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Background aims When cells are exposed to stresses such as mechanical stimuli, they release growth factors and adapt to the surrounding environment H ere, we demonstrated that mechanical stimulation during culture affects the production of osteogenic and angiogenic factors. Methods Human bone marrow derived mesenchymal stromal cells (hMSCs) and human periodontal ligament fibroblasts (HPLFs ) were cultured under cyclic stretch stimulation for 24 h. Collected of the cells and conditioned media (CM), the gene and protein expression levels of osteogenic and angiogenic factors were evaluated. CM was also evaluated for angiogenic activity and calc ification ability. In in vivo study, CM was administered to a mouse calvarial defect model and histologically and radiologically evaluated. Results Quantitative real time polymerase chain reaction results showed that the expression of bone morphogenetic pro tein 2, 4 (BMP 2, 4), vascular endothelial growth factor A (VEGF A), and platelet derived growth factor AA (PDGF AA) was upregulated in the cyclic stretch stimulation group in comparison with the non stretch group in each cell type. Enzyme linked immunosor bent assay results revealed that the expression of BMP 2,4, VEGF A was upregulated in the cyclic stretch group in comparison with the non stretch group in each cell type. Only HPLFs showed significant difference in PDGF AA expression between the cyclic str etch and the non stretch group. Tube formation assay and Alizarin Red S staining results showed that angiogenic activity and calcification ability of CM was upregulated in the cyclic stretch stimulation group in comparison with the non stretch group in eac h cell type. CM was administered to the mouse calvarial defect model. Histological and radiological examination showed that the bone healing was promoted by CM from the cyclic stretch culture group. Immunohistological staining revealed that CM from cyclic stretch group have greater angiogenic effect than CM from the non stretch group. Conclusions These results indicate that osteogenesis was promoted by CM obtained under cyclic stretch stimulation through the increase of angiogenesis in the mouse calvarial defect model.

Dentigerous cysts (DC) are the second most common odontogenic cysts of the jaw and are usually associated with the crowns of impacted or unerupted teeth. In particular, DCs tend to occur around unerupted mandibular third molars and maxillary mesiodens, which are supernumerary teeth. Such cases account for 5% of all DCs. However, DCs related to the supernumerary (fourth) molar distal to the mandibular third molar are uncommon, occurring in 0.04–0.175% of investigated populations. This article reports the case of a 43-year-old woman with a DC associated with a mandibular supernumerary (fourth) molar.

Guided bone regeneration (GBR) is an established treatment. However, the mechanisms of GBR are not fully understood. Recently, a GBR membrane was identified that acts as a passive barrier to regenerate bone via activation and migration of macrophages (Mps) and bone marrow stem cells (BMSCs). Atmospheric pressure plasma treatment of the titanium membrane (APP-Ti) activated macrophages. The purpose of this study was to analyze whether macrophages attached to an APP-Ti membrane affected differentiation of BMSCs in a GBR model. Human THP-1 macrophages (hMps) were cultured on non-treated Ti (N-Ti) and APP-Ti membrane. Macrophage polarization was analyzed by RT-PCR and immunocytochemistry. Secreted proteins from hMps on N-Ti and APP-Ti were detected by LC/MS/MS. hBMSCs were co-cultured with hMps on N-Ti or APP-Ti and analyzed by osteogenic differentiation, Alizarin red S staining, and alkaline phosphatase (ALP) activity. N-Ti and APP-Ti membrane were also implanted into bone defects of rat calvaria. hMps on APP-Ti were polarized M2-like macrophages. hMps on N-Ti secreted plasminogen activator inhibitor-1 and syndecan-2, but hMps on APP-Ti did not. hBMSCs co-cultured with hMps on APP-Ti increased cell migration and gene expression of osteogenic markers, but suppressed mineralization, while ALP activity was similar to that of hMps on N-Ti in vitro. The volume of newly formed bone was not significantly different between N-Ti and APP-Ti membrane in vivo. M2 polarized hMps on APP-Ti suppressed osteogenic induction of hBMSCs in vitro. The indirect role of hMps on APP-Ti in newly formed bone was limited.

Successful osseointegration is essential for dental implants. However, the complete molecular mechanism of osseointegration remains to be elucidated. In this study, we focused on the proteoglycan (PG)-rich layer between titanium oxides (TiOx) and bone, and chondroitin-4-sulfate transferase-1 (C4ST-1), which forms the sugar chain in PGs. Human bone marrow mesenchymal stem cells (hBMSCs) depleted of C4ST-1 were cultured on titanium (Ti) plates, and the interface between hBMSCs and TiOx was analyzed using transmission electron microscopy. Immunotolerance, proliferation, initial adhesion, and calcification of the cells were analyzed in vitro. At 14 days of cultivation, a PG-rich layer was observed between hBMSCs and TiOx. However, the PG-rich layer was reduced in C4ST-1-depleted hBMSCs on TiOx. Real-time RT-PCR showed that conditioned media increased the levels of expression of M1-macrophage markers in human macrophages. However, depletion of C4ST-1 did not affect calcification, cell proliferation, or initial cell adhesion on Ti plates. These results suggested that C4ST-1 in hBMSCs affects their immunotolerance and alters the formation of PG-rich layer formation on TiOx.

