AMPK activation enhances osteoblast differentiation on a titanium disc via autophagy.

PURPOSE: The acquisition of osseointegration during implant therapy is slower and poorer in patients with diabetes compared with healthy persons. The serum concentration of adiponectin in patients with type II diabetes is lower than that of healthy persons via the suppression of AMP-activated protein kinase (AMPK). Therefore, we hypothesized that the AMPK activation enhances bone formation around implants, resulting in the improved acquisition of osseointegration. The purpose of this study was to evaluate the impact of AMPK activation on osteoblast differentiation and its mechanism of downstream signaling on titanium disc (Ti). METHODS: Confluent mouse pre-osteoblasts (MC3T3-E1) cells (1 × 105 cells/well) were cultured with BMP-2 for osteoblast differentiation, in the presence or absence AICAR, an AMPK activator. We examined the effects of AMPK activation on osteoblast differentiation and the underlying mechanism on a Ti using a CCK8 assay, a luciferase assay, quantitative RT-PCR, and western blotting. RESULTS: Although the proliferation rate of osteoblasts was not different between a Ti and a tissue culture polystyrene dish, the addition of AICAR, AMPK activator slightly enhanced osteoblast proliferation on the Ti. AICAR enhanced the BMP-2-dependent transcriptional activity on the Ti, leading to upregulation in the expression of osteogenesis-associated molecules. AICAR simultaneously upregulated the expression of autophagy-associated molecules on the Ti, especially LC3-II. AdipoRon, an adiponectin receptor type1/type2 activator activated AMPK, and upregulated osteogenesis-associated molecules on Ti. CONCLUSIONS: AMPK activation enhances osteoblast differentiation on a Ti via autophagy, suggesting that it promotes the acquisition of osseointegration during implant therapy.

The effect of AMP kinase activation on differentiation and maturation of osteoblast cultured on titanium plate.

Background/purpose: 5' Adenosine monophosphate-activated protein kinase (AMPK) is known as an enzyme that maintains intracellular homeostasis and has various biological activity. The purpose of this study is evaluation effect of AMPK activation on implant prognosis. Materials & methods: MC3T3-E1 osteoblast-like cells were cultured on titanium using a 24-well plate. The experimental group was divided into the following 3 groups: (1) the normal culture group (control group), (2) the osteogenic induction group, and (3) the osteogenic induction + AMPK activation group. The cell counts were measured; real-time PCR was used to assess the expression of ALP and Osterix as osteogenic related genes at Day 0,7,14 and 21 after experiments. Additionally, ALP activity and calcification were assessed. Results: The results of the real-time PCR assessments revealed that the expression of ALP, which is a marker for the initial stages of calcification, was significantly increased by AMPK activation compared to the normal culture or osteogenic induction. A significant increase was also observed in the expression of Osterix, which is a marker for the later stages of calcification. Because significant increases were observed in ALP activity and calcification potential, this suggested that AMPK activation could elicit an increase in osteoblast calcification potential. Conclusion: AMPK activation promotes implant peripheral osteoblast differentiation and maturation and enhances calcification. Our results suggest that AMPK activation may help to maintain implant stability.

Space-making effect for new bone formation by suppressing scar contraction of mucosal epithelium of rat tooth extraction wound using diode laser and CO2 laser treatment.

Background/purpose: As an extraction wound closes, the mucosal epithelium of the tooth extraction wound impedes the space for new bone formation by invading into the extraction socket. Thus, the height of the alveolar crest decreases, causing significant depression of the alveolar mucosa. In this study, we created a rat tooth extraction model and examined the effects of laser irradiation by CO2 and diode on the dynamics of myofibroblast expression through α-SMA, and TGF-ß1. Materials and methods: After tooth extraction of five-week-old male Wistar rats, they were divided into two laser treatment groups (CO2 laser or diode laser was irradiated into tooth extraction socket) and non-laser treatment group (control group). Surrounding tissues, including the extraction socket, were removed at 3, 5, 7, and 21 days after tooth extraction and the expression of α-SMA and TGF-ß1 was verified using immunohistological techniques (6 animals in each group and each period, 72 animals in total). Results: α-SMA-positive cells and TGF-ß1-positive areas were significantly lower in the two laser treatment groups than in the control group. Particularly, the diode group almost had no TGF-ß1-positive areas on the 21st day when healing after tooth extraction was deemed to be completed. Conclusion: Both CO2 and diode laser irradiation of tooth extraction wounds decreases α-SMA-positive cells and TGF-ß1-positive areas. Further, it causes a decrease in myofibroblast expression and suppresses the invasion of mucosal epithelium into the extraction socket. Therefore, laser irradiation may exert a space-making effect for new bone formation and also contribute to socket preservation.

Effects of ytterbium laser surface treatment on the bonding of two resin cements to zirconia.

Monolithic zirconia crowns bonded to zirconia abutments have become more commonly used in the construction of cement-retained implant superstructures. The present study aimed to examine the effects of laser surface treatments on the bond strength of two resin cements to zirconia. Three types of surfaces were examined: untreated, alumina blasted, and ytterbium laser treated; and two types of resin cements: 4-META/MMA-TBB resin cement and composite resin cement. Half of the specimens were subjected to a thermocycling process. Subsequently, a shear bond test was carried out. In addition, surface roughness was measured for each surface type. The results showed that laser treatment increased zirconia surface roughness and that laser treatment significantly increased shear bond strength after the thermocycling of both cement types compared to no treatment. Our experimental results suggested that ytterbium laser surface treatment of zirconia increased the bond strength of resin cements.

Bond durability and surface states of titanium, Ti-6Al-4V alloy, and zirconia for implant materials.

