Craniofacial and olfactory sensory changes after long-term unilateral nasal obstruction-an animal study using MMP-3-LUC transgenic rats.

Nasal obstruction exerts considerable physiological effects on the respiratory system and craniofacial morphology during the developmental stage. This study used MMP-3-LUC transgenic rats for in vivo tracking of long-term expression in the rat nasal region after unilateral nasal obstruction. Skeletal changes of the craniofacial, nasal, and sinus regions were measured through micro-computed tomography examination and analysis with 3D image processing and calculation. Matrix metalloproteinase-3 and olfactory marker protein expression were also investigated through immunohistochemistry (IHC). Unilateral nasal obstruction significantly reduced the MMP-3 signal in the nasal region of MMP-3-LUC transgenic rats, which was mainly expressed in the respiratory epithelium. Long-term obstruction also caused morphological changes of the craniofacial hard tissue, such as nasal septal deviation, longer inter-jaw distance, and increased maxillary molar dental height. It also caused compensatory growth in olfactory nerve bundles and the olfactory epithelium, as confirmed by IHC. In our study, long-term unilateral nasal obstruction caused nasal septal deviation toward the unobstructed side, hyper divergent facial development including longer molar dental height, and reduced MMP-3 production. However, further investigation is necessary to explore the mechanism in depth.

Orthodontic correction of acquired open bite with TMJ degeneration: A retrospective study of outcomes and stability.

BACKGROUND/PURPOSE: Newly developed temporary anchorage devices (TADs) serve a strong orthodontic anchorage to intrude molars for correction of anterior open bite (AOB). We measured cephalometric changes in skeletal open bite patients which developed subsequently to temporomandibular joint disorders with bilateral point contacts at terminal molars. METHODS: We retrospectively recruited 32 patients who had been treated their TMD before orthodontic correction (overbite: -3.14 ± 1.86 mm). Partial orthodontic appliances were used to intrude posterior teeth using TADs until positive OB obtained (T1). Full fixed appliances were then used to achieve proper overjet and overbite (T2). We collected lateral cephalograms before (T0), during (T1) and after (T2) treatment, and at follow-ups (T3). Using ANOVA, we analyzed the differences among these time points to determine treatment changes and stability of orthodontic results. RESULTS: In this group predominantly comprising young adult women, orthodontic treatment with TADs significantly reduced upper posterior dental heights (T2-T0:-1.84 ± 0.66 mm) and facilitated the retraction and uprighting upper incisors (T2-T0: -9.92 ± 1.72°), to achieve appropriate OJ (T2-T0: -3.21 ± 0.49 mm) and OB (T2-T0: 4.10 ± 0.28 mm) with p < 0.05. Except upper posterior dental height, most of cephalometric changes including OJ, OB, and upper incisal axis remained significant at follow-ups with retention time of 3.7 ± 2.6 years. Only three out of 30 patients experienced small amount of open bite at T3. CONCLUSION: Orthodontic correction of OJ remained relatively stable among 90 % of patients with TMJ degeneration by intrusion via TADs. This modern but conservative orthodontic approach can improve occlusal functions in skeletal open bites.

Molecular signaling and mechanisms of low-level laser-induced gene expression in cells involved in orthodontic tooth movement.

BACKGROUND: The study aimed to observe molecular signaling, including reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨm), to evaluate the alteration of gene expression by low-level laser therapy (LLLT) and the correlation between its mechanisms and the NF-kB pathway in cells involved in orthodontic tooth movement. METHODS: Osteoblast-like cells (MG63), immortalized periodontal ligament cells (iPDL), and M1 macrophage-like cells were irradiated by 980-nm LLLT with energy densities of 1 and 10 J/cm2 ΔΨm and intracellular ROS were monitored using fluorescent probes. The changes of mRNA expression were assessed using reverse transcription polymerase chain reaction (RT-PCR). NF-kB inhibitor, ROS scavenger, and ΔΨm suppressor were used to analyze signals associated with the regulation of gene expression. Finally, Western blot analysis was performed to confirm NF-kB signaling after LLLT. RESULTS: We found the increases of ΔΨm and ROS in all three cell types after LLLT, but no significant difference was observed between 1 and 10 J/cm2 LLLT. Regarding gene expression, some target genes were upregulated in MG63 6 h, 12 h, and 1 day after LLLT and in iPDL cells 12 h and 1 day after LLLT. However, no changes occurred in M1 cells. The inhibitor that significantly reduced most changes in gene expression was NF-kB inhibitor. Western blot analysis showed the increase in p-IkBα level after LLLT in iPDL and MG63, but not in M1. CONCLUSION: The 980-nm LLLT increased ΔΨm and ROS production in all three cell types. However, changes in gene regulation were found only in MG63 and iPDL cells, which related to the NF-kB pathway.

