Ameloblasts serum-free conditioned medium: bone morphogenic protein 4-induced odontogenic differentiation of mouse induced pluripotent stem cells.

Induced pluripotent stem (iPS) cells possess the ability of self-renewal and can differentiate into cells of the three germ layers, both in vitro and in vivo. Here we report a new method to efficiently induce differentiation of mouse iPS cells into the odontogenic lineage. Using ameloblasts serum-free conditioned medium (ASF-CM), we successfully generated ameloblast-like cells from mouse iPS cells. Importantly, culturing mouse iPS cells in ASF-CM supplemented with BMP4 (ASF-BMP4) promoted odontogenic differentiation, which was evident by the upregulation of ameloblast-specific as well as odontoblast-specific genes. On the other hand, culturing mouse iPS cells in ASF-CM supplemented with noggin (ASF-noggin), an inhibitor of BMP4, abrogated this effect. These results suggest that mouse iPS cells can be induced by ASF-BMP4 to differentiate into ameloblast-like and odontoblast-like cells. The results of our study raise the possibility of using patient-specific iPS cells for tooth regeneration in the future. Copyright © 2016 John Wiley & Sons, Ltd.

The Effects of Diamond-Like Carbon Films on Fretting Wear Behavior of Orthodontic Archwire-Bracket Contacts.

This study aims to assess the effects of diamond-like carbon (DLC) films on fretting wear behavior of orthodontic archwire-bracket contacts. 'Mirror-confinement-type electron cyclotron resonance (MCECR) plasma sputtering' was utilized to deposit carbon films on stainless steel archwires and brackets. Nanostructure of carbon films such as the bonding structure, cross-sectional thickness and surface roughness were studied. The fretting wear behavior of various archwire-bracket contacts were investigated by using a self-developed tester in ambient air and artificial saliva. The results indicated that DLC-coated wires showed significantly low friction coefficient than the uncoated wires independently of the applied environments. Nevertheless, the DLC-coated and uncoated brackets showed no significant differences in the friction coefficient. Microscopic analysis showed that low wear took place for the DLC-coated surfaces. It is proposed that the application of DLC coating on archwires can decrease the orthodontic fretting wear and coefficient of friction. Unfortunately it does not affect the frictional properties for brackets at present.

Combined degenerative and regenerative remodeling responses of the mandibular condyle to experimentally induced disordered occlusion.

INTRODUCTION: The purposes of this research were to investigate the long-term responses of mandibular condylar cartilage to experimentally induced disordered occlusion and to evaluate changes in the expression of the SDF-1/CXCR4 axis. METHODS: Experimentally induced disordered occlusions were created in 8-week-old female Sprague-Dawley rats by orthodontic methods. After 24 weeks, remodeling of the mandibular condylar cartilage was assessed by hematoxylin and eosin staining. Protein and mRNA expression of SDF-1, CXCR4, MMP9, IL6, OPG, and RANKL were investigated by means of immunohistochemical staining and real-time polymerase chain reaction. RESULTS: Obvious cartilage degenerative remodeling responses were observed; they appeared as uneven distributions of cellular disposition, loss of cartilage surface integrity, and cell-free areas. Regenerative responses presenting as thickening of the whole and the calcified cartilage layers in the experimental group were also observed. Compared with the age-matched controls, the protein and mRNA levels of SDF-1, CXCR4, MMP9, IL6, and OPG, but not RANKL, were increased in the experimental group (all, P <0.05). In addition, the mRNA level of RANKL/OPG showed a decreasing trend in the experimental group compared with the age-matched controls (P = 0.052). CONCLUSIONS: This study demonstrated that long-term experimentally induced disordered occlusion leads to a combined response in degeneration and regeneration of mandibular cartilage, accompanied by active interaction of the SDF-1/CXCR4 axis and local upregulation of MMP9, IL6, and OPG.

No significant association between p53 codon 72 Arg/Pro polymorphism and risk of oral cancer.