PURPOSE: Screw-retained implant crowns used as dental implants comprise a zirconia coping and titanium base bonded using resin cement. These devices are prone to debonding failures. This study investigated the bond characteristics of implant materials based on shear bond strength (SBS) and surface characteristics. METHODS: Chemically pure (CP) titanium grade-4 (Ti), Ti-6Al-4V alloy (Ti-6Al-4V), and tetragonal polycrystalline zirconia (zirconia) were evaluated as adherent materials. Plates of each material were polished, primed for the respective resin cements, and cemented using either methyl methacrylate-based resin cement (Super-Bond) or composite-based resin cement (Panavia). The cemented samples were subjected to 10,000 thermocycles alternating between 5 and 55 °C, and the SBS were obtained before and after thermocycling. The sample surfaces were characterized based on surface observations, roughness, and free energy (SFE). RESULTS: The SBSs of all materials bonded using Panavia were significantly compromised during thermocycling and reached zero. Although the SBSs of Ti and Ti-6Al-4V bonded using Super-Bond were not significantly affected by thermocycling, those of zirconia decreased significantly. The bond durability between zirconia and Super-Bond was improved via alumina air-abrasion, which caused no significant loss of SBS after thermocycling. Surface analyses of the air-abraded zirconia validated these results and confirmed that its surface roughness and SFE were significantly increased. CONCLUSION: The bond durability between resin cement and zirconia was lower than that between Ti and Ti-6Al-4V. The alumina air-abrasion pretreatment of zirconia improved the SFE and surface roughness, thereby enhancing bond durability.

Three-dimensional spheroids of dedifferentiated fat cells enhance bone regeneration.

INTRODUCTION: Mesenchymal stromal/stem cells (MSCs) are multipotent, self-renewing cells that are extensively used in tissue engineering. Dedifferentiated fat (DFAT) cells are derived from adipose tissues and are similar to MSCs. Three-dimensional (3D) spheroid cultures comprising MSCs mimic the biological microenvironment more accurately than two-dimensional cultures; however, it remains unclear whether DFAT cells in 3D spheroids possess high osteogenerative ability. Furthermore, it is unclear whether DFAT cells from 3D spheroids transplanted into calvarial bone defects are as effective as those from two-dimensional (2D) monolayers in promoting bone regeneration. METHODS: We compared the in vitro osteogenic potential of rat DFAT cells cultured under osteogenic conditions in 3D spheroids with that in 2D monolayers. Furthermore, to elucidate the ability of 3D spheroid DFAT cells to promote bone healing, we examined the in vivo osteogenic potential of transplanting DFAT cells from 3D spheroids or 2D monolayers into a rat calvarial defect model. RESULTS: Osteoblast differentiation stimulated by bone morphogenetic protein-2 (BMP-2) or osteogenesis-inducing medium upregulated osteogenesis-related molecules in 3D spheroid DFAT cells compared with 2D monolayer DFAT cells. BMP-2 activated phosphorylation in the canonical Smad 1/5 pathways in 3D spheroid DFAT cells but phosphorylated ERK1/2 and Smad2 in 2D monolayer DFAT cells. Regardless of osteogenic stimulation, the transplantation of 3D DFAT spheroid cells into rat calvarial defects promoted new bone formation at a greater extent than that of 2D DFAT cells. CONCLUSIONS: Compared with 2D DFAT cells, 3D DFAT spheroid cells promote osteoblast differentiation and new bone formation via canonical Smad 1/5 signaling pathways. These results indicate that transplantation of DFAT cells from 3D spheroids, but not 2D monolayers, accelerates bone healing.

Enhancement of jaw bone regeneration via ERK1/2 activation using dedifferentiated fat cells.

BACKGROUND AIMS: Mesenchymal stem/stromal cells (MSCs) are multipotent and self-renewing cells that are extensively used in tissue engineering. Adipose tissues are known to be the source of two types of MSCs; namely, adipose tissue-derived MSCs (ASCs) and dedifferentiated fat (DFAT) cells. Although ASCs are sometimes transplanted for clinical cytotherapy, the effects of DFAT cell transplantation on mandibular bone healing remain unclear. METHODS: The authors assessed whether DFAT cells have osteogenerative potential compared with ASCs in rats in vitro. In addition, to elucidate the ability of DFAT cells to regenerate the jaw bone, the authors examined the effects of DFAT cells on new bone formation in a mandibular defect model in (i) 30-week-old rats and (ii) ovariectomy-induced osteoporotic rats in vivo. RESULTS: Osteoblast differentiation with bone morphogenetic protein 2 (BMP-2) or osteogenesis-induced medium upregulated the osteogenesis-related molecules in DFAT cells compared with those in ASCs. BMP-2 activated the phosphorylation signaling pathways of ERK1/2 and Smad2 in DFAT cells, but minor Smad1/5/9 activation was noted in ASCs. The transplantation of DFAT cells into normal or ovariectomy-induced osteoporotic rats with mandibular defects promoted new bone formation compared with that seen with ASCs. CONCLUSIONS: DFAT cells promoted osteoblast differentiation and new bone formation through ERK1/2 and Smad2 signaling pathways in vitro. The transplantation of DFAT cells promoted new mandibular bone formation in vivo compared with that seen with ASCs. These results suggest that transplantation of ERK1/2-activated DFAT cells shorten the mandibular bone healing process in cytotherapy.

Intramedullary injury combined with osteoporosis therapeutics regulates targeted local osteogenesis.

Bone marrow ablation prompts transient bone formation in nearly the entire medullary cavity before marrow regeneration occurs. Here, we establish a procedure to direct bone formation in a desