Chihiro Masaki

The Effects of Low-Intensity Pulsed Ultrasound Exposure on Gingival Cells

BACKGROUND Low-intensity pulsed ultrasound (LIPUS) has been used in fracture treatment to shorten the time needed for biologic wound healing. Clinical trials applying LIPUS in implant dentistry have reported accelerated soft-tissue healing and osseointegration. However, details of the clinical effects of LIPUS have not been well characterized. Connective tissue growth factor (CCN2/CTGF) plays an important role in wound healing and angiogenesis in periodontal tissue. In this study, we focus on the effect of LIPUS on gingival epithelial cells and the role of CCN2/CTGF therein. METHODS Gingival epithelial cells (GE1) were cultured in six-well cell-culture plates for 24 hours at 37°C with 5% CO(2), and then exposed to LIPUS for 15 minutes at 3-MHz frequency and 40-mW/cm(2) power. Total RNA was extracted after LIPUS exposure and analyzed by real-time polymerase chain reaction to detect CCN2/CTGF. Additionally, total protein from each sample after LIPUS exposure was immunoblotted with anti-CCN2/CTGF antibody. RESULTS LIPUS exposure increased the mRNA level of CCN2/CTGF on exposure, and the level was significantly greater at 0 and 15 minutes after LIPUS exposure compared to the control. Western blotting analysis showed intense staining of CCN2/CTGF for 60 minutes after LIPUS exposure. The results demonstrate that LIPUS exposure accelerates soft-tissue healing by increasing CCN2/CTGF on exposure, in addition to its effects on bone formation. CONCLUSION Our findings demonstrate that LIPUS exposure accelerates soft-tissue healing by increasing connective tissue growth factors via a mitogen-activated protein kinase signaling pathway on exposure.

Promotion of insulin-induced glucose uptake in C2C12 myotubes by osteocalcin.

A close relationship between the bone and systemic glucose metabolism has recently been the center of attention, since the uncarboxylated form of osteocalcin (GluOC), a bone-derived protein, but not the γ-carboxylated form, is involved in glucose metabolism. However, the analysis of GluOC effect using isolated organs and related cell lines are required to understand its roles in a whole systemic metabolic status. In the present study, we examined the effect of GluOC on cell lines derived from skeletal muscle to explore the mechanisms by which GluOC regulates glucose uptake. In the differentiated C2C12 myotubes, GluOC dose-dependently induced the phosphorylation of ERK without affecting intracellular cAMP and Ca(2+) levels. This effect was inhibited by U0126, an inhibitor of ERK kinase (MEK). Additionally, U73122, an inhibitor of phospholipase C tended to inhibit it as well. Furthermore, cell treatment with GluOC for a long period promoted insulin-induced Akt phosphorylation and glucose uptake in the myotubes, which was abolished by ERK signaling inhibition. These results indicate that GluOC does not triggered Akt phosphorylation and glucose uptake by itself but promotes insulin-induced glucose uptake in myotubes, probably by up-regulating Akt signaling through ERK activation.

Strategies for alveolar ridge reconstruction and preservation for implant therapy

PURPOSE In dental implant treatment, ridge preservation and immediate or early implant placement are recommended to minimize bone resorption after tooth extraction and achieve esthetic outcomes. However, there is no consensus concerning the efficacy of this surgical method. There is also no consensus on the efficacy of bone and soft tissue grafts and surgical methods for alveolar ridge reconstruction. STUDY SELECTION This paper reports ridge alteration in the anterior maxilla after tooth extraction, and summarizes the efficacy of various ridge preservation methods and immediate or early implant placement as alveolar ridge preservation methods to minimize bone resorption after tooth extraction. The advantages and complications of alveolar ridge reconstruction methods, and the efficacy and surgical method of soft tissue graft are reviewed. RESULTS The anterior maxilla is in the esthetic zone, and the thickness of the bone on the labial side around the natural tooth is less than 1mm in many cases. Therefore, it is impossible to prevent bone resorption completely, even if ridge preservation and immediate or early implant placement are performed after tooth extraction. It is necessary to obtain stable and long-term esthetics by combining connective tissue and free gingival grafts, in addition to hard tissue augmentation. CONCLUSIONS It is important to consider the burden and level of satisfaction of patients, such as in terms of donor site morbidity in hard and soft tissue grafting, and to pay attention to appropriate indications to avoid overtreatment.

Low-intensity pulsed ultrasound enhances palatal mucosa wound healing in rats

PURPOSE Low-intensity pulsed ultrasound (LIPUS) has been used in fracture treatment to shorten the time needed for biological wound healing. However, the influence of LIPUS exposure on oral wound healing has not been sufficiently investigated. This study was conducted to evaluate low-intensity pulsed ultrasound on wound healing in palatal excisional wounds of rats. METHODS Excisional wounds, 5 mm in diameter, were made in the center of the palate of rats. Animals were divided into four experimental and control groups (1-week after LIPUS exposure, 1-week control, 2-week after LIPUS exposure, and 2-week control). The affected area in the experimental group was exposed to LIPUS, daily frequency: 3 MHz, intensity: 160 mW, exposure time: 15 min. Specimens were fixed in 10% neutral formalin solution immediately after sacrifice. The wound was measured histologically. RESULTS Wound width in the LIPUS group tended to be smaller than that of the control group. The experimental group in both 1-week and 2-week groups showed that unhealed areas were significantly smaller by LIPUS than those in the control groups (P<0.05). CONCLUSION Our results suggest that the use of LIPUS on palatal excisional wounds was effective in promoting epithelial and connective tissue closure.