Tolosa-Hunt syndrome (THS) is a type of painful ophthalmoplegia characterized by unilateral orbital pain and oculomotor paresis. This report describes a case of painful ophthalmoplegia considered to be THS. The patient was a 72-year-old woman. After dental implant placement in the left maxillary first molar site, she was referred to our hospital for persistent left periorbital and facial pain, diplopia, and taste disorder. With an initial diagnosis of trigeminal neuralgia, diplopia, and taste disorder, we began administration of carbamazepine and polaprezinc, and asked the department of ophthalmology in our hospital to examine her diplopia. With the increase in serum zinc levels, the taste disorder resolved, but her periorbital pain and diplopia were aggravated, and she had to be hospitalized. As a result of extensive examinations, we suspected her painful ophthalmoplegia was due to cavernous sinus abnormalities; finally, THS was diagnosed. There was significant improvement in the periorbital pain within 48 h after the start of corticosteroid therapy. Since then, she has never suffered from painful ophthalmoplegia. This case indicates that careful evaluation and accurate diagnosis are critical in cases of painful ophthalmoplegia, and the possibility of THS should always be kept in mind.

Shuhei Tsuchiya,1 Keisuke Sugimoto,2 Hisanobu Kamio,2 Kazuto Okabe,1 Kensuke Kuroda,3 Masazumi Okido,3 Hideharu Hibi2 1Department of Oral and Maxillofacial Surgery, Nagoya University Hospital, Nagoya, Japan; 2Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine, Nagoya, Japan; 3Institute of Materials and Systems for Sustainability, Nagoya University, Nagoya, Japan Background: Surface modification of titanium dioxide (TiO2) implants promotes bone formation and shortens the osseointegration period. Kaempferol is a flavonoid that has the capacity to promote osteogenic differentiation in bone marrow stromal cells. The aim of this study was to promote bone formation around kaempferol immobilized on TiO2 implants. Methods: There were four experimental groups. Alkali-treated TiO2 samples (implants and discs) were used as a control and immersed in Dulbecco’s phosphate-buffered saline (DPBS) (Al-Ti). For the coprecipitation sample (Al-cK), the control samples were immersed in DPBS containing 50µg kaempferol/100% ethanol. For the adsorption sample (Al-aK), 50µg kaempferol/100% ethanol was dropped onto control samples. The surface topography of the TiO2 implants was observed by scanning electron microscopy with energy-dispersive X-ray spectroscopy, and a release assay was performed. For in vitro experiments, rat bone marrow stromal cells (rBMSCs) were cultured on each of the TiO2 samples to analyze cell proliferation, alkaline phosphatase activity, calcium deposition, and osteogenic differentiation. For in vivo experiments, TiO2 implants placed on rat femur bones were analyzed for bone–implant contact by histological methods. Results: Kaempferol was detected on the surface of Al-cK and Al-aK. The results of the in vitro study showed that rBMSCs cultured on Al-cK and Al-aK promoted alkaline phosphatase activity, calcium deposition, and osteogenic differentiation. The in vivo histological analysis revealed that Al-cK and Al-aK stimulated new bone formation around implants. Conclusion: TiO2 implant-immobilized kaempferol may be an effective tool for bone regeneration around dental implants. Keywords: kaempferol, titanium implant, surface treatment, biomaterial

Osseointegration can be defined as a direct connection, both structural and functional, between living bone and the surface of an artificial implant. Indeed, the word comes from the Greek term for 'bone' and 'to make whole'. In dentistry, once dental implants are placed, the body will react with osseointegration, enabling the implants to become a permanent part of the jaw. There are many benefits to this type of implant, compared with traditional tooth replacement options, not least that dental implants mimic the strength and functionality of a natural tooth. Dr Shuhei Tsuchiya is a researcher based in the Division of Oral and Maxillofacial Surgery at Nagoya University, Japan, who is interested in a range of areas, including regenerative medicine and the extracellular matrix. One of his key preoccupations, though, is shedding light on osseointegration. He and his team are working to unravel the mysteries of the mechanism.

Osteoradionecrosis of the jaws (ORNJ) is a well-known complication of head and neck radiotherapy that may severely impair patient quality of life. Not only dentoalveolar surgery but also root canal treatment may also damage the alveolar bone. This report describes a case of ORNJ in the maxilla following treatment for periapical periodontitis. The patient was treated with chemotherapy and radiotherapy consisting of the course of cisplatin and etoposide, and a total dose of 60 Gy/30Fr for small cell carcinoma of the right-maxillary gingiva. In April 2015, after irradiation therapy, he received root canal treatment in the right-maxillary first premolar because of periapical periodontitis. Six months after root canal treatment, he had developed ORNJ in the right maxilla with acute inflammation, and was thus treated with antibiotics. In April 2016, after treatment of ORNJ, T1- and T2-weighted magnetic resonance images showed a hypointense area surrounding the right-maxillary first premolar. These findings suggest that root canal treatment for dental pulp necrosis may be triggered to cause the ORNJ.

