Bone marrow-derived mesenchymal stem cells (BMSCs) have considerable potential for self-renewal and multi-differentiation. Tumor necrosis factor-α (TNF-α) is an inflammatory cytokine and is involved in tissue regeneration during wound healing. It was already reported that cultured human dental pulp cells acquire stem cell properties following short-term stimulation by TNF-α. However, it has not been clarified if BMSCs acquire stem cell properties after TNF-α treatment. The purpose of this study was to investigate the effect of short-term stimulation with TNF-α on BMSCs. Rat BMSCs were cultured up to 60% confluence and then incubated with 1–100 ng/ml of recombinant rat TNF-α (rTNF-α) for a further 2 days. After reaching subconfluence, cells were passaged once to remove rTNF-α completely before subsequent assays. Cells in the control group were passaged without stimulation. Expression levels of Nanog and Oct4 stem cell markers were significantly increased in the rTNF-α 10 ng/ml stimulation group. rTNF-α stimulation did not affect cell proliferation compared with the control group. However, rTNF-α stimulation led to a delay in cell differentiation. This study suggests that short-term TNF-α stimulation of BMSCs significantly increased their stem cell phenotype, but delayed their osteogenic cell differentiation.

Intracapsular condylar fracture in children and adolescents may damage the condylar cartilage and hinder the growth of mandibular branch, but the treatment of the pediatric/children’s intracapsular fracture is still an academic controversy. In this study, we build the intracapsular condylar fracture models in miniature pigs and make the three-dimensional computed tomography analysis based on the datas and then make a series of histopathologic evaluation .The results indicated that the growth of mandibular branch is restrained and there are statistical significances in experimental and control groups,moreover the most serious degenerative change was found in experimental groups. The results will provide the theoretical basis for clinical treatment.

Human periodontal ligament stem cells (HPLSCs) contribute to the regeneration of periodontal tissue because of their multilineage potential. Although monolayer cultures are commonly used in cell cultures, they inadequately overcome their low differentiation capacity. The use of spheroid cultures is expected to overcome the problem of mimicking the in vivo microenvironment. In this study, we assessed whether HPLSC spheroids are susceptible to osteogenic differentiation through the canonical Wnt/β-catenin signaling pathway. HPLSC spheroids were generated using low-binding plates. Osteogenic differentiation of monolayer- and spheroid-derived HPLSCs was induced by osteogenic induction medium. Increased expression levels of osterix and Runx2 and intense staining of alkaline phosphatase (ALP) activity were observed in spheroid-derived HPLSCs, as compared with monolayer-derived cells. During spheroid formation, the integrin pathway of cells composed of spheroids was activated through focal adhesion kinase (FAK), suggesting that this activation may induce susceptibility of the Wnt pathway in HPLSC spheroids. Wnt 3a stimulation increased the expression levels of β-catenin and T-cell factor (TCF), but decreased that of glycogen synthase kinase-3β. Wnt 3a-induced the expression of β-catenin and TCF was effectively decreased by Dickkopf-1 (Dkk-1), a Wnt antagonist. Wnt 3a stimulation also increased the expression levels of osterix and Runx2 which was accompanied by intense ALP staining, in HPLSC spheroids, whereas the addition of Dkk-1 decreased both expression levels and ALP staining. These findings indicate that HPLSC spheroids enhance osteogenic differentiation because cells, composed of spheroids, induce susceptibility of the canonical Wnt pathway, which is mediated by activation of the integrin/FAK pathway during spheroid formation.

To elucidate the crystallo-chemical reaction between the enamel and carbonated soft drink, a micro-X-ray diffraction analysis (micro-XRD) was conducted using the human tooth sections soaked in the drink (Sprite^®) for 1 and 7 days. In this study, 4 typically and heavily dissolved teeth were selected from 20 teeth, and the divergence of the crystallographic properties of the enamel was analyzed.

All of the untreated human tooth enamels exhibited only the apatitic XRD patterns. We confirmed that the carbonated soft drink changed the macro- and microscopic morphological features of individual tooth enamel. The following 4 results were obtained from our study: i) the unit cell dimensions and crystallinity of the untreated enamel apatite varied between the layers and the individual teeth; ii) after the soaking experiment, the crystallographic properties of the enamels changed remarkably and displayed non-uniformity; iii) no relationship was discerned between the enamel’s crystallographic properties and the sensitivity of the carious attack; and iv) the intermediate reaction product of soaking was uncovered; however, it was not identified at this time. The presence or absence of occurrence and variations in the amount of intermediate product reflected the complex chemical and crystallo-chemical reactions between the decalcification solution and the human tooth enamel crystallites.