PAX9 mutations and genetic synergism in familial tooth agenesis.

Familial tooth agenesis (FTA) is one of the most common craniofacial anomalies in humans. Loss-of-function mutations in PAX9 and WNT10A have been known to cause FTA with various expressivity. In this study, we identified five FTA kindreds with novel PAX9 disease-causing mutations: p.(Glu7Lys), p.(Val83Leu), p.(Pro118Ser), p.(Ser197Argfs*23), and c.771+4A>G. Concomitant PAX9 and WNT10A pathogenic variants found in two probands with severe phenotypes suggested an effect of mutational synergism. All overexpressed PAX9s showed proper nuclear localization, excepting the p.(Pro118Ser) mutant. Various missense mutations caused differential loss of PAX9 transcriptional ability. PAX9 overexpression in dental pulp cells upregulated LEF1 and AXIN2 expression, indicating a positive regulatory role for PAX9 in canonical Wnt signaling. Analyzing 176 cases with 63 different mutations, we observed a distinct pattern of tooth agenesis for PAX9-associated FTA: Maxillary teeth are in general more frequently affected than mandibular ones. Along with all second molars, maxillary bicuspids and first molars are mostly involved, while maxillary lateral incisors and mandibular bicuspids are relatively less affected. Genotypically, missense mutations are associated with fewer missing teeth than frameshift and nonsense variants. This study significantly expands the phenotypic and genotypic spectrums of PAX9-associated disorders and reveals a molecular mechanism of genetic synergism underlying FTA variable expressivity.

Occlusal function and electromyographic activity of masticatory muscles in skeletal Class III patients with different patterns of mandibular asymmetry.

BACKGROUND: Although jaw asymmetry is commonly seen in skeletal Class III patients, its correlation with occlusal function and masticatory muscle activity has not been fully elucidated. OBJECTIVES: The purpose of this study was to investigate the occlusal function and masticatory muscle activity in skeletal Class III patients with various patterns of mandibular asymmetry. METHODS: Forty-two patients and 10 normal participants were examined. The patients were categorised into three groups. Groups 1 and 2 exhibited menton and ramus deviation to the same side. Menton deviation was larger than ramus deviation in Group 1, whereas Group 2 showed the inverse relation. Group 3 patients showed menton and ramus deviation in opposite directions. Occlusal contact area (OCA), relative bite force (RBF), and temporalis anterior (TA) and masseter muscle (MM) activity at maximum clenching were measured using T-Scan Novus system and Bio-EMG-III. Statistical analysis was performed using the t-test, one-way analysis of variance with Bonferroni correction and Spearman correlation (α = .05). RESULTS: Compared with normal participants, the patients had smaller OCA and greater asymmetry in the distribution of masticatory muscle activity. Greater ramus deviation was associated with smaller OCA in Group 1 but with larger OCA in Group 3. In Group 1, greater menton deviation was related to stronger TA activity on the non-deviation side. In Group 2, greater ramus deviation was related to stronger MM activity on the deviation side. CONCLUSION: Deviation of the menton and ramus was individually related to OCA and masticatory muscle activity, and this relationship varied according to the pattern of mandibular asymmetry.

Inducing cathepsin L expression/production, lysosomal activation, and autophagy of human dental pulp cells by dentin bonding agents, camphorquinone and BisGMA and the related mechanisms.