The genetic polymorphism of p53 codon 72 Arg/Pro has been implicated in oral cancer risk, but the results of previous studies remain controversial and ambiguous. To estimate the effect of the p53 codon 72 Arg/Pro polymorphism on the risk of oral cancer, a meta-analysis was performed. Based on a comprehensive search in PubMed, Embase, Web of Science, and China National Knowledge Infrastructure (CNKI) databases, we identified all available publications assessing the association between p53 codon 72 Arg/Pro polymorphism and oral cancer risk. The pooled odds ratio (OR) with its corresponding 95 % confidence interval (CI) was calculated to assess the association. Subgroup analyses by ethnicity and study quality were performed to further identify the correlation. Totally, 17 studies with 2,975 cases and 3,413 controls were included into this meta-analysis. There was no statistically significant association between the p53 codon 72 Arg/Pro polymorphism and oral cancer risk in all genetic contrast models (OR(Pro allele vs. Arg allele) = 1.05, 95 % CI 0.94-1.18, P(OR) = 0.379; OR(Pro/Pro vs. Arg/Arg) = 1.11, 95 % CI 0.89-1.40, P(OR) = 0.356; OR(Pro/Arg vs. Arg/Arg) = 1.10, 95 % CI 0.93-1.30, P(OR) = 0.256; OR(Pro/Arg + Pro/Pro vs. Arg/Arg) = 1.10, 95 % CI 0.93-1.31, P(OR) = 0.263; and OR(Pro/Pro vs. Arg/Arg + Pro/Arg) = 1.03, 95 % CI 0.90-1.18, P(OR) = 0.647). In the subgroup analysis of high-quality studies, we failed to find the susceptibility of p53 codon 72 Arg/Pro polymorphism to oral cancer. Moreover, the results were similar among Asians, Caucasians, and mixed populations when stratifying by ethnicity. Sensitivity analysis further confirmed the stability of the results. The present meta-analysis of currently available data shows no association between the p53 codon 72 Arg/Pro polymorphism and oral cancer risk.

Variability in permanent tooth size of three ancient populations in Xi'an, northern China.

OBJECTIVES: This paper compares permanent dental dimensions between three ancient populations that belonged to the same biological population throughout a temporal range of 2000 years to detect temporal trends and metric variation in dentition. MATERIALS AND METHODS: The samples analysed were dental remains of 4502 permanent teeth from 321 individuals, which were excavated from three archaeological sites: Chang'an (1000-1300 years BP), Shanren (2200 years BP) and Shaolingyuan (3000 years BP) in the Xi'an region (northern China). For each tooth three standard measurements were taken: Mesiodistal (MD) diameter of crown, labiolingual or buccolingual (BL) diameter of crown and length of root (LR). RESULTS: Three ancient population samples generally displayed the same dental dimensions (p>0.05), whereas some tooth types varied. The Shaolingyuan had larger canine and the smallest maxillary second molars and the Chang'an had the largest mandibular first molars in the MD dimension. The Shanren had the smallest maxillary third molars and mandibular central incisors, and the Chang'an had the smallest maxillary lateral incisors in the BL dimension. In the LR measures, statistically significant differences of five tooth types showed that the Chang'an were smaller than the Shaolingyuan and the Shanren. Comparisons of coefficients of variation for teeth showed that the length of root and third molar usually displayed greater variation. CONCLUSIONS: Decreasing or increasing trend for crown size does not occur between the ancient populations, while changes in crown size of a few tooth types fluctuate. The root size is more variable than the crown size and is likely to reflect a degenerated trend in a few tooth types.

Osteochondral angiogenesis in rat mandibular condyles with osteoarthritis-like changes.

OBJECTIVE: To investigate angiogenesis at the osteochondral junction and changes in expression of pro- and anti-angiogenic factors in rat mandibular condyles with osteoarthritis-like changes. METHODS: In order to evoke osteoarthritis-like lesions in mandibular condyles, disordered occlusion was created experimentally in rats. Osteochondral vascularity was assessed histologically at 20 and 24 weeks. Protein and mRNA levels of pro-angiogenic factors including vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF) and matrix metalloproteases 9 (MMP9), and anti-angiogenic factor chondromodulin-I (CHM-I) were investigated by means of immunohistochemical staining and real-time PCR. RESULTS: Osteochondral angiogenesis was demonstrated as increased numbers of vascular channels terminating in the calcified cartilage and non-calcified cartilage in 20- and 24-week experimental groups compared with controls (all P<0.05). In the experimental groups, VEGF, CTGF and MMP9 were highly expressed in the tissues adjacent to the osteochondral junction. However, CHM-I was more expressed in the superior but not deep hypertrophic chondrocytes. Compared to their age-matched controls, the protein levels of VEGF and CTGF were higher in 20-week experimental group, and the protein and mRNA levels of CTGF, MMP-9, and CHM-I increased in the 24-week experimental group (all P<0.05). CONCLUSION: In the present rat model, osteochondral angiogenesis was observed in mandibular condyles with osteoarthritis-like changes, accompanied with local upregulation of VEGF, CTGF and MMP9. Although the increase in CHM-I may moderate pro-angiogenic factors effects in the superior cartilage, the deficiency of deep hypertrophic chondrocytes to express CHM-I may permit vascular invasion into condylar cartilage.