We clearly established that the untreated human tooth enamels differed in their crystallographic properties and did not react in the same manner, which resulted in varied apatitic structures after soaking. These results necessitate reconsideration of the generally accepted caries protection methods that are applied as common standards for all individuals and tooth enamels.

Many studies have been conducted to evaluate the timing of brushing and methods to suppress or halt the progression of acid erosion and initial caries formation, and to restore tooth enamel. However, the analyses in these studies were done from a macroscopic point of view, such as evaluating the change in the hardness of the enamel surface and the change in X-ray permeability, the morphological change due to crystal deposits on the enamel surface has not been elucidated. The purpose of this study was to investigate how crystals on the enamel surface differ when they are in contact with oral cavity liquids such as saliva versus no contact with these liquids. In addition, we assessed the condition of initial caries in which no substantial defect is observable, from the morphological perspective by using elemental analysis and observations with a transmission electron microscope (TEM). We also considered the causes of crystal formation on the enamel surface. As a result, the outermost layer in the pre-eruptive enamel contained a large amount of Mg, which is a stabilizer of amorphous calcium phosphate, suggesting that the layer contains acidic calcium phosphates. It was suggested that tricalcium phosphate, dicalcium phosphate dihydrate, octacalcium phosphate, etc. form a layer on the surface of post-eruptive enamel with no caries and in post-eruptive enamel with caries exposed to saliva in the oral cavity and the layer containing calcium phosphate have higher acid resistance than HAp. The information from this study can be applied to basic clinical research on remineralization therapy and non-invasive treatments of initial caries.

The development of intervertebral disc degeneration (IDD) is largely influenced by disorders in the nucleus pulposus cells (NPCs) and collagen loss. This study aimed to investigate the in vitro effects of the overexpression of the platelet-derived growth factor (PDGF)-B on the biological functions and chondrogenic gene expression in the NPCs. PDGF-B was overexpressed in primary cultured rat NPCs using lentivirus. The CCK8 method was used to assess in vitro cell viability that indicates the proliferation activity. Real-time polymerase chain reaction (PCR) and western blotting were applied to observe the matrix synthesis and expression of the chondrogenic genes. Cell viability was significantly increased in the PDGF-B groups compared to the negative controls in a time- and dose-dependent manner. PDGF-B enhanced mRNA expression of collagen II and aggrecan, and suppressed that of collagen X. On day 7, the number of collagen II-positive cells significantly increased in the PDGF-B group. Taken together, PDGF-B overexpression could promote the proliferation of NPCs and enhance matrix synthesis in vitro. Thus, PDGF-B can be a promising target to prevent and reverse IDD.

The present study aimed to classify variations in tooth root cross-sectional morphology after conventional endodontic microsurgery in the maxillary first molars and to accurately predict the risk of concealed isthmus deeper in the root based on the observed morphology. Using micro-CT data, tooth root cross-sections obtained at 3–6 mm from the apex were classified as Types I–V according to the isthmus classifications of Hsu and Kim. The rates of mismatch between isthmus classifications in the cross-sections at 3 mm from the apex and those obtained deeper in the root at 4–6 mm from the apex were calculated. High rates of match were observed between classifications in tooth root cross-sections at 3 mm from the apex and those deeper in the tooth root (4–6 mm) in the distobuccal (83%) and paratal (90%) roots, while the rate of match in the mesiobuccal root was low (36%). In mesiobuccal roots with incomplete isthmus at 3 mm from the apex, the probability of complete isthmus deeper in the root was 90% or higher. Accessory root canals and lateral branches were often found not only close to the apex, but also concealed deeper in the root more than 3 mm from the apex in the mesiobuccal roots of the maxillary first molars. Thus, as with cases of complete isthmus, treatment requires enlargement of the root canal to create a cavity that encompasses the main root canal and any smaller structures, followed by retrograde filling.

Fluoride has a wide range of physiological activities that may contribute to its beneficial effects against inflammation-related diseases. However, the molecular mechanisms underlying the anti-inflammatory activity of fluoride are not completely characterized, and many features remain to be elucidated. In this study, we investigated the molecular basis for the down-regulation of toll-like receptor 4 (TLR4) signal transduction by low-level fluoride (NaF) in periodontal ligament cells. In addition, the expression of asporin was investigated by quantitative real-time PCR as well as by immunohistochemical analysis of periodontal ligament cells, with or without fluoride treatment. P. gingivalis-derived lipopolysaccharide (LPS) markedly elevated the mRNA and protein expression levels of DEC1, a regulator of TLR signaling. LPS-induced expression of TLR4, DEC1 and Notch1 was inhibited by low-level NaF. Asporin and periostin were expressed in periodontal ligament cells as expected. Treatment with LPS decreased the expression levels of asporin and periostin in periodontal ligament cells and treatment with low-level NaF increased those levels both in vivo and in vitro. These novel findings provide new insights into understanding the regulatory mechanisms of the TLR4 signaling pathway and the pharmacological role of low-level NaF in the inflammatory response against the development and progression of periodontal disease.