Camphorquinone (CQ) and resin monomers are included in dentin bonding agents (DBAs) and composite resin to restore tooth defects due to abrasion, crown fracture, or dental caries. DBAs, CQ, and bisphenol A-glycidyl methacrylate (BisGMA) applications influence the biological activities of the dental pulp. The current investigation aimed to delineate the effect of DBAs, CQ, and BisGMA on cathepsin L production/expression, lysosomal activity, and autophagy induction in human dental pulp cells (HDPCs). HDPCs were exposed to DBAs, CQ, or BisGMA with/without inhibitors for 24 h. Enzyme-linked immunosorbent assay was employed to determine the cathepsin L level in culture medium. The cell layer was utilized to measure cell viability by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl -tetrazolium bromide (MTT) assay. Real-time PCR was used to evaluate the mRNA expression. Western blotting or immunofluorescent staining was used to study protein expression. Lysosomal density was evaluated by lysotracker red staining. We found that DBAs, CQ, and BisGMA stimulated cathepsin L mRNA, protein expression, and production in HDPCs. In addition, CQ and BisGMA induced lysosomal activity, Beclin1, ATG12, LC3B, Bax, and p53 expression in HDPCs, indicating the stimulation of autophagy. Glutathione (GSH) prevented CQ- and BisGMA-induced cytotoxicity. Moreover, E64d, cathepsin L inhibitor (two cathepsin inhibitors), and Pifithrin-α (a p53 inhibitor) showed little preventive effect toward CQ- and BisGMA-induced cytotoxicity. Autophagy inhibitors (NH4Cl, Lys05) mildly enhanced the CQ- and BisGMA-induced cytotoxicity. These results indicate that DBAs stimulated cathepsin L, possibly due to their content of CQ and BisGMA that may induce cathepsin L in HDPCs. CQ and BisGMA stimulated lysosomal activity, autophagy, and apoptosis, possibly via induction of Beclin 1, ATG12, LC-3B, Bax, and p53 expression. In addition, CQ and BisGMA cytotoxicity was related to redox change and autophagy. These events are important role in pulpal changes after the restoration of tooth decay using CQ- and BisGMA-containing DBAs and resin composite.

Novel Application of Photo-Crosslinked Urocanic-Acid-Modified Chitosan in Corneal Wounds.

In the last few years, the use of tissue adhesives in corneal perforation has gained immense popularity in clinical practices. The present study aimed to devise a new application of urocanic-acid-modified chitosan (CS) with methylene blue (MB) as a photosensitizer for the development of a photo-crosslinked tissue adhesive. In particular, the curing time was controlled with the aid of a 650 nm red diode. Under the same irradiation condition, the mechanical properties were tuned using the photosensitizer at different concentrations. In vitro tests revealed that the gel was ductile and biocompatible. The application of the gel to a perforated cornea model stopped the leakage of aqueous humor, immediately after the gel was photo-crosslinked. The blue appearance of the gel provided high precision when applied to corneal wounds. Importantly, the crosslinked gel became transparent within 24 h, owing to the dissipation of MB from tears, and the gel spontaneously sloughed off without artificial removal. Altogether, the study reported the development of a novel photo-crosslinkable urocanic-acid-modified CS gel that exhibited significant potential to be utilized in the healing of corneal perforation.

Biological properties of human periodontal ligament cell spheroids cultivated on chitosan and polyvinyl alcohol membranes.

BACKGROUND/PURPOSE: Multicellular spheroid cultures have attracted increasing attention in the field of periodontal regeneration. However, very few studies have reported the periodontal ligament (PDL) cell spheroid formation via biomaterials-induced processes. This study investigated the biological characteristics of human PDL cell spheroids formed on two hydrophilic polymer-based biomaterials, namely chitosan and polyvinyl alcohol. METHODS: The expressions of periostin, paxillin, hypoxia-inducible factor 1-α (HIF-1α), and vascular endothelial growth factor (VEGF) were analyzed. Cell migration ability was assessed using a scratch assay. Furthermore, PDL cell spheroids were cultured in 3D-printed polylactic acid scaffolds to evaluate mineralizing capability. RESULTS: Western blot analysis revealed increased expressions of periostin, HIF-1α, and VEGF in the 3D spheroids. After the spheroids were reseeded, the cells gradually migrated outward from the spheroids and time-dependent distribution of paxillin was observed. The cells migrating outward from the 3D spheroids demonstrated greater migration ability than that of 2D monolayer cells. Compared to the dissociated cells from a monolayer culture, the cell spheroids formed on the chitosan membrane exhibited elevated alkaline phosphatase activity and an increase in mineralized matrix deposition. CONCLUSION: The biomaterial-induced formation of PDL cell spheroids suggests a novel strategy for cell delivery in research and clinical applications of periodontal regeneration.

Orthodontic force regulates metalloproteinase-3 promoter in osteoblasts and transgenic mouse models.

BACKGROUND/PURPOSE: Previously we demonstrated up-regulation of matrix metalloproteinase-3 (MMP-3) in human osteoblasts under compression and in bony specimens of experimental orthodontic tooth movement (OTM). Here, we studied the temporal characteristics of compression stimulation in human and mouse osteoblast cell lines, and generated a transgenic mouse model for assessing the MMP-3 expression during OTM. MATERIALS AND METHODS: We investigated MMP-3 expressions in human and murine osteoblasts through RT-PCR and luciferase assay, after compressive force loading. Inhibitors were added to identify the possible mechanisms for signal transduction. A human MMP-3 promoter was isolated, cloned and transfected to generate a transgenic mouse with a green fluorescent protein reporter. OTM was then initiated to observe the location and time course of transcriptional regulation of MMP-3 signals. RESULTS: We found changes in the transcription of MMP-3 in response to mechanical force applied to both human and mouse osteoblast cell lines, suggesting that the response is positive across species. Cloned human MMP-3 promoter may cause the response of luciferase to 1% compression. Moreover, p38 inhibitor exerted a down-regulatory effect on MMP-3 promoter expression, although the inhibitory effect didn't reach a significant level. In the transgenic mouse OTM model, we again found increased expression of MMP-3 in response to mechanical force loading around the periodontal ligament. CONCLUSION: Mechanical force can stimulate MMP-3 expression, possibly through the p38 MAPK pathway, with its strongest signal occurring at 24 h. The mechanical responsiveness in MMP-3 promoter regions can be observed in both humans and rodents in vitro and in vivo.

Differences of condylar changes after orthognathic surgery among Class II and Class III patients.

BACKGROUND/PURPOSE: The nature of susceptibility to condylar resorption after orthognathic surgery can be different between skeletal Class II and Class III populations, which was addressed by few in the past. The aim of the present study was to use cone-beam computed tomographic (CBCT) images to investigate the displacement and morphological changes of temporomandibular joints (TMJs) in patients received orthodontic treatment combined with orthognathic surgery. METHODS: Both Class III (n = 34) and Class II (n = 17) patients were compared through overall and regional superimpositions of the initial and posttreatment CBCTs. Two-sample t-test was used to identify significance between group differences. Pearson's correlation coefficient was used to address changes of TMJ and the amount of setback or advancement. RESULTS: The axial ramal angle increased significantly in Class III group and decreased in Class II groups after orthognathic surgery (p < FDR_p). For condylar dimensions, significant widths and lengths reductions were noted only in Class II group. However, no significant difference was found after comparing subgroup differences according to one-jaw and two-jaw options, nor any significant correlation found between the condylar changes and the amount of surgical movements. CONCLUSION: The nature of condylar susceptibility could result more from different skeletal patterns than the amount of surgical movements. However, the direction of mandibular surgery may contribute to different changes of condylar angle in axial section.

Architectural changes in alveolar bone for dental decompensation before surgery in Class III patients with differing facial divergence: a CBCT study.