Osseointegration is the structural and functional connection between bone tissues and implants such as titanium dioxide (TiO2). The bone-TiO2 interface is thought to contain proteoglycans. However, exhaustive analysis of the proteins in this layer has not been performed. In this study, we evaluated the bone protein adhered on the surface of TiO2 comprehensively. Pig bone protein was extracted by sequential elutions with guanidine, 0.1 M EDTA, and again with guanidine. The proteins obtained from these extractions were allowed to adhere to an HPLC column packed with TiO2 and were eluted with 0.2 M NaOH. The eluted proteins were identified by LC/MS/MS and included not only proteoglycans but also other proteins such as extracellular matrix proteins, enzymes, and growth factors. Calcium depositions were observed on TiO2 particles incubated with bone proteins, guanidine-extracted proteins adhered to TiO2 displayed significantly high amounts of calcium depositions., Highlights • We identified bone proteins adhered on the surface of TiO2. • Chromatography was an useful tool for investigating the layer adhered on the TiO2. • The bone-TiO2 interface contained not only proteoglycans but also other proteins. • Some of the adhered proteins showed mineralization capacity.

Suppurative osteomyelitis of the jaw is known to usually present in patients with decreased immunity, and molecular targeting drugs used for immunosuppressive treatment in autoimmune disorders have the side effect of making the patient an immunocompromised host. Infliximab is a molecular targeting drug that is used to treat Crohn's disease, rheumatoid arthritis, and Behcet's disease. In patients receiving infliximab, the oral cavity may act as a bacterial reservoir leading to unwanted local or systemic complications. This report describes a case of suppurative osteomyelitis derived from periapical periodontitis in a patient who was receiving treatment with infliximab. This patient was referred by the otolaryngology department and consulted us for an infection focus in submandibular region derived from oral cavity. Suppurative osteomyelitis was diagnosed on the basis of clinical and radiologic examinations and the patient was treated with antibacterial agents for acute inflammation. After the acute stage, the left mandibular second molar that was the infection focus was extracted. The extracted socket followed a normal healing process without bone exposure. Computed tomography (CT) and magnetic resonance imaging (MRI) examinations were performed at 6 and 18 months after tooth extraction. CT images showed remnant osteosclerosis around the tooth socket until 18 months after tooth extraction, while T1- and T2-weighted MRI images showed a hypointense area surrounding the tooth socket. These findings suggested that the suppurative osteomyelitis could be attributed to the immunosuppressive activity of the molecular targeting drugs and acute periapical periodontitis, such as typical and classical suppurative osteomyelitis.

Distraction osteogenesis (DO) is a surgical procedure that involves skeletal tissue regeneration without cell transplantation. A DO model consists of the following three phases: the latency phase after osteotomy and placement of the external distractor; the distraction phase, wherein the separated bone ends are gradually and continuously distracted; and the consolidation phase. This custom-made distractor used for DO is comprised of two incomplete acrylic resin rings and an expansion screw. The process was initiated by making a mold with silicone impression material and then creating the custom-made distractor. Dental resin was poured into the formwork made of silicone impression material, and it was allowed to polymerize to create the incomplete resin rings required for the custom-made distractor. These rings were fixed with an expansion screw using transparent resin. The custom-made distractor created via this approach was attached to the tibia of mice. The tibia was fixed to the device using one pair of 25 G needles proximally, one pair of 27 G needles distally, and acrylic resin. After a latency period of 5 days, distraction was initiated at a rate of 0.2 mm/12 h. The lengthening was continued for 8 days, resulting in a total gap of 3.2 mm. The mice were sacrificed 4 weeks after distraction. Bone formation in the distraction gap was confirmed using both radiography and histology.

Maxillomandibular bone defects arise from maxillofacial injury or tumor/cyst removal. While the standard therapy for bone regeneration is transplantation with autologous bone or artificial bone, these therapies are still unsatisfactory. Autologous bone harvesting is invasive and occasionally absorbed at the implanted site. The artificial bone takes a long time to ossify and it often gets infected. Therefore, we have focused on regenerative therapy consisting of autologous bone marrow-derived mesenchymal cells (BM-MSCs), which decreases the burden on patients. Based on our previous research in patients with maxillomandibular bone defects or alveolar bone atrophy using a mixture of BM-MSCs, platelet-rich plasma (PRP), thrombin, and calcium, we confirmed the efficacy and acceptable safety profile of this treatment. In this investigator-initiated clinical study (the TEOM study), we intended to add β-tricalcium phosphate (β-TCP) owing to large defect with patients. The TEOM study aimed to evaluate the efficacy and safety of bone regeneration using mixtures of BM-MSCs in patients with bone defects resulting from maxillofacial injury, and tumor/cyst removal in the maxillomandibular region. The TEOM study is an open-label, single-center, randomized controlled study involving a total of 83 segments by the Federation Dentaire Internationale numbering system in maxillomandibular bone defects that comprise over 1/3 of the maxillomandibular area with a remaining bone height of ≤10 mm. The primary endpoint is rate of procedure sites with successful bone regeneration defined as a computed tomography (CT) value of more than 400 and a bone height of more than 10 mm. Our specific hypothesis is that the number of required regions was calculated assuming that the rate of procedure sites with successful bone regeneration is similar and the non-inferiority margin is 15.0%. The TEOM study is the first randomized controlled study of regenerative treatment using BM-MSCs for large maxillomandibular bone defects. We will evaluate the efficacy and safety in this study to provide an exploratory basis for the necessity of BM-MSCs for these patients. This trial was registered at the University Hospital Medical information Network Clinical Trials Registry (UMIN-CTR Unique ID: UMIN000020398; Registration Date: Jan 15, 2016; URL: https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000016543 ).

Purpose: To investigate whether bone regeneration can be accelerated by using a conditioned medium (CM) and guided bone regeneration (GBR) technique. Materials and Methods: CM was harvested from rat bone marrow stromal cells (BMSCs). The components of CM were immobilized using a polylactide-co-glycolide (PLGA) membrane treated with and without 0.5 mol/L sodium hydroxide (NaOH) to elevate the hydrophilicity. Four experimental groups were prepared: PLGA membrane treated with (1) phosphate-buffered saline (PBS; PBS-M), (2) PBS and 0.5 mol/L NaOH (hydrophilic treatment; PBS-HM), (3) CM (CM-M), and (4) CM and 0.5 mol/L NaOH (CM-HM). These experimental membranes were observed using scanning electron microscopy. Proteins derived from BMSCs immobilized on the PLGA membrane were detected with liquid chromatography–tandem mass spectrometry (LC/MS/MS). Cell proliferation and alkaline phosphatase (ALP) activity were measured to analyze the effect of CM on the BMSCs. Experimental membranes were transplanted into a rat calvarial bone defect model. Microcomputed tomography and histologic analyses were performed 4 and 8 weeks after transplantation. Results: The CM derived from BMSCs can be immobilized on the PLGA membrane. Hydrophilic treatment of the PLGA membrane enhanced the amount of CM immobilization. LC/MS/MS analysis showed that the immobilized proteins on the surface of PLGA membrane were extracellular matrix, such as collagen, decorin, and fibronectin. The proteins in the CM, which were released from the PLGA membrane, enhanced cell proliferation and ALP activity in rat BMSCs. Newly formed bone area at the bone defects that had been treated with CM-HM was significantly high compared with those at bone defects treated with the other membranes. Conclusion: The PLGA membrane treated with 0.5 mol/L NaOH and CM promoted bone regeneration in this rat calvarial defect model.

PURPOSE To enhance the stability of titanium (Ti) implants using conditioned medium (CM) derived from rat bone marrow stromal cell (BMSC). MATERIALS AND METHODS BMSCs were isolated from rat femurs and grown in culture, and the culture medium was used as CM. The CM was immobilized on the surface of Ti implants with calcifying solution. The topology of the Ti implants after immobilization of CM was observed by scanning electron microscopy (SEM). The Ti-immobilized CM was analyzed by liquid chromatography with tandem mass spectrometry. The adhesiveness and the osteogenic differentiation of BMSCs grown on CM-coated discs were analyzed by reverse-transcription polymerase chain reaction. Ti implants with specimen-immobilized CM labeled with quantum dots (QDs) were placed into rat femurs. The localization of the CM was detected by in vivo imaging at 1, 7, 14, and 28 days after implantation. The removal torque test and histologic bone implant contact (BIC) were also analyzed. RESULTS Rat BMSC-CM was successfully immobilized on Ti implants. The immobilized CM contained about 2000 proteins, including collagen type I, bone sialoprotein, fibronectin, and vascular endothelial growth factor that are important in new bone formation. CM promoted cell adhesion and osteocalcin gene expression of rat BMSCs. The labeled CM remained associated with the Ti implant at 1, 7, 14, and 28 days postimplantation. The removal torque value and BIC of Ti implants with immobilized CM were higher than those of control implants on days 1, 7, and 14 after implantation. CONCLUSION Immobilized CM components on the surface of Ti implants promoted integration into bone during an early stage.

Dentin sialophosphoprotein (Dspp) is critical for proper dentin biomineralization because genetic defects in DSPP cause dentin dysplasia type II and dentinogenesis imperfecta types II and III. Dspp is processed by proteases into smaller subunits; the initial cleavage releases dentin phosphoprotein (Dpp). We incubated fluorescence resonance energy transfer (FRET) peptides containing the amino acid context of the Dpp cleavage site (YEFDGKSMQGDDPN, designated Dspp-FRET) or a mutant version of that context (YEFDGKSIEGDDPN, designated mutDspp-FRET) with BMP-1, MEP1A, MEP1B, MMP-2, MMP-8, MMP-9, MT1-MMP, MT3-MMP, Klk4, MMP-20, plasmin, or porcine Dpp and characterized the peptide cleavage products. Only BMP-1, MEP1A, and MEP1B cleaved Dspp-FRET at the G–D peptide bond that releases Dpp from Dspp in vivo. We isolated Dspp proteoglycan from dentin power and incubated it with the three enzymes that cleaved Dspp-FRET at the G–D bond. In each case, the released Dpp domain was isolated, and its N-terminus was characterized by Edman degradation. BMP-1 and MEP1A both cleaved native Dspp at the correct site to generate Dpp, making both these enzymes prime candidates for the protease that cleaves Dspp in vivo. MEP1B was able to degrade Dpp when the Dpp was at sufficiently high concentration to deplete free calcium ion concentration. Immunohistochemistry of developing porcine molars demonstrated that astacins are expressed by odontoblasts, a result that is consistent with RT-PCR analyses. We conclude that during odontogenesis, astacins in the predentin matrix cleave Dspp before the DDPN sequence at the N-terminus of Dpp to release Dpp from the parent Dspp protein. © 2011 American Society for Bone and Mineral Research.