Soft Tissue Biological Response to Zirconia and Metal Implant Abutments Compared With Natural Tooth: Microcirculation Monitoring as a Novel Bioindicator

Introduction:Zirconia is often used for implant abutments for esthetics. The aim of this clinical study was to compare the effects of zirconia and metal abutments on periimplant soft tissue. Materials and Methods:Ten maxillary anterior implant patients, 5 with metal abutments and 5 with zirconia abutments, were enrolled in this trial. The soft tissue around the implant abutments was evaluated by 2-dimensional laser speckle imaging and thermography. The blood flow in soft tissue around natural teeth was also measured to correct for differences among the subjects. Results:Significantly greater blood flow was detected in the zirconia abutment group (95.64 ± 5.17%) relative to the metal abutment group (82.25 ± 8.92%) in free gingiva (P = 0.0317). Reduced blood flow (by almost 18%) was detected in the tissue surrounding metal abutments compared with the tissue surrounding natural teeth. The surface temperature showed no significant difference for all measurements. Conclusions:These results suggest that blood flow in tissue surrounding zirconia abutments is similar to that in soft tissue around natural teeth. Moreover, zirconia abutments could be advantageous for the maintenance of immune function by improving blood circulation.

Cevimeline-Induced Monophasic Salivation from the Mouse Submandibular Gland: Decreased Na+ Content in Saliva Results from Specific and Early Activation of Na+/H+ Exchange

Cevimeline and pilocarpine are muscarinic agonists used clinically to treat dry mouth. In this study, we explored fluid secretion from mouse submandibular glands to determine the mechanism of cevimeline, pilocarpine, and an experimentally used agent carbachol. Cevimeline evoked almost the same amount of secretion at concentrations from 30 μM to 1 mM. Pilocarpine also induced secretion at a concentration as low as 1 μM and was the most powerful secretagogue at 10 μM. Secretion was induced by carbachol at 0.1 μM, with maximum secretion at 1.0 μM. Cevimeline induced monophasic secretion at all concentrations tested, whereas higher concentrations of pilocarpine and carbachol induced secretion with variable kinetics, i.e., an initial transient high flow rate, followed by decreased secretion after 2 to 3 min. In the presence of an epithelial Na+ channel blocker, amiloride, neither carbachol nor pilocarpine affected the Na+ level of secreted saliva; however, it significantly increased the Na+ content of cevimeline-induced saliva. The intracellular Ca2+ response of acinar cells was almost identical among all three agents, although recovery after drug removal was slower for cevimeline and pilocarpine. A profound decrease in intracellular pH was observed during pilocarpine and carbachol treatment, whereas intracellular acidification induced by cevimeline was only seen in the presence of a Na+/H+ exchange inhibitor. When external HCO3− was removed, cevimeline-induced saliva significantly decreased. These findings suggest that cevimeline specifically activates Na+/H+ exchange and may promote Na+ reabsorption by stabilizing epithelial sodium channel activity.

Bone Aging by Advanced Glycation End Products A Multiscale Mechanical Analysis

The quality and quantity of mandibular bone are essential prerequisites for osseointegrated implants. Only the Hounsfield unit on preoperative computed tomography is currently used as a clinical index. Nevertheless, a considerable mismatch occurs between bone quality and the Hounsfield unit. Loss of bone toughness during aging has been accepted based on empirical evidence, but this concept is unlikely evidence based at the level of mechanical properties. Nonenzymatic bone matrix cross-links associated with advanced glycation end products predominate as a consequence of aging. Thus, loss of tissue integrity could diminish the bone toughening mechanism. Here, we demonstrate an impaired bone toughening mechanism caused by mimicking aging in rabbits on a methionine-rich diet, which enabled an enhanced nonenzymatically cross-linked bone matrix. A 3-point bending test revealed a greater reduction in femoral fracture resistance in rabbits on a methionine-rich diet, despite higher maximum and normalized breaking forces (287.3 N and 88.1%, respectively), than in rabbits on a normal diet (262.2 N and 79.7%, respectively). In situ nanoindentation on mandibular cortical bone obtained from rabbits on a methionine-rich diet did not enable strain rate–dependent stiffening and consequent large-dimensional recovery during rapid loading following constant displacement after a rapid-load indentation test as compared with those in rabbits on a normal diet. Such nanoscale structure-function relationships dictate resistance to cracking propagation at the material level and allow for the overall bone toughening mechanism to operate under large external stressors. The strain-dependent stiffening was likely associated with strain-energy transfer to the superior cross-linked bone matrix network of the normal diet, while the reduction in the enzymatically cross-linked matrix in bone samples from rabbits on a methionine-rich diet likely diminished the intrinsic bone toughening mechanism. The present study also provides a precise protocol for evaluating bone mechanical properties at the material level based on observations from a series of nanoindentation experiments.

