Kosuke Takahashi

Characteristics and osteogenic differentiation of stem/progenitor cells in the human dental follicle analyzed by gene expression profiling.

The dental follicle is an ectomesenchymal tissue that surrounds developing tooth germ and that contains osteoblastic-lineage-committed stem/progenitor cells. We examined the osteogenic potential of human dental follicle cells (hDFC) by microarray analysis. We first compared the characteristics of hDFC with those of human bone marrow mesenchymal stem cells (hMSC). Like hMSC, hDFC expressed stem cell markers such as STRO-1 and Notch-1 and differentiated not only into the osteoblastic lineage, but also into the adipogenic lineage. We analyzed the gene expression profiles of hDFC and hMSC that were not differentiated toward the osteogenic lineage. The expression of cell markers and growth factor receptors by hDFC and hMSC was similar, whereas the expression pattern of homeobox genes differed between hDFC and hMSC. Next, we investigated gene expression in hDFC during osteogenic differentiation. Gene expression profiles were analyzed in hDFC cultured in osteogenic induction medium (OIM) or in growth medium (GM) for 3 and 10 days. Many genes whose expression was regulated under these conditions were functionally categorized as “transcription” genes. Osteogenic markers were up-regulated in hDFC during osteogenic differentiation, whereas neurogenic markers were down-regulated. The genes whose expression was regulated in hDFC during osteogenic differentiation were further analyzed by ingenuity pathway analysis and real-time polymerase chain reaction. Bone morphogenetic protein and transforming growth factor-β signaling pathways were activated in hDFC cultured in OIM for 3 days. This study indicates that the dental follicle contains stem cells and/or osteoblastic progenitor cells and is a potential cellular resource for bone regeneration therapy.

Bone morphogenetic protein 6 stimulates mineralization in human dental follicle cells without dexamethasone

OBJECTIVE The aim of this study is to investigate the osteogenic differentiation human dental follicle cells (hDFCs) cultured with in osteogenic induction medium (OIM) without dexamethasone (DEX), and to analyze the gene expression profile during osteogenic differentiation. METHODS hDFCs, which isolated from dental follicle tissue from impacted third molar teeth, were cultured with OIM with or without DEX. Osteogenic differentiation of hDFCs was examined using Alkaline phosphatase activity and Arizarin red staining. Gene expression analysis was performed by Microarray and real time-PCR. RESULTS We showed that hDFCs have the capacity to differentiate into osteogenic lineages in osteogenic induction medium lacking DEX. We also analyzed gene expression profiling of hDFCs during osteogenic differentiation. BMP6 is up-regulated in both the presence and absence of DEX. In addition, BMP6 enhances gene expression levels of DLX-5, Runx2, and Osterix, which are transcription factors associated with osteogenic differentiation. BMP6 also stimulates phosphorylation of Smad1/5/8 which are transcription factors associated with BMP signalling at protein levels. Additionally BMP6 stimulates mineralization of hDFCs monolayers examined by Arizarin red S staining. CONCLUSION These findings suggest that hDFCs can differentiate to osteogenic lineage cells osteogenic induction medium without DEX, and BMP6 is a key gene in the osteogenic differentiation of hDFCs, and has therapeutic utility for bone regeneration and bone research.

Capacity of Human Dental Follicle Cells to Differentiate into Neural Cells In Vitro

The dental follicle is an ectomesenchymal tissue surrounding the developing tooth germ. Human dental follicle cells (hDFCs) have the capacity to commit to differentiation into multiple cell types. Here we investigated the capacity of hDFCs to differentiate into neural cells and the efficiency of a two-step strategy involving floating neurosphere-like bodies for neural differentiation. Undifferentiated hDFCs showed a spindle-like morphology and were positive for neural markers such as nestin, β-III-tubulin, and S100β. The cellular morphology of several cells was neuronal-like including branched dendrite-like processes and neurites. Next, hDFCs were used for neurosphere formation in serum-free medium containing basic fibroblast growth factor, epidermal growth factor, and B27 supplement. The number of cells with neuronal-like morphology and that were strongly positive for neural markers increased with sphere formation. Gene expression of neural markers also increased in hDFCs with sphere formation. Next, gene expression of neural markers was examined in hDFCs during neuronal differentiation after sphere formation. Expression of Musashi-1 and Musashi-2, MAP2, GFAP, MBP, and SOX10 was upregulated in hDFCs undergoing neuronal differentiation via neurospheres, whereas expression of nestin and β-III-tubulin was downregulated. In conclusion, hDFCs may be another optimal source of neural/glial cells for cell-based therapies to treat neurological diseases.

Plasma rich in growth factors stimulates proliferation, migration, and gene expression associated with bone formation in human dental follicle cells

Background/purpose Plasma rich in growth factors (PRGFs), which is prepared from autologous blood from patients, has been reported with regards to bone regeneration for dental implants. Human dental follicle cells (hDFCs) have the capacity to commit to multiple cell types such as the osteoblastic lineage. The aim of this study is to evaluate the effects of PRGFs for mineralization in hDFCs. Materials and methods PRGFs was prepared from whole blood centrifuged at 460g for 8 minutes. hDFCs isolated from the dental follicle with collagenase/dispase were cultured with growth medium or osteogenic induction medium (OIM) containing PRGFs or fetal bovine serum. Concentrations of the growth factors were examined using an enzyme-linked immunosorbent assay kit. A cell migration assay was used for two-dimensional movement. Gene expressions were examined with real-time polymerase chain reaction using a DyNAmo SYBR Green quantitative polymerase chain reaction kit. Results The platelet concentration in PRGF Fraction 2 was 2.14-fold higher than in whole blood. White blood cells were not detected in PRGFs. Transforming growth factor-β levels were higher than insulin-like growth factor-1, platelet-derived growth factor-AB and -BB, and vascular endothelial growth factors in PRGF Fraction 2. Proliferation and migration by hDFCs increased in OIM supplemented with PRGFs in a dose-dependent manner and were higher in hDFCs cultured in OIM plus 10% PRGFs compared with OIM plus 10% fetal bovine serum. PRGFs upregulated the gene expression of type I collagen, osteomodulin, alkaline phosphatase, bone morphogenic protein-4, and transforming growth factor-β in hDFCs. Conclusion PRGFs may promote bone regeneration due to it including high levels of growth factors.

Applicability of human dental follicle cells to bone regeneration without dexamethasone: an in vivo pilot study

The aim of this study was to investigate the capacity of human dental follicle cells (hDFCs) for bone formation in vivo. hDFCs were obtained from wisdom teeth extracted from patients aged 14 and 22 years. hDFCs from the 5th to 8th passages were grown in three-dimensional (3D) culture using gelatin sponges. Cells were transplanted onto the calvaria of F344/NJcl-rnu/rnu male rats (immunodeficient rats). Haematoxylin and eosin (HE) staining and immunohistochemistry were performed, and newly formed bone was evaluated by micro-computed tomography (micro-CT). HE staining showed newly formed bone in 3D culture. Immunohistochemistry showed bone morphogenetic protein 2 (BMP-2), runt-related transcription factor 2 (RUNX2), and osterix staining in areas with newly formed bone. Furthermore, micro-CT showed that, in comparison to controls, transplanted hDFCs promoted better bone quality and bone mineral density (BMD 582 ± 131.1 vs. 300.5 ± 77.7 mg/cm(3); P=0.039), bone mineral content (BMC 5.6 ± 1.1 vs. 2.1 ± 0.4 mg; P = 0.006), bone volume (BV 9.7 ± 0.5 × 10(-3) vs. 7.0 ± 0.4 × 10(-3) cm(3); P = 0.002), BMC/total volume (TV) (399.9 ± 76.3 vs. 147.7 ± 30.8 mg/cm(3); P = 0.006), and BV/TV (69.1 ± 3.6% vs. 49.6 ± 3.1%; P=0.002). This suggests that human dental follicles are potentially useful for regenerative therapy.

Peri-implant squamous cell carcinoma

Peri-implant squamous cell carcinoma is an uncommon pathological manifestation, whereas peri-implantitis is commonly found in association with dental implants. Both present similarly with loss of supporting soft and hard tissue around dental implants; therefore, a careful differential diagnosis is required. The present case concerns a 62-year-old Japanese man who had a dental implant which had been in the left maxillary incisor region for 4 years who apparently developed peri-implantitis. This did not respond to localized therapy and antibiotics so was referred for specialist surgical management. A biopsy confirmed it to be a squamous cell carcinoma rather than an inflammatory lesion. A literature review shows that this is an unusual presentation without a previous history of malignancy, mucosal disease or risk factors for cancers. Although rare, the possibility of peri-implant squamous cell carcinoma should be borne in mind by all practitioners who monitor implant patients.