Summary Since cells isolated from the developing tooth possess an enormous regenerative potential, it has been proposed that they could be applied to the in vivo regeneration of teeth in dental practice. The generation of the entire tooth structure depends upon the developmental stage of tooth when the cells are harvested. This review focuses on the performance of postnatal and adult dental cells that have been used for generating teeth. Their ability to contribute to tooth development was assessed in the omentum or in the tooth socket. Adult dental cells were limited in their potential owing to various parameters. From these results described, new approaches for regenerated teeth are proposed in this review. One strategy to replace teeth is tooth root engineering using tissue from postnatal teeth. Since the enamel organ epithelium disappears after tooth maturation, the epithelial rest cells of Malassez were evaluated to determine their capacity to generate enamel. From these results, it is suggested that erupted mature teeth have cell sources with the capacity to produce tooth root. The development of biological approaches for tooth root regeneration using postnatal dental cells is promising and remains one of the greatest challenges in the dental field in the years to come.

This study examined the effect of extracellular matrix (ECM) on the osteogenic differentiation capacity and osteogenesis of dental follicle cells. Single cell-derived porcine dental follicle cells (DFC-I) obtained at the early stage of crown formation in tooth were subcultured and characterized using periodontal ligament cells (PDLC) and bone marrow-derived mesenchymal stem cells (BMSC) as comparison cell populations. The effect of ECM constituents including collagen type I, fibronectin, laminin, and collagen type IV on the differentiation of DFC-1 into osteogenic-lineage cells was evaluated in vitro. In addition, the DFC-1, PDLC, and BMSC populations were compared for osteogenic capacity in vitro by Alizarin red staining and in vivo by transplantation. DFC-I showed different features from PDLC and BMSC. Different components of ECM had different effects on the differentiation of DFC-1 into osteogenic-lineage cells in vitro. Alkaline phosphatase activity and matrix mineralization as early- and late-stage markers of osteogenesis, respectively, supported the differentiation of DFC-1 into osteogenic-related cells in vitro. All three cell types showed equivalent osteogenic capacity in vivo at 4 weeks postoperatively. There were no statistically significant differences among the cell populations with respect to capacity for bone formation. These results suggest a potential application for dental follicle cells in bone-tissue engineering.

Objectives Bacterial metabolites demineralize dental hard tissues, and soluble factors lead to tertiary dentinogenesis in the area of the dentin-pulp complex. However, it is unclear whether the oral bacteria are directly involved in the differentiation of dental pulp cells. In this study, we evaluated the effect of oral bacterial extracts on cellular differentiation in human dental pulp–derived cells (hDPC). Study design The hDPC were obtained from third molar teeth, and the cells were subcultured. The sonicated extracts were obtained from Porphyromonas gingivalis (gram-negative) and Streptococcus mutans (gram-positive). The effect of bacterial extracts on cellular growth and differentiation in hDPC were tested. Results Alkaline phosphatase activity and bone sialoprotein (BSP) gene expression were increased in hDPC exposed to low concentrations of both sonicated extracts, whereas the activity was decreased upon exposure to high concentrations of sonicated extracts from P. gingivalis . Conclusion This is the first evidence that oral bacteria have a positive effect on cellular differentiation in hPDC.

Mmp-20 and Klk4 are the two key enamel proteases. Can both enzymes process amelogenin to generate the major cleavage products that accumulate during the secretory stage of amelogenesis? We isolated Mmp-20 and Klk4 from developing pig teeth and used them to digest the tyrosine-rich amelogenin polypeptide (TRAP), the leucine-rich amelogenin protein (LRAP), and 5 fluorescence peptides. We characterized the digestion products by LC-MSMS, SDS-PAGE, and C18 RP-HPLC monitored with fluorescence and UV detectors. Mmp-20 cleaves amelogenin sequences after Pro162, Ser148, His62, Ala63, and Trp45. These cleavages generate all of the major cleavage products that accumulate in porcine secretory-stage enamel: the 23-kDa, 20-kDa, 13-kDa, 11-kDa, and 6-kDa (TRAP) amelogenins. Mmp-20 cleaves LRAP after Pro45 and Pro40, producing the two LRAP products previously identified in tooth extracts. Among these key cleavage sites, Klk4 was able to cleave only after His62. We propose that Mmp-20 alone processes amelogenin during the secretory stage.

Epithelial cell rests of Malassez (ERM) are quiescent epithelial remnants of Hertwig's epithelial root sheath (HERS) that are involved in the formation of tooth roots. After completion of crown formation, HERS are converted from cervical loop cells, which have the potential to generate enamel for tooth crown formation. Cervical loop cells have the potential to differentiate into ameloblasts. Generally, no new ameloblasts can be generated from HERS, however this study demonstrated that subcultured ERM can differentiate into ameloblast-like cells and generate enamel-like tissues in combination with dental pulp cells at the crown formation stage. Porcine ERM were obtained from periodontal ligament tissue by explant culture and were subcultured with non-serum medium. Thereafter, subcultured ERM were expanded on 3T3-J2 feeder cell layers until the tenth passage. The in vitro mRNA expression pattern of the subcultured ERM after four passages was found to be different from that of enamel organ epithelial cells and oral gingival epithelial cells after the fourth passage using the same expansion technique. When subcultured ERM were combined with subcultured dental pulp cells, ERM expressed cytokeratin14 and amelogenin proteins in vitro. In addition, subcultured ERM combined with primary dental pulp cells seeded onto scaffolds showed enamel-like tissues at 8 weeks post-transplantation. Moreover, positive staining for amelogenin was observed in the enamel-like tissues, indicating the presence of well-developed ameloblasts in the implants. These results suggest that ERM can differentiate into ameloblast-like cells.