[Orthodontic retention and adjustment of the occlusion after orthognathic surgery].

OBJECTIVE: To investigate the orthodontic retention and adjustment of the occlusion after orthognathic surgery. METHODS: 18 patients were divided into three groups. Group I: Cases with skeletal Class I bimaxillary protrusion treated by subapical osteotomy; Group II: Cases with skeletal Class II mandibular retrusion treated by sagittal split mandibular advancement surgery; Group III: Cases with skeletal Class III treated by Le Fort I osteotomy on maxilla and sagittal split osteotomy on mandible. There were 6 patients in each group. Three kinds of orthodontic elastic tractions were used based on different categories of malocclusion and different types of operation. RESULTS: 18 patients attained functional occlusion after the orthodontic occlusion adjustment. There was no relapse and malocclusion by surgery. CONCLUSION: Intermaxillary elastics based on different categories of occlusion and different kinds of surgery can improve the occlusion after orthognathic surgery and attatin the functional occlusion.

Porcine tooth germ cell conditioned medium can induce odontogenic differentiation of human dental pulp stem cells.

It is suggested that the differentiation of tooth-derived stem cells is modulated by the local microenvironment in which they reside. Previous studies have indicated that tooth germ cell-conditioned medium (TGC-CM) holds the potential to induce dental pulp stem cells (DPSCs) to differentiate into the odontogenic lineage. Nevertheless, human TGC-CM (hTGC-CM) is not feasible in practical application, so we conjectured that xenogenic TGC-CM might exert a similar influence on human dental stem cells. In this study, we chose swine as the xenogenic origin and compared the effect of porcine tooth germ cell-conditioned medium (pTGC-CM) with its human counterpart on human DPSCs. Morphological appearance, colony-forming assay, in vitro multipotential ability, protein and gene expression of the odontogenic phenotype and the in vivo differentiation capacity of DPSCs were evaluated. The results showed that pTGC-CM exerted a similar effect to hTGC-CM in inducing human DPSCs to present odontogenic changes, which were indicated by remarkable morphological changes, higher multipotential capability and the expression of some odontogenic markers in gene and protein levels. Besides, the in vivo results showed that pTGC-CM-treated DPSCs, similar to hTGC-CM-treated DPSCs, could form a more regular dentine-pulp complex. Our data provided the first evidence that pTGC-CM is able to exert almost the same effect on DPSCs with hTGC-CM. The observations suggest that the application of xenogenic TGC-CM may facilitate generating bioengineered teeth from tooth-derived stem cells in future.

A novel possible strategy based on self-assembly approach to achieve complete periodontal regeneration.

Limitations of current regeneration modalities underscore the importance of restoring the three-dimensional (3D) microenvironment of periodontal development, which is able to elicit the intrinsic capacity of mesenchymal stem cells to proceed to engage in a redevelopment-like program. With increased attention for the potential therapeutic applications of periodontal ligament stem cells (PDLSCs) in periodontal regeneration, it has been proposed that bone marrow mesenchymal stem cells (BMMSCs) are very likely another cell source of physiological repair of periodontal tissues. With this in mind, enlightened from the research targeting the fabrication of laminar structures such as liver and kidney with heterotypic stratification of cell sheets, we proposed a novel possible strategy based on self-assembly approach, which is akin to the physiological phenomenon that occurs during organogenesis, to enhance complete reconstruction of functional complex periodontium-organ systems. We assumed that in this strategy, using the intrinsic capacity of monodispersed cells to self-assemble into a microtissue such as a 3D spheroid, bilayered cell pellet constructs comprising calcified bone-forming cell pellets (i.e., BMMSCs) and cementum/PDL-forming cell pellets (i.e., PDLSCs) would be fabricated in vitro in a tissue-mimicking way and then implanted into periodontal defects. We hypothesize that this novel strategy might open new options to reconstruct extended periodontal defects and then achieve the ultimate goal of predictable and complete regeneration of the periodontium.