Fundamental studies on biocompatible materials were conducted using the mechanism of dentin formation as a model. Hydroxyapatite and fluorinated apatite were synthesized in a gelatin solution. Synthesized apatite was pressed under vacuum, and the mechanical strength was investigated. A hardened mold was prepared by adding amorphous calcium phosphate (ACP). Then, changes in the crystals of the hardened mold dipped in distilled water or 1 ppm fluoride solution were observed with a transmission electron microscope. The results showed that the crystal growth of apatite synthesized in 1% gelatin was suppressed. Moreover, small and homogenous granular apatite was formed in the precipitate synthesized with fluorine. Gelatin contributed to the reduction of brittleness of the hardened mold, and thus a high cutting resistance value was obtained. Furthermore, acceleration of crystal growth and aggregation of fine crystals were observed in the hardened mold when ACP was added to the solution.

Bone tissue engineering is a complex process that requires concerted functions of biomaterials, cells, and bioactive growth factors. A porous nano-hydroxyapatite (nHA)/collagen (Col)-bone morphogenetic protein (BMP)-9/gelatin microsphere (GM) scaffold was fabricated by a combination of blending, crosslinking, and freeze-drying technologies. The nHA/Col-BMP-9/GM scaffold was well manufactured with a three-dimensional structure and had good mechanical properties. Rat bone marrow mesenchymal stem cells (BMMSCs) were isolated and cultured in vitro, and then identified by flow cytometry. An indirect Cell Counting Kit-8 assay demonstrated that the scaffolds favored BMMSC proliferation, indicating their cytocompatibility. In vitro, BMMSCs were seeded onto the nHA/Col-BMP-9/GM scaffolds to mimic a natural microenvironment. Scanning electron microscopy observation and adhesion rate determination were performed to evaluate the adherence and growth of BMMSCs on the composite scaffolds. Cells cultured in the scaffolds tended to maintain good morphology, attachment, and proliferation, confirming that the scaffolds were biocompatible and showed no cytotoxicity in vitro. Alkaline phosphatase expression was examined to assess the effect of the composite scaffolds on the osteogenic differentiation of rat BMMSCs. The results revealed that the nHA/Col-GM scaffolds, especially the BMP-9-loaded nHA/Col-GM scaffolds, significantly promoted the osteogenic differentiation of BMMSCs. For in vivo determination, acute toxicity, pyrogenic, and implantation tests were performed and verified that the composite scaffolds possessed excellent biocompatibility. This novel biocompatible scaffold has good application prospects for bone repair and regeneration.

An increased knowledge of specific cellular response and function has led to the development of numerous treatment modalities based on the utilization of growth factors. The present controlled clinical study was undertaken to evaluate the effectiveness of autologous platelet rich fibrin (PRF) in combination with HA and beta-TCP in treatment of human class II furcation defects and to compare it with HA and beta-TCP alone. A total of 24 interproximal defects in 24 chronic periodontitis patients were included in the study. The test group was treated by an open flap debridement in combination with autologous platelet rich fibrin (PRF) in combination with hydroxyapatite beta tricalcium phosphate, while the control group was treated by an open flap debridement (OFD) along with hydroxyapatite and beta tricalcium phosphate. At 12 months, both the test and control groups showed significant mean PPD reduction and CAL gain. There was statistically significant (p<0.05) greater probing depth reduction of 1.50 mm for the test group compared to the control. The mean Clinical Attachment Level (CAL) gains of 3.0 ± 0.95 mm was observed in the test group, while the control group displayed mean CAL gains of 2.00 ± 0.85 mm. The observed differences between baseline CAL and 12 months CAL were found to be statistically significant in both the groups (p<0.05). The mean CAL gain observed in the test group was significantly greater than the control group. Horizontal probing depth were significantly reduced in test group (3.33 ± 0.83 mm) compared to control group (1.75 ± 1.21 mm). Frequency analysis of furcation changes revealed complete furcation closure in 50% sites in test groups than control group which showed only 16.66% sites of complete resolution of furcation defects. The treatment with PRF in combination with HA and β-TCP group resulted in a significantly higher CAL gain, PPD and HPD reduction in comparison with hydroxyapatite and beta tricalcium phosphate.