This study aimed to investigate alveolar bone change around mandibular anterior teeth during orthodontic decompensation in patients with skeletal Class III malocclusion and different vertical facial patterns. The records of 29 consecutive Class III patients selected from those pending two-jaw orthognathic surgery were divided into low (≤ 28°), average (30°-37°), and high (≥ 39°) mandibular plane angle (MPA) groups. The DICOM files of CBCT scans and STL files of digital dental models, taken before (T1) and after (T2) presurgical orthodontic treatment, were imported into Dolphin imaging software to reconstruct dentoskeletal images. T1 and T2 images were superimposed and analyzed for bone thickness and height at the level of root apex on each mid-sagittal slice of six mandibular anterior teeth. Differences between T1 and T2 were analyzed by non-parametric tests and mixed-effect model analysis. The results showed that the measurements of alveolar bone height generally decreased after treatment, regardless of MPA. The facial divergence, incisor irregularity, tooth site, treatment time, and change in proclination were identified as the significant factors affecting alveolar bone thickness and height during treatment. The presurgical orthodontic treatment to decompensate mandibular anterior teeth should be very careful in all MPA groups.

Hyaluronic acid on the urokinase sustained release with a hydrogel system composed of poloxamer 407: HA/P407 hydrogel system for drug delivery.

Pleural empyema is an inflammatory condition characterized by accumulation of pus inside the pleural cavity, which is usually followed by bacterial pneumonia. During the disease process, the pro-inflammatory and pro-fibrotic cytokines in the purulent pleural effusion cause proliferation of fibroblasts and deposition of extracellular matrix, which lead to fibrin deposition and fibrothorax. Urokinase instillation therapy through a chest drainage tube is frequently used for fibrinolysis in patients with empyema. However, urokinase treatment requires multiple instillation (2-3 times per day, for 4-8 days) and easily flows out from the chest drainage tube due to its high water solubility. In this in vitro study, we developed a thermo-responsive hydrogel based on poloxamer 407 (P407) combined with hyaluronic acid (HA) for optimal loading and release of urokinase. Our results show that the addition of HA to poloxamer gels provides a significantly more compact microstructure, with smaller pore sizes (**p < 0.001). The differential scanning calorimetry (DSC) profile revealed no influence on the micellization intensity of poloxamer gel by HA. The 25% poloxamer-based gel was significantly superior to the 23% poloxamer-based gel, with slower gel erosion when comparing the 16th hour residual gel weight of both gels (*p < 0.05; **p < 0.001). The 25% poloxamer-HA gel also exhibited a superior urokinase release profile and longer release time. A Fourier-transform infrared spectroscopy (FT-IR) study of the P407/HA hydrogel showed no chemical interactions between P407 and HA in the hydrogel system. The thermoresponsive P407/HA hydrogel may have a promising potential in the loading and delivery of hydrophilic drugs. On top of that, in vitro toxicity test of this combination demonstrates a lower toxicity.

Analysis of root canal system of maxillary first and second molars and their correlations by cone beam computed tomography.

BACKGROUND/PURPOSE: Understanding the root canal systems of molars and the association of root canal system in adjacent or contralateral molars is important for dental practice. This study aimed to use cone-beam computed tomography (CBCT) to analyze the morphology similarity of root canal systems in the maxillary first and second molars. METHODS: CBCT images of 1741 maxillary molars in a total of 519 patients were blindly examined to analyze the correlation of root canal systems between maxillary first and second molars as well as the bilateral first and second molars. RESULTS: The most common type in maxillary first molars is 3R4C (3 roots/4 canals), whereas in maxillary second molars is 3R3C.The symmetry in type of root canals in bilateral maxillary first and second molars were 87.36% and 79.85%, respectively. The similarities of root canal system in adjacent maxillary first and second molars were 53.07% (right side) and 52.58% (left side). The concurrence of MB2 canal in bilateral maxillary first molars is 77.8%, and 35.97% in maxillary second molars. In the 110 patients with MB2 canal in bilateral maxillary second molars, the chance of bilateral MB2 canals in their maxillary first molar is almost 100%. CONCLUSION: Maxillary first molars have higher prevalence of 3R4C than second molars. The symmetry in bilateral maxillary molars is higher than the similarity in adjacent maxillary first and second molars. Application of CBCT analysis of root canal system can improve endodontic treatment outcomes. The correlation of root canal system between teeth is useful for genetic linkage.

Clinical outcomes of adjunctive indocyanine green-diode lasers therapy for treating refractory periodontitis: A randomized controlled trial with in vitro assessment.

