation potential. The proliferation was decreased after cultivation in chemical inductive medium as cultivation modes 3 and 5. The cells could present neural-like cell morphology after cultivation with human epidermal growth factor (EGF) and fibroblast growth factor-basic (bFGF) as cultivation modes 4 and 5. The vast majority of DFSCs gene expression levels in mode 4 on the third day was upregulated significantly. In conclusion, our data suggested that different dental stem cells exhibited different neural differentiation potentials. DFSCs might be the better candidate cell type. Furthermore, cultivation mode 4 and timing of the third day may promote differentiation into neurogenic cell lineages more effectively before transplantation to treat neurological diseases.

Cryopreserved dentin matrix as a scaffold material for dentin-pulp tissue regeneration.

Cryopreservation has been identified as an efficient approach to preserve tissue engineered products for a long term. Our prior studies have suggested that the treated dentin matrix (TDM) could be an ideal bioactive scaffold for dental tissue regeneration. In this study, we hypothesize that the cryopreservation could effectively maintain the survival and viability of dentinogenesis-related proteins of TDM and the cryopreserved dentin matrix (CDM) would provide the suitable biological scaffold and inductive microenvironment for the regeneration of dentin-pulp like tissue. CDM-3 and CDM-6 were prepared by cryopreserving TDM in liquid nitrogen (-196 °C) with cryoprotectant for 3 months and 6 months, respectively. Various biological characteristics of CDM, including mechanical properties, cell proliferation, and odontogenesis ability, were investigated. To further evaluate the inductive capacity of CDM, human dental follicle cells were encapsulated within CDM, and implanted the scaffold into a mouse model for 8 weeks, and the grafts were harvested and assessed histologically. The CDM showed superior mechanical properties than TDM. Compared to TDM, CDM can release more dentinogenesis-related proteins due to the larger pore diameter. Cell proliferation with the addition of CDM extract liquid was similar to that of TDM in the first five days. Human dental follicle cells, under the effect of CDM extract liquid, highly expressed bone sialoprotein, collagen-1, alkaline phosphatase, indicating that CDM, regarded as the inductive microenvironment, plays an important role in odontogenesis. Most importantly, in vivo, CDM could induce dental follicle cells to regenerate new dentin-pulp like tissues, such as dentinal tubules, predentin, collagen fibers, nerves, and blood vessels which were positive for dentin sialophosphoprotein, dental matrix protein-1, Tubulin, and collagen-1. In conclusion, CDM is an ideal biological scaffold material for human dentin-pulp like tissue regeneration. These findings indicated that TDM could be preserved as the tissue engineering scaffold that is readily available for patient treatments. Furthermore, the success of cryopreservation of TDM may also provide an insight into preserving other bioactive scaffold materials of tissue engineering.

TGF-ß1 and FGF2 stimulate the epithelial-mesenchymal transition of HERS cells through a MEK-dependent mechanism.

Hertwig's epithelial root sheath (HERS) cells participate in cementum formation through epithelial-mesenchymal transition (EMT). Previous studies have shown that transforming growth factor beta 1 (TGF-ß1) and fibroblast growth factor 2 (FGF2) are involved in inducing EMT. However, their involvement in HERS cell transition remains elusive. In this study, we confirmed that HERS cells underwent EMT during the formation of acellular cementum. We found that both TGF-ß1 and FGF2 stimulated the EMT of HERS cells. The TGF-ß1 regulated the differentiation of HERS cells into periodontal ligament fibroblast-like cells, and FGF2 directed the differentiation of HERS cells into cementoblast-like cells. Treatment with TGF-ß1 or FGF2 inhibitor could effectively suppress HERS cells differential transition. Combined stimulation with both TGF-ß1 and FGF-2 did not synergistically accelerate the EMT of HERS. Moreover, TGF-ß1/FGF2-mediated EMT of HERS cells was reversed by the MEK1/2 inhibitor U0126. These results suggest that TGF-ß1 and FGF2 induce the EMT of HERS through a MAPK/ERK-dependent signaling pathway. They also exert their different tendency of cellular differentiation during tooth root formation. This study further expands our knowledge of tooth root morphogenesis and provides more evidence for the use of alternative cell sources in clinical treatment of periodontal diseases.

Hertwig's epithelial root sheath cells regulate osteogenic differentiation of dental follicle cells through the Wnt pathway.

The development of periodontal ligament-cementum complex (PLCC) originates from the interaction between epithelial cells of Hertwig's epithelial root sheath (HERS) and mesenchymal cells of the dental follicle. While previous studies have suggested that the Wnt pathway is involved in osteogenic differentiation of dental follicle cells (DFCs) during tooth root development, its involvement in the interaction between DFCs and HERS cells (HERSCs) in tooth root mineralization remains unclear. Here, we investigated the hypothesis that HERSCs control osteogenic differentiation of DFCs via the Wnt pathway. We found that during co-culture with HERSCs, DFCs exhibited a greater tendency to form mineralized nodules. Moreover, under these conditions, DFCs expressed high levels of cementoblast/osteoblast differentiation-related markers, such as bone sialoprotein (BSP) and osteocalcin (OCN), the periodontal ligament phenotype-related gene type I collagen (COL1), and ß-catenin (CTNNB1), a core player in the canonical Wnt pathway. In contrast, expression in DFCs of alkaline phosphatase (ALP) was greatly decreased in the presence of HERSCs. Expression of CTNNB1 in DFCs was stimulated by Wnt3a, a representative canonical member of the Wnt family of ligands, but suppressed by Dickkopf1 (DKK1), a Wnt/CTNNB1 signaling inhibitor. Furthermore, in the presence of treated dentin matrix (TDM), differentiation of DFCs was enhanced by Wnt3a when they were in direct contact with HERSCs, but was curtailed by DKK1. Taken together, these results indicate that during tooth root formation, HERSCs induce osteogenic differentiation of DFCs in a process involving the Wnt pathway and the dentin matrix. Our study not only contributes to our understanding of tooth root development and diseases of tooth root mineralization, but also proffers a novel potential strategy for controlling mineralization during tooth root regeneration.