DNA/protamine complex has been reported to enhance bone formation in animal experiments. Another notable property of the complex is that it becomes a paste after being knead with water. Conventionally, DNA/protamine paste is prepared by kneading the DNA/protamine complex with water; however, conventional DNA paste has problems with regards to the preparation method, handling, moldability, etc. In the present study, we developed a new method for preparing DNA pastes, called the on-the-spot preparation method. DNA/protamine paste was prepared by mixing DNA into viscous liquid protamine or mixing gelatin into viscous liquid DNA. The viscosity of the DNA/protamine and DNA/gelatin paste was better than that of the conventional DNA/protamine-complex paste. The DNA/protamine and DNA/gelatin pastes were injected into the extracted sockets of rat maxillary molars. Microfocus X-ray computed tomography (micro-CT) and histological observation of nondecalcified sections after 2 weeks of injection revealed that the DNA/protamine and DNA/gelatin paste enhanced the formation of new bone in the extracted sockets. Quantitative analysis for calcification degree and newly formed bone mass showed there was a significant difference between DNA/protamine paste- and DNA/gelatin paste-injected specimens. The bone formation ability of DNA/gelatin paste is better than that of the DNA/protamine paste in the extracted sockets of rat maxillary molars. It is suggested that DNA paste prepared by on-the-spot preparation method will be useful candidate materials for bone regeneration.

This study aimed to investigate the changes of histomorphology and bone mineral density (BMD) change in different parts of ovariectomized osteoporosis rats. The six-month SD rats were randomly divided into the sham operation group and operation group, each consisting of 30 rats and an osteoporosis model was established by bilateral ovariectomy. The rats were harvested at 8 weeks, 12 weeks and 16 weeks after operation. The changes of bone morphology were observed by Masson staining on lumbar spine 3-5, right tibia and femur. BMD was measured at lumbar spine, left tibia and femur by Dual Energy X-ray Absorptiometry (DXA). Compared with sham operation group, the trabecular bones of the model group were sparse, irregularly arranged, unevenly distributed, the trabecular bone spacing was enlarged. A significantly increased amount of lipid droplets in the medullary cavity. The bone mass changes were most significant at 16 weeks after operation, reflected by the significant trabecula loss in the proximal tibia and distal femur, while in distal tibia and lumbar spine trabecula loss is less significant; BMD declines in all the four parts (P<0.01) and the differences in BMD, in descending order, are the proximal tibia, distal femur, proximal femur, and lumbar spine. In ovariectomized rats, the most sensitive areas to bone mass change are the proximal tibia and distal femur, which are major weight-bearing bones; non-weight-bearing areas show the most significant trabecula loss, and there is a positive correlation between the degree of trabecula loss and lipid accumulation in the medullary cavity, which makes non-weight-bearing areas optimal for osteoporosis research on postmenopausal rats.

The osteogenic effect of newly developed bisphosphonate (Disodium Dihydrogen-4-[(methylthio) phenylthio] Methane- Bisphosphonate (MPMBP)) was examined for clinical application in bone damage. A bone defect was made in the femur of ninety-seven male Wistar rats. 30µl of MPMBP (experimental group) or saline (control group) was injected around defect once every 3 days. The osteogenic effect to 4 weeks was performed at 1, 2, 3 and 4 weeks (experiment 1). Long-term observation was performed at 12 and 24 weeks after discontinuation of administration of MPMBP at 3 weeks (experiment 2). Newly formed bone was examined radiographically and histologically in both experiments. The elemental analysis was performed to investigate the quality of bone by energy-dispersive X-ray analysis. In experiment 1, newly bone formation is observed in the bone marrow cavity and cortical defect in both groups. The bone volume at 4 weeks was decreased in control group and maintained in experimental groups. In experiment 2, newly formed bone in bone marrow cavity was absorbed at 12 weeks in control group, and showed gradually decrease to 24 weeks in experimental group. The Ca/P of newly formed bone showed little difference between control and experimental groups. It was confirmed that the MPMBP administration had local effects, showing the promotion of new bone formation and long-term bone remodeling.