Comparison of plaque accumulation and soft‐tissue blood flow with the use of full‐arch implant‐supported fixed prostheses with mucosal surfaces of different materials: a randomized clinical study

OBJECTIVE The aims of this clinical study were to determine differences in plaque accumulation and to compare the effects of reinforced composite resin and titanium on peri-implant soft-tissue and residual-ridge inflammation. MATERIAL AND METHODS A total of 19 subjects were enrolled in this clinical trial; 10 jaws had implant-supported fixed prostheses with composite resin mucosal surfaces, 11 jaws had titanium prostheses fabricated by computer-aided design/computer-aided manufacture (CAD/CAM), and 6 jaws had acrylic resin prostheses. Plaque area indexes (PAIs) were calculated on the mucosal surfaces of prostheses, and blood flow in the mucosa was captured with two-dimensional laser speckle imaging to evaluate residual-ridge inflammation. Subjects were educated about oral hygiene and reevaluated after 3 months. RESULTS The PAI was significantly lower on titanium mucosal surfaces than on reinforced composite resin surfaces at the initial and second measurements (initial, P = 0.0052; second, P = 0.0044). Self-curing acrylic resin surfaces did not show any significant difference when compared with reinforced resin or titanium. Blood flow was significantly lower in mucosa contacting titanium surfaces than in mucosa contacting reinforced composite resin surfaces at the initial measurement (P = 0.0330). Although subjects were instructed about plaque control after the initial measurement, PAIs indicated that the difference between the two materials could not be overcome. CONCLUSION In terms of oral hygiene and mucosal inflammation, titanium was superior to reinforced composite resin in implant-supported fixed prostheses for edentulous subjects, and the short-term use of acrylic resin was superior to the use of reinforced composite resin.

Non-randomized controlled prospective study on perioperative levels of stress and dysautonomia during dental implant surgery

PURPOSE The purpose of this study was to compare pre- and postoperative autonomic activities and changes in salivary stress biomarkers between patients who received only local anesthesia and those who received local anesthesia together with intravenous sedation in dental implant surgery. METHODS A total of 21 patients were enrolled in this non-randomized controlled prospective study; 7 subjects underwent implant surgery under local anesthesia with intravenous sedation and 14 subjects underwent surgery under only local anesthesia. Stress was evaluated by measuring salivary levels of chromogranin A (CgA) and a spectral analysis of heart rate variability (HRV) at baseline (on a day other than the day of surgery), 1h preoperatively, and 1h postoperatively. HRV analysis yields low- (LF) and high-frequency (HF) components, the LF/HF ratio, and the component coefficient of variance (CCV[HF]), which provide indices of sympathetic and parasympathetic regulatory activity. RESULTS CgA levels were significantly higher (p<0.05) at baseline in patients who received sedation than those who did not, but CgA levels did not differ prior to surgery. Also, the values of most parameters, including LF, HF, LF/HF (L/H), and CCV(HF), did not significantly differ between groups or among the three time points. Only ΔL/H and ΔCCV(HF) were significantly lower (p<0.05) at 1h preoperatively in patients who received sedation than those who received only local anesthesia. CONCLUSIONS CgA levels were high in both groups immediately before surgery, and thus CgA values immediately before surgery may not be a reliable indicator of the need for intravenous sedation. Also, spectral analysis of HRV, especially ΔL/H and ΔCCV(HF), could be useful for assessing tension and anxiety.

Na+-K+-2Cl− cotransporter-mediated fluid secretion increases under hypotonic osmolarity in the mouse submandibular salivary gland

Water-handling epithelia are sensitive to the osmotic environment. In this study, the effects of a hypo-osmotic challenge on carbachol (CCh)-induced fluid secretion was investigated using an ex vivo submandibular gland perfusion technique and intracellular pH and Ca(2+) measurements. The osmolality of the perfusion solution was altered to examine the response of the gland to a hypotonic challenge. The flow rate was increased by 34% with a 30% hypotonic solution (225 mosmol/kgH2O), although the Ca(2+) response was unchanged. The lowering of the external Cl(-) by 50% abolished this increase in the 30% hypotonic solution. Furthermore, bumetanide, an inhibitor of the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1), completely inhibited the fluid secretion increase caused by the 30% hypotonic solution, and both the total amount of fluid and the flow rate were identical to those of the isotonic solution. This finding was confirmed by measuring the NKCC1 bumetanide-dependent NH4 (+) transport; Na(+)-K(+)-2Cl(-) transport was upregulated >40% by a 30% hypotonic challenge. Therefore, the increase in CCh-induced fluid secretion in response to hypotonic conditions can be attributed, to a large extent, to the specific activation of the NKCC1.