Increased COX2 in the trigeminal nucleus caudalis is involved in orofacial pain induced by experimental tooth movement.

Pain is among the major problems during orthodontic treatment. Recent studies have shown that central Cyclooxygenase2 (COX2) pathway was involved in several pain models. The present study investigated whether inducible COX2 within the trigeminal nucleus caudalis (Vc) contributed to experimental tooth movement pain in freely moving rats. Elastic rubber bands were inserted between the first and second maxillary molars bilaterally to establish tooth movement model. The directed mouth wiping behavior was used to evaluate the pain during tooth movement. COX2 distribution in Vc was studied by immunohistochemistry and the changes of COX2 expression were detected by Western blot at different time point after rubber band insertion. Our results showed that tooth movement significantly increased COX2 expression in Vc and the time spent on mouth wiping, reaching a maximum at 1 day and then decreasing gradually. Furthermore, the rhythm change of COX2 expression in Vc and the mouth wiping behavior were much correlative with each other. All of the COX2-immunoreactive structures in Vc exhibited NeuN-immunopositive staining and most of these COX2-immunoreactive neurons were Fos-immunopositive. Importantly, the mouth wiping behavior could be attenuated by intracisternal injection of NS-398 (a selective COX2 inhibitor) but not by periodontal administration of NS-398. All these results suggested that increased COX2 in Vc was involved in tooth movement pain and thus may be a central target for orthodontic pain treatment.

[Treatment of skeletal Class III adult patients with facemask protraction].

OBJECTIVE: To investigate the clinical effects of facemask protraction on skeletal Class III adult patients. METHODS: Totally 15 skeletal Class III patients (male 7, female 8, aged 18 - 24 years) were included in the study. Removable and fixed appliances were used in the upper or lower arches. Facemask protraction was used at night for about 7 months and Class III elastics were worn during the day. The total treatment time was 24 mouths on average. Cephalometric analysis was carried out before and after treatment. RESULTS: The profile was greatly improved and Class I molar relationship was achieved. SNA angle was increased from (79.6 ± 3.7)° to (81.1 ± 3.8)°. SNB angle was decreased from (83.5 ± 3.3)° to (82.6 ± 3.6)°. ANB angle was increased from (-4.1 ± 2.0)° to (-1.5 ± 1.8)°. CONCLUSIONS: Protraction was effective in the treatment of skeletal Class III adult patients.

Effects of BMP-2 and dexamethasone on osteogenic differentiation of rat dental follicle progenitor cells seeded on three-dimensional beta-TCP.

The aim of this study was to investigate the effects of BMP-2 and dexamethasone (Dex) on osteogenic differentiation of rat dental follicle progenitor cells (RDFCs) seeded on three-dimensional beta-TCP. The alkaline phosphatase (ALP), the calcium and phosphonium, the osteocalcin in media of the third passage RDFCs on biomaterial beta-TCP after 1-3, 3-7, 7-14 days of culture were examined respectively. The growth of cells on the scaffolds was observed by scanning electron microscope (SEM) after 3, 7 days of culture and by implanting in the backs of severe combined immunodeficient (SCID) mice for bone regeneration. The third passage RDFCs could be seen adhered, extended and proliferated on the beta-TCP by scanning electron microscopy. The ALP activity, the calcium and phosphoniums and the osteocalcin content of dexamethasone (10(-8) M) or/and BMP-2 (100 ng ml(-1)) were significantly higher than their existence in the control group. They were the significantly highest among four groups after joint application of BMP-2 and dexamethasone. After 8 weeks of implantation, the percentage of the new bones formed area in the RDFCs+beta-TCP+BMP-2+Dex group was significantly higher than that in the RDFCs+beta-TCP+BMP-2 group. In contrast, beta-TCP, RDFCs+beta-TCP+Dex and control constructs lacked new bone formation by histological staining and histomorphometric analysis. The BMP-2+Dex could significantly promote osteogenic differentiation of RDFCs on beta-TCP. beta-TCP supported fast cellular adhesion, proliferation and differentiation of RDFCs. The feasibility of its application in periodontal tissue engineering was also proved.