Biocompatible scaffolding materials play an important role in bone tissue engineering. This study sought to assess the biocompatibility and osteoinductivity of a chitosan (CS)/collagenI(ColI)/multi-walled carbon nanotube (MWCNT) composite scaffold blended with bone morphogenetic protein-6 (BMP-6) and seeded with bone marrow mesenchymal stem cells (BMMSCs). The composite was fabricated using varying quantities of MWCNT to determine the appropriate combination, and combined with BMP-6 by blending and vacuum freeze-drying. Scanning electron microscopy (SEM) was used to observe the morphology of the CS/ColI/MWCNT (CCM) scaffolds. The porosity and mechanical properties of the composite scaffolds were measured. Scaffolds were seeded with BMMSCs in vitro and analyzed using SEM, CCK-8 assay and alkaline phosphatase (ALP) activity. To assess the in vivo utility of the composite scaffolds, critical-sized calvarial bone defects were prepared in 15 Sprague–Dawley rats and implanted with pre-seeded scaffolds (the CCM scaffold+BMMSCs group and the BMP-6/CCM scaffold+BMMSCs group) or left untreated (control). Three-dimensional computerized tomography and hematoxylin–eosin (HE) staining were used to evaluate bone formation at 8 weeks postoperatively. We found that higher concentrations of MWCNT decreased the porosity of the scaffold but increased its mechanical strength. In vitro, we found that BMP-6/CCM scaffolds provided the best conditions for BMMSCs proliferation and osteogenic differentiation. Similarly, BMP-6/CCM scaffolds seeded with BMMSCs could more effectively induce new bone formation in critical-sized calvarial bone defects compared with the other three groups. The BMP-6/CCM scaffold possesses good biocompatibility and induces bone formation in vitro and in vivo.

The mandible is commonly disturbed by medication-related osteonecrosis of the jaws (MRONJ), and preservation of inferior alveolar nerve (IAN) function is critical after surgery. A piezoelectric device is an ultrasound instrument that can remove the bone without soft tissue damage. We evaluated the usefulness of the piezoelectric device for the removal of a sequestrum involving the IAN in MRONJ patients with comparison to a conventional device such as rotary burs. Piezoelectric device group (n=12) and conventional device group (n=8) were compared for age, sex, disease, medication, cause, stage, operation time, hospitalization, prognosis, and subjective neurological dysfunction of the mental region with a question. In the piezoelectric device group, the neurological function was also assessed objectively with neurosensory testing. Both groups were similar in age distributions and MRONJ stage. The piezoelectric device group tended to be associated with female patients with osteoporosis and those receiving oral bisphosphonates; however, no significant differences. A comparison of the clinical outcomes showed a tendency of the reduction in the operation time and hospitalization period in the piezoelectric device group; however, no significant difference. Almost good prognosis was obtained in both groups. Subjective neurosensory dysfunction of the mental region was observed in both groups at 1 week postoperation, but the incident rate was lower in the piezoelectric device group. At 8 weeks postoperation, no patients in the piezoelectric device group showed dysfunction while 2 patients in the conventional device group showed dysfunction. In the piezoelectric device group, objective neurosensory tests of the IAN showed 2 patients of dysfunction at 1 week postoperation, but improved within 4 weeks postoperation. Piezoelectric surgery safely removed the sequestrum adjacent to the IAN with good prognosis in MRONJ patients. Our results indicate that piezoelectric surgery may be helpful in shortening the operation and hospitalization time, and reducing the risk of nerve injury.

The aim of this study was to evaluate the effect of marsupialization therapy for unicystic ameloblastoma by using three-dimensional (3-D) volumetric analysis with OsiriX software. This retrospective study assessed patients with unicystic ameloblastoma treated using marsupialization. Four patients (age, 29.00 ± 21.65 years) were included in this study, all of whom had mandibular ameloblastoma. The period between computed tomography before and after marsupialization was 5.75 ± 1.71 (4–8) months. In 3-D volumetric analysis using OsiriX software, the mean volume of the lesions before marsupialization was 22.91 ± 28.74 (6.23–65.86) cm³, and that after marsupialization was 11.28 ± 15.60 (2.19–34.62) cm³. The mean reduction in volume was 11.63 ± 13.16 (4.03–31.24) cm³, and the volumetric reduction ratio was 56.42 ± 7.60 (47.43–64.76)%. Additional conservative treatments were performed after marsupialization; however, radical resection with surgical margins was avoided because of the reduction in the volume of the ameloblastoma, owing to marsupialization, in all cases. In conclusion, marsupialization contributed to reducing the volume of unicystic ameloblastoma and avoiding radical resection, which reduces patients’ quality of life. The results suggested that marsupialization should be favored over other methods in the treatment of unicystic ameloblastoma.