Aim To investigate the presence of side population (SP) cells by the Hoechst exclusion method in human adult dental pulp tissue. Methodology Human adult dental pulp-derived cells were generated from third molar teeth. The cells were stained with Hoechst 33342 and sorted into SP cells or non-SP cells [main population (MP) cells]. Both cell types were compared with cell growth and RT–PCR analyses. Results SP cells that express ABCG2, Nestin, Notch-1 and α-smooth muscle actin were found at frequencies ranging from 0.67% to 1.02%. This SP profile disappeared in the presence of verapamil. These SP cells expressed dentine sialophosphoprotein and dentine matrix protein-1 when cultured in osteogenic medium. Conclusion Human adult dental pulp tissue contains SP cells that differentiate into odontoblast-like cells.

Introduction Surface modification of titanium (Ti) implants promotes bone formation and shortens the osseointegration period. The aim of this study was to promote bone regeneration and stability around implants using atmospheric pressure plasma (APP) pretreatment. This was followed by immobilization of stem cells from human exfoliated deciduous teeth-conditioned medium (SHED-CM) on the Ti implant surface. Methods Ti samples (implants, discs, powder) were treated with APP for 30 seconds. Subsequently, these were immobilized on the treated Ti surface, soaked and agitated in phosphate-buffered saline or SHED-CM for 24 hours at 37 °C. The surface topography of the Ti implants was observed using scanning electron microscopy with energy dispersive X-ray spectroscopy. In vivo experiments using Ti implants placed on canine femur bone were then conducted to permit histological analysis at the bone-implant boundary. For the in vitro experiments, protein assays (SDS-PAGE, Bradford assay, liquid chromatography-ion trap mass spectrometry) and canine bone marrow stromal cell (cBMSC) attachment assays were performed using Ti discs or powder. Results In the in vitro study, treatment of Ti implant surfaces with SHED-CM led to calcium phosphate and extracellular matrix protein immobilization. APP pretreatment increased the amount of SHED-CM immobilized on Ti powder, and contributed to increased cBMSC attachment on Ti discs. In the in vivo study, histological analysis revealed that the Ti implants treated with APP and SHED-CM stimulated new bone formation around implants. Conclusions Implant device APP pretreatment followed by SHED-CM immobilization may be an effective application to facilitate bone regeneration around dental implants.

Tooth structure can be regenerated by seeding dissociated tooth cells onto polyglycolic acid fiber mesh, although the success rate of tooth production is low. The present study was designed to compare the performance of collagen sponge with polyglycolic acid fiber mesh as a 3-D scaffold for tooth-tissue engineering. Porcine third molar teeth at the early stage of crown formation were enzymatically dissociated into single cells, and the heterogeneous cells were seeded onto collagen sponge or the polyglycolic acid fiber mesh scaffolds. Scaffolds were then cultured to evaluate cell adhesion and ALP activity in vitro. An in vivo analysis was performed by implanting the constructs into the omentum of immunocompromised rats and evaluating tooth production up to 25 weeks. After 24h, there were a significantly higher number of cells attached to the collagen sponge scaffold than the polyglycolic acid fiber mesh scaffold. Similarly, the ALP activity was significantly higher for the collagen sponge scaffold was than the polyglycolic acid fiber mesh scaffold after 7 days of culture. The area of calcified tissue formed in the collagen sponge scaffold was also larger than in the polyglycolic acid fiber mesh scaffold. The results from in vivo experiments show conclusively that a collagen sponge scaffold allows tooth production with a higher degree of success than polyglycolic acid fiber mesh. Taken together, the results from this study show that collagen sponge scaffold is superior to the polyglycolic acid fiber mesh scaffold for tooth-tissue engineering.

An investigation on effects of a focused laser pulse for combustion enhancement characteristics was conducted for supersonic airstreams with a transverse hydrogen-fuel injection. In order to estimate behaviors of a laser-induced plasma, temporal variations of its emission distribution were measured with an ICCD camera and photodiodes. From these measurements, characteristic time scales of an absorption process of an incident laser pulse and a plasma formation process were also estimated. Numerical simulations using a time-dependant Navier–Stokes equation with finite-rate chemistry were also conducted to elucidate laser-induced ignition and mixing enhancement characteristics in supersonic airstreams with a transverse hydrogen injection. From the results, it was confirmed that, depending on the laser energy density and focal points, radicals, a shock wave, and shock-induced local turbulences were being formed through laser irradiation. Then, recirculation zones with flamelets were induced. Moreover, it was shown that these flamelets were growing in moving upstream and downstream from the focal point up to 1 ms. Consequently, it was shown that laser-induced plasmas could be effective in both combustion reaction and mixing enhancements for the supersonic combustion.