BACKGROUND/PURPOSE: It is still challengeable to treat periodontal pockets refractory to mechanical debridement. This study is to evaluate the potential of indocyanine green (ICG)-diode laser-based photothermal therapy (PTT) for solving this dilemma. METHODS: Bone marrow-derived mesenchymal stem cells (BMSCs) and periodontal ligament cells (PDLCs) were incubated with phosphate-buffered saline, chlorhexidine, or ICG, non-irradiated or irradiated with 810-nm diode lasers, and the cell viability was evaluated. Patients with teeth refractory to mechanical periodontal debridement on different quadrants were recruited. At baseline (T0), all examined teeth received scaling and root planing, and those on the test quadrant (PTT group) received ICG-diode laser treatment. The outcome was evaluated using clinical parameters and cytokines in the gingival crevicular fluids at 4-6 weeks (T1) and 6 months (T2). RESULTS: In ICG-treated cultures, the viability of BMSCs and PDLCs was recovered on day 4, and laser irradiation inhibited the metabolic activities of BMSCs. 22 patients with 30 control teeth and 35 PTT-treated teeth were examined. All examined teeth showed modest reductions in probing pocket depth (PPD), clinical attachment loss (CAL), bleeding upon probing (BOP), and plaque score at T1 and T2 and significant reductions in IL-1ß and MMP-8 at T2. Compared with controls, BOP was reduced more prominently, IL-1ß and MMP-8 were significantly lower, and reductions in PPD and CAL were slightly greater in the PTT group at T1 (0.05-0.19 mm). CONCLUSION: ICG-diode laser-based PTT is compatible to periodontium and assists in faster resolution of gingival inflammation in periodontal pockets refractory to mechanical debridement.

Cytotoxicity and genotoxicity of DMABEE, a co-photoinitiator of resin polymerization, on CHO-K1 cells: Role of redox and carboxylesterase.

The 4-dimethylaminobenzoic acid ethyl ester (DMABEE) is an important co-initiator for resin polymerization in dental resinous materials. As a radical forming chemical with high lipophilicity, the genotoxicity and cytotoxicity of DMABEE deserve prudent investigation. In this study, we found that DMABEE reduced the viability and proliferation of Chinese hamster ovary (CHO-K1) cells in a dose-dependent manner, and altered cell morphology at higher concentrations. G0/G1 cell cycle arrest was induced by DMABEE at 0.25-0.75 mM, and cell proportion of sub-G0/G1 phase was significantly elevated at 1 mM while cell apoptosis was observed. Genotoxic effect was noted when cells were treated by 0.1 mM DMABEE, as revealed by increase of micronucleus formation. Reactive oxygen species overproduction was observed as cells treated with 0.75 and 1 mM, while elevation of intracellular glutathione was noticeable since 0.1 mM. Contrary to our expectation, pretreatment by N-acetyl-l-cysteine enhanced the toxicity of DMABEE on CHO-K1 cells. Catalase mildly reduced the toxic effect and carboxylesterase showed obvious ability to reverse the toxicity of DMABEE. These findings highlight the mechanism of DMABEE toxicity and provide clues for safety improvement of its application in clinical dental treatment.

Airway increase after open bite closure with temporary anchorage devices for intrusion of the upper posteriors: Evidence from 2D cephalometric measurements and 3D magnetic resonance imaging.

OBJECTIVE: The aim of this study was to analyse morphological changes in the upper airways in patients with anterior open bite treated with temporary anchorage devices for intrusion of upper posterior teeth. MATERIALS AND METHODS: Twelve nonobese (body mass index: <25) anterior open bite patients between the ages of 19 and 44 years (mean age: 22.83 ± 8.19 years) were recruited for this study. Cephalometric radiographs and magnetic resonance imaging (MRI) scans before and after anterior bite closure without bracketing on anterior teeth were used to measure the upper airway, which was divided into retropalatal and retroglossal regions. RESULTS: The mandibular plane angle and lower facial height were significantly reduced by intrusion of the upper posteriors and autorotation of the mandible. The retroglossal airway width (AW2) and retroglossal area (RG area) measured on cephalometric radiographs both increased significantly after treatment. Retroglossal volume increased and the retroglossal width/length ratio decreased significantly in MRI analysis. All other measurements were not significantly changed. However, no statistically significant correlations were observed between all measurements in 2D and 3D images, with the exception of the AW2 linear measurement in 2D images correlating with the AP length in MRI axial view images (r = 0.56, P = 0.0430). CONCLUSION: Counterclockwise rotation of the mandible after anterior open bite closed using orthodontic treatment changed the airway morphology. Retroglossal volume significantly increased and the airway shape became less elliptical after bite closure.