[Effects of the first premolar extraction on the third molar angulation].

OBJECTIVE: To analyze the effects about inclinations of the second and the third molars in patients treated with or without premolar extractions. METHODS: Fifty-six adolescents were chosen and divided into the first premolar extraction and non-extraction groups, 30 and 26 patients respectively. The pre-treatment and post-treatment panoramic radiographs were made. Angles between long axis of the third molar and the occlusal plane (the second molar alike), and long axis of the second and the third molar were measured and evaluated. RESULTS: The maxillary and mandibular third molar angulations were all improved after treatment in two groups. Compared with non-extraction group, the average changes of angle between long axis of the third molar and the occlusal plane increased significantly in maxilla and mandible (P < 0.05). Average changes of angles between long axis of the second and the third molar decreased and had statistically significant difference (P < 0.05). The change of angle between long axis of mandibular second molar and the occlusal plane had statistically significant difference (P < 0.05), but there was no statistically significant difference in maxillary second molar (P > 0.05). CONCLUSION: The first premolar extraction in orthodontic treatment could improve the third molar angulations and it would promote the eruption of the third molar.

Osteogenic-related gene expression profiles of human dental follicle cells induced by dexamethasone.

AIM: Human dental follicle cells (hDFC) have the ability to differentiate into mineralized tissue-forming cells during root and periodontal development or osteogenic induction in vitro. The present study aimed to validate the osteogenic induction of hDFC by dexamethasone (DEX) and to explore the changes of related genes responsible for the osteogenic differentiation process. METHODS: Passage-cultured hDFC were induced by DEX and analyzed for mineralization activity by morphological observation, alkaline phosphatase (ALP) activity, and alizarin red S staining. GEArray Q series human osteogenesis gene array was used to describe large-scale gene expression in treated hDFC compared to the control group. Quantitative real-time RT-PCR was performed to confirm the microarray data by analyzing the expression of 7 critical transcripts. RESULTS: Osteogenic differentiation of hDFC was confirmed by morphological change, elevated ALP activity and calcified nodules. In 96 genes investigated through the microarray analysis, 20 genes were upregulated and 8 genes were downregulated more than 2-fold. The results of the real-time RT-PCR correlated with the microarray analysis. The expression of the transforming growth factor-beta superfamily showed varying degrees of increase, and fibroblast growth factors exhibited a differential changing trend of expression. The expression of most types of collagen genes representative of extracellular matrixes increased under DEX treatment while small mothers against decapentaplegic 6 and 7 expressions significantly decreased. CONCLUSION: Our results demonstrated that hDFC displayed osteoblastic features in both phenotypic and genotypic traits induced by DEX in vitro.

[Study on the height of marginal ridge to cusp in posterior teeth and its effect on brackets placement].

OBJECTIVE: To measure the vertical height of mesio-distal marginal ridge to cusp in posterior teeth, which may be helpful to brackets positioning. METHODS: The study groups comprised of 60 patients (30 men, 30 women, mostly aged 12-14 years) who underwent orthodontic treatment without tooth extraction and matched the Andrews normal occlusion standard after treatment. Study model of each patient was made. Three-dimensional laser measurer was used to evaluate the vertical height of mesio-distal marginal ridge to mesial cusp in posterior teeth. The data were stored in a personal computer and submitted to statistical analysis of paired t test. RESULTS: No statistical significant difference was found in the same teeth between men and women. Not only in maxilla but also in mandible, there was no significant difference between the left and the right (P>0.05). The average vertical height of maxillary first premolars was (1.70+/-0.50) mm, the maxillary second premolars was (1.24+/-0.45) mm, and for maxillary first molars, the result was (0.83+/-0.40) mm. The difference between each result was statistically significant (P9< 0.01). The average vertical height of mandibular first premolars was (2.25+/-0.45) mm, the mandibular second premolars was (1.55+/-0.45) mm, and for mandibular first molars, the result was (1.18+/-0.40) mm. The difference between each result was statistically significant (P<0.0 1). CONCLUSION: The vertical height of brackets position in posterior teeth should be considered to guarantee that mesio-distal marginal ridges of deferent posterior teeth located in the same plane, so that satisfying goal could be achieved, If the vertical height in the first molar was X mm, the vertical height in the second premolar should be (X+0.5) mm, and (X+1.0) mm might be suit for the first premolar.

[Orthodontic treatment of Class III patients with mandibular asymmetry].

OBJECTIVE: To investigate the treatment outcome of Class III patients with dental, functional and mild skeletal mandibular asymmetry. METHODS: Thirty-five patients (14 males and 21 females) with dental, functional and mild skeletal mandibular asymmetry were selected. The age range of the patients was 7 - 22 years with a mean age of 16.5 years. Dental mandibular asymmetry was treated with expansion of maxillary arch to help the mandible returning to normal position. Functional mandibular asymmetry was treated with activator or asymmetrical protraction and Class III elastics. Mild skeletal mandibular asymmetry was treated with camouflage treatment. RESULTS: Good occlusal relationships were achieved and facial esthetics was greatly improved after orthodontic treatment in patients with dental and functional mandibular asymmetry. However, patients with skeletal mandibular asymmetry should be treated with both extraction and genioplasty. CONCLUSIONS: Orthodontic treatment was suitable for patients with dental and functional mandibular asymmetry, while combined orthodontics and surgery could get good results in patients with skeletal mandibular asymmetry.

Expression of vascular endothelial growth factor in cultured human dental follicle cells and its biological roles.

AIM: To investigate the expression of vascular endothelial growth factor (VEGF) in cultured human dental follicle cells (HDFC), and to examine the roles of VEGF in the proliferation, differentiation, and apoptosis of HDFC in vitro. METHODS: Immunocytochemistry, ELISA, and RT-PCR were used to detect the expression and transcription of VEGF in cultured HDFC. The dose-dependent and the time-course effect of VEGF on cell proliferation and alkaline phosphatase (ALP) activity in cultured HDFC were determined by MTT assay and colorimetric ALP assay, respectively. The effect of specific mitogen-activated protein kinase (MAPK) inhibitors (PD98059 and U0126) on the VEGF-mediated HDFC proliferation was also determined by MTT assay. The effect of VEGF on HDFC apoptosis was measured by flow cytometry. RESULTS: VEGF was transcribed and expressed in cultured HDFC. VEGF at 10-300 microg/L significantly increased HDFC proliferation and ALP activity compared to the control. Following 1, 3, 5, or 7 d of stimulation, VEGF induced a significant increase in HDFC proliferation compared with the corresponding control, while VEGF was effective at increasing ALP activity at the incubation time point of 3, 5, or 7 d. PD98059 and U0126 could attenuate the VEGF-mediated HDFC proliferation. Fewer apoptotic cells were observed in the VEGF-treated groups compared to the controls, although the difference was not statistically significant. CONCLUSION: VEGF is expressed in cultured HDFC, and at a proper concentration range can stimulate HDFC proliferation, induce HDFC to differentiate in a "cementoblast/osteoblast" pathway and protect HDFC from apoptosis. The MAPK signaling pathway might be involved in the VEGF-mediated HDFC proliferation.

[Tooth movement in tissue-engineered bone].

OBJECTIVE: To explore the effect and practical value of repairing the alveolar defects with tissue engineering technique and investigate the influence of the generated new bone on the orthodontic tooth movement. METHODS: The marrow stromal cells of rats were separated, cultured in vitro and induced to osteoblast-like cells. The osteoblast-like cells were implanted onto the ceramic bovine bone. Then the complex was implanted into the alveolar defect in one side of the rat's mandible. The other side of the mandible served as control. Eight weeks later, the orthodontic appliances were placed between the first molar and incisors of SD rats to move the first molar forward. The tooth movement and root resorption of the molar were observed. RESULTS: We found that the tooth movement in the experimental area was faster than that in the normal alveolar bone (P < 0.05). The root resorption and the alveolar bone height loss were less than that in the control area (P < 0.05). CONCLUSIONS: The tissue-engineered bone did not have negative influences on tooth movement. The repair of alveolar bone defect by tissue engineering approach may be used in craniofacial surgery and orthodontics.