Cell scaffold-based tooth engineering research was started by 2000 at Forsyth Institute corroborated with Dr. Vacanti's team at Massachusetts General Hospital. The first work was published in 2002 in Journal of Dental Research, in which we particularly focused on cells from postnatal tooth because of its clinical application. However, making a functional tooth from postnatal cells is still ways away. Alternatively, we formulated a partial replacement of the tooth by engineering the root of the tooth. Here, we describe a new technique in which the root of the third molar is used to replace missing teeth.

The aim of this paper was to determine whether the interaction between IGF, IGFBP, and VN modulates the functions of porcine EOE cells. Enamel organs from 6-month-old porcine third molars were dissociated into single epithelial cells and subcultured on culture dishes pretreated with VN, IGF-I, and IGFBP-3 (IGF-IGFBP-VN complex). The subcultured EOE cells retained their capacity for ameloblast-related gene expression, as shown by semiquantitative reverse transcription-polymerase chain reaction. Amelogenin expression was detected in the subcultured EOE cells by immunostaining. The subcultured EOE cells were then seeded onto collagen sponge scaffolds in combination with fresh dental mesenchymal cells and transplanted into athymic rats. After 4 weeks, enamel-dentin-like complex structures were present in the implanted constructs. These results show that EOE cells cultured on IGF-IGFBP-VN complex differentiated into ameloblasts-like cells that were able to secrete amelogenin proteins and form enamel-like tissuesin vivo. Functional assays demonstrated that the IGF/IGFBP/VN complex significantly enhanced porcine EOE cell proliferation and tissue forming capacity for enamel. This is the first study to demonstrate a functional role of the IGF-IGFBP-VN complex in EOE cells. This application of the subculturing technique provides a foundation for further tooth-tissue engineering and for improving our understanding of ameloblast biology.

Cell scaffold-based tooth engineering research was started by 2000 at Forsyth Institute corroborated with Dr. Vacanti's team at Massachusetts General Hospital. The first work was published in 2002 in Journal of Dental Research, in which we particularly focused on cells from postnatal tooth because of its clinical application. However, making a functional tooth from postnatal cells is still ways away. Alternatively, we formulated a partial replacement of the tooth by engineering the root of the tooth. Here, we describe a new technique in which the root of the third molar is used to replace missing teeth.

Purpose : This study is to evaluate the bone quality of surrounding areas of implants with bone marrow stromal cells (BMSCs) transplantation to rat femur, which have become osteoporosis-induced models. Materials and methods : The Sprague-Dawley rats were divided into 3 groups: the first group where their ovaries were removed (OVX group), the second group where a sham surgery was given (SHAM group), and the third group where BMSCs were transplanted to an OVX group (OVX-BMSCs group). In the OVX-BMSCs group, 1 × 10 BMSCs were transplanted into femur with implant. Each value of the bone to implant contact and the bone area of each cortical bone and cancellous bone was obtained. Bone density of the width of 500 μm from the implants was measured. Results : Each ratio of bone to implant contact, bone area, and bone density in the OVX-BMSCs group was significantly higher than those of OVX group as to the cancellous bone. Conclusion : The BMSCs transplantation therapy improved local bone healing in the cancellous bone surrounding implants and also significantly improved bone binding with implants.

Adult stem cells are multipotent and can be induced experimentally to differentiate into various cell lineages. Such cells are therefore a key part of achieving the promise of tissue regeneration. The most studied stem cells are those of the hematopoietic and mesenchymal lineages. Recently, mesenchymal stem cells were demonstrated in dental tissues, including dental pulp, periodontal ligament, and dental follicle. The dental follicle is a loose connective tissue that surrounds the developing tooth. Dental follicle stem cells could therefore be a cell source for mesenchymal stem cells. Indeed, dental follicle is present in impacted teeth, which are commonly extracted and disposed of as medical waste in dental practice. Dental follicle stem cells can be isolated and grown under defined tissue culture conditions, and recent characterization of these stem cells has increased their potential for use in tissue engineering applications, including periodontal and bone regeneration. This review describes current knowledge and recent developments in dental follicle stem cells and their application.

Objective Stem cells isolated from human dental follicles as a potential cell source for bone-tissue engineering were examined for correcting a critical bone defect. Study design Impacted third molars were collected and single cell–derived cell populations were cultivated in growth medium. Single cell–derived cell lines were examined in terms of cell shape, gene expression patterns, differentiation capacity in vitro, and osteogenic potential in vivo. Results Three distinct cell populations were identified with different morphologies, patterns of gene expression, and differentiation capacity. All 3 cell populations promoted bone formation when transplanted into surgically created critical-size defects in imunodeficient rat calvaria, compared with control animals without cell transplantation, although one of these populations showed a weak capacity for osteogenetic differentiation in vitro. Conclusions Human dental follicle can derive at least 3 unique cell populations in culture, all of which promote bone formation in vivo.

This study investigated the characteristics of two dental pulp cell populations during the early stages of crown formation in porcine teeth. A transplantation method was developed to reproduce epithelial cell-mesenchymal cell interactions during odontogenesis (tooth development). The technique allowed two types of cells/tissue to be combined in vivo. Populations of cells localized in the cervical loop epithelium region, dental pulp horn, and dental pulp core chambers were isolated and dissociated into single cells. Each population was examined for its gene-expression pattern using both semiquantitative and quantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses, and for its tissue-formation capability by combining the cervical loop epithelial cells with either pulp horn cells or pulp core cells on biodegradable collagen scaffolds that were subsequently examined using histology and immunohistology. Gene-expression patterns showed that pulp horn cells were more mature than pulp core cells. Cervical loop epithelial cells combined with pulp horn cells mainly reconstituted dentin-cementum structures. By contrast, cervical loop epithelial cells combined with pulp core cells reconstituted enamel-dentin structures. These results suggest that mesenchymal cells residing in a specific location of the pulp possess a specific tissue-formation potential when combined with epithelial cells.