Aggregation of human dental pulp cells into 3D spheroids enhances their migration ability after reseeding.

Multicellular three-dimensional (3D) spheroids allow intimate cell-cell communication and cell-extracellular matrix interaction. Thus, 3D cell spheroids better mimic microenvironment in vivo than two-dimensional (2D) monolayer cultures. The purpose of this study was to evaluate the behaviors of human dental pulp cells (DPCs) cultured on chitosan and polyvinyl alcohol (PVA) membranes. The protein expression of hypoxia-inducible factor 1-α (HIF-1α) and vascular endothelial growth factor (VEGF), and the migration ability of the DPCs from 2D versus 3D environments were investigated. The results showed that both chitosan and PVA membranes support DPCs aggregation to form multicellular spheroids. In comparison to 2D cultures on tissue culture polystyrene, DPC spheroids exhibited higher protein expression of HIF-1α and VEGF. The treatment with YC-1 (inhibitor to HIF-1α) blocked the upregulation of VEGF, indicating a downstream event to HIF-1α expression. When DPC spheroids were collected and subjected to the transwell assay, the cells growing outward from 3D spheroids showed greater migration ability than those from 2D cultures. Moreover, DPCs aggregation and spheroid formation on chitosan membrane were abolished by Y-27632 (inhibitor to Rho-associated kinases), whereas the inhibitory effect did not exist on PVA membrane. This suggests that the mechanism regulating DPCs aggregation and spheroid formation on chitosan membrane is involved with the Rho-associated kinase signaling pathway. In summary, the multicellular spheroid structure was beneficial to the protein expression of HIF-1α and VEGF in DPCs and enhanced the migration ability of the cells climbing from spheroids. This study showed a new perspective in exploring novel strategies for DPC-based research and application.

970 nm low-level laser affects bone metabolism in orthodontic tooth movement.

OBJECTIVE: During orthodontic tooth movement (OTM), the speed of movement depends on the rate of bone turnover. In this study, we used a rat model to investigate the effect of 970 nm low-level laser therapy (LLLT) on OTM under different dose and frequency protocols. METHODS: We first compared the OTM rates between the OTM only control and the OTM + LLLT group (1250 J/cm2) in Experiment 1 and showed that LLLT significantly increased OTM. In Experiment 2, we employed 3 different LLLT protocols: the low-dose group and the high-dose group receiving 5 doses of 750 J/cm2 and 15,000 J/cm2 of LLLT every 3 days, respectively, and the early high-dose group which received 5 daily doses at 15,000 J/cm2 at the beginning of the experiments. The OTM-only control group received no LLLT. Tooth movement rate was measured through sequential silicone impressions. MicroCT was also performed to evaluate bone de-mineralization rate. Bone histmorphometry was used to compare the bone turnover rate between LLLT group and control group. Finally, TRAP, Osteocalcin, and VEGF expression is evaluated by immunohistochemistry (IHC) in tissue sections. RESULTS: When LLLT treatment was given every three days, both the 1250 J/cm2 and 15,000 J/cm2 groups showed significantly increased OTM compared to the control group. No significant difference was observed in the 750 J/cm2 group, or in the early irradiation group, when compared with controls, although 750 J/cm2 showed the same trend of accelerating OTM. The MicroCT result of rat maxilla demonstrated that LLLT increased bone remodeling and showed decreased bone mineral density and bone volume/total volume in the furcation areas of the maxillary first molars at the end of experiment. LLLT without OTM increased bone turnover as evidenced by fluorochrome incorporation. Immunohistochemistry analyses revealed high osteocalcin expression at later stages of OTM in the LLLT group, while VEGF expression was highly induced in the LLLT + OTM group at an early stage. CONCLUSION: Our results suggest that the 970 nm LLLT increases the rate of OTM in a dose-sensitive and frequency-dependent manner. Further animal and human studies are needed to determine the optimal timing and dosage of LLLT for OTM acceleration.

Periostin inhibits mechanical stretch-induced apoptosis in osteoblast-like MG-63 cells.

BACKGROUND/PURPOSE: Appropriate mechanical stress plays an important role in regulating the proliferation and differentiation of osteoblasts, whereas high-level mechanical stress may be harmful and compromise cell survival. Periostin, a matricellular protein, is essential in maintaining functional integrity of bone and collagen-rich connective tissue in response to mechanical stress. This study investigated whether or not high-level mechanical stretch induces cell apoptosis and the regulatory role of periostin in mechanical stretch-induced apoptosis in osteoblastic cells. METHODS: Osteoblast-like MG-63 cells were seeded onto Bio-Flex I culture plates and subjected to cyclic mechanical stretching (15% elongation, 0.1 Hz) in a Flexercell tension plus system-5000. The same process was applied to cells pre-treated with exogenous human recombinant periostin before mechanical stretching. We used a chromatin condensation and membrane permeability dead cell apoptosis kit to evaluate the stretch-induced cell responses. Expression of caspase-3 and cPARP was examined by immunofluorescent stain and flow cytometry. RESULTS: The expression of periostin in MG-63 cells is involved in the TGF-ß signaling pathway. High-level cyclic mechanical stretch induced apoptotic responses in MG-63 osteoblastic cells. The percentages of apoptotic cells and cells expressing cPARP protein increased in the groups of cells subjected to mechanical stretch, but these responses were absent in the presence of exogenous periostin. CONCLUSION: Our study revealed that high-level mechanical stretch induces apoptotic cell death, and that periostin plays a protective role against mechanical stretch-induced apoptosis in osteoblastic cells.

Basic Fibroblast Growth Factor Regulates Gene and Protein Expression Related to Proliferation, Differentiation, and Matrix Production of Human Dental Pulp Cells.

INTRODUCTION: Basic fibroblast growth factor (bFGF) plays differential effects on the proliferation, differentiation, and extracellular matrix turnover in various tissues. However, limited information is known about the effect of bFGF on dental pulp cells. The purposes of this study were to investigate whether bFGF influences the cell differentiation and extracellular matrix turnover of human dental pulp cells (HDPCs) and the related gene and protein expression as well as the role of the mitogen-activated protein kinase (MEK)/extracellular-signal regulated kinase (ERK) signaling pathway. The expression of fibroblast growth factor receptors (FGFRs) in HDPCs was also studied. METHODS: The expression of FGFR1 and FGFR2 in HDPCs was investigated by reverse-transcription polymerase chain reaction. HDPCs were treated with different concentrations of bFGF. Cell proliferation was evaluated using the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Cell differentiation was evaluated using alkaline phosphatase (ALP) staining. Changes in messenger expression of cyclin B1 and tissue inhibitor of metalloproteinase (TIMP) 1 were determined by reverse-transcription polymerase chain reaction. Changes in protein expression of cdc2, TIMP-1, TIMP-2, and collagen I were determined by Western blotting. U0126 was used to clarify the role of MEK/ERK signaling. RESULTS: HDPCs expressed both FGFR1 and FGFR2. Cell viability was stimulated by 50-250 ng/mL bFGF. The expression and enzyme activities of ALP were inhibited by 10-500 ng/mL bFGF. At similar concentrations, bFGF stimulates cdc2, cyclin B1, and TIMP-1 messenger RNA and protein expression. bFGF showed little effect on TIMP-2 and partly inhibited collagen I expression of pulp cells. U0126 (a MEK/ERK inhibitor) attenuated the bFGF-induced increase of cyclin B1, cdc2, and TIMP-1. CONCLUSIONS: bFGF may be involved in pulpal repair and regeneration by activation of FGFRs to regulate cell growth; stimulate cdc2, cyclin B1, and TIMP-1 expression; and inhibit ALP. These events are partly associated with MEK/ERK signaling.