We investigated porcine dental follicle cells at the early crown-formation stage and examined the behavior of cells grown in a collagen type I (Col-I) matrix. Clone-porcine dental follicle cells (DFC-I) and controls, viz., dental follicle itself, nonclone-dental follicle cells, periodontal ligament cells (PDLC), and bone marrow stromal cells, were obtained from 6-month-old pigs. DFC-I showed a different gene expression pattern from controls by reverse-transcription polymerase chain reaction analysis. In addition, Col-I treatment enhanced DFC-I proliferation and increased their alkaline phosphatase activity compared with nontreated DFC-I. The expression of periostin, biglycan, and osteocalcin (OCN) in cells growing on collagen was upregulated, similar to the pattern seen in PDLC. DFC-I with and without Col-I treatment were combined with beta-tricalcium phosphate particles and implanted into immunodeficient mice. Significant differences were found in the gene expression patterns of bone sialoprotein, OCN, and periostin in both treated and non-treated implants at 2 and/or 4 weeks. The results showed that Col-I induced the mineralization pathway in these cells. Hard tissue formation was observed in both implant types at 8 weeks. Our results suggest that Col-I facilitates the differentiation of DFC-I along the mineralization process.

Progress is being made toward regenerating teeth by seeding dissociated postnatal odontogenic cells onto scaffolds and implanting them in vivo, but tooth morphology remains difficult to control. In this study, we aimed to facilitate tooth regeneration using a novel technique to sequentially seed epithelial cells and mesenchymal cells so that they formed appropriate interactions in the scaffold. Dental epithelium and mesenchyme from porcine third molar teeth were enzymatically separated and dissociated into single cells. Mesenchymal cells were seeded onto the surface of the scaffold and epithelial cells were then plated on top so that the two cell types were in direct contact. The cell-scaffold constructs were evaluated in vitro and also implanted into immunocompromised rats for in vivo analysis. Control groups included constructs where direct contact between the two cell types was prevented. In scaffolds seed using the novel technique, alkaline phosphatase activity was significantly greater than controls, the tooth morphology in vivo was developed in similar to that of natural tooth, and only one tooth structure formed in each scaffold. These results suggest that the novel cell-seeding technique could be useful for regulating the morphology of regenerated teeth.

CFD simulations on effects of a focused laser pulse for combustion enhancement characteristics were conducted for supersonic airstreams with a transverse hydrogen-fuel injection. In order to estimate behaviors of a laser-induced plasma, temporal variations of its emission, and laser absorption processes were measured with photodiodes. From these measurements, characteristic time scales of the absorption process of an incident laser pulse and a plasma formation process were estimated. Numerical simulations using a time-dependent Navier-Stokes equation with finite-rate chemistry were also conducted to elucidate laser-induced ignition and mixing enhancement characteristics in supersonic airstreams with a transverse hydrogen injection. From the results, it was confirmed that, depending on the laser energy density and focal points, radicals, a shock wave, and shock-induced local turbulences were being formed through the laser irradiaton. Then recirculation zones with flamelets were induced. Moreover it was shown that these flamelets were growing in moving upstream and downstream from the focal point up to 1 msec. Consequently, it was shown that laser-induced plasmas could be effective in both combustion reaction and mixing enhancements for the supersonic combustion.

An investigation on effects of a focused laser pulse for combustion enhancement characteristics was conducted for supersonic airstreams with a transverse hydrogen‐fuel injection. Numerical simulations using a time‐dependant Navier‐Stokes equation with finite‐rate chemistry were conducted to elucidate laser‐induced ignition and mixing enhancement characteristics. From the results, it was confirmed that, depending on the laser energy density and focal points, radicals, a shock wave, and shock‐induced local turbulences were being formed through the laser irradiation. Then recirculation zones with flamelets were induced. Moreover it was shown that these flamelets grew in moving upstream and downstream from the focal point up to 1 msec. From these results, it was shown that laser‐induced plasmas could be effective in both combustion reaction and mixing enhancements for the supersonic combustion.

Effects of laser-induced plasmas on ignition and flame holding characteristics of a hydrogen diffusion flame in supersonic airstreams were investigated. Temporal observation down to nanosecond resolution for behaviors of the laser-induced plasma was conducted with an ICCD camera. It was confirmed that laser ignition processes were characterized into several time scales; (I) absorption of an incident laser pulse (10 -9 ~ 10 8 sec), (II) plasma formation process (10 -8 ~ 10 -7 sec), (III) ignition process (10 -6 ~ 10 -4 sec), and (IV) shock-flow interaction and (V) convective diffusion processes (10 -5 sec ~ ). From the numerical simulation, it was found that positions of plasma kernels induced by laser irradiation affected ignition and flame stabilization characteristics of the flame and also induction process of local turbulence augmenting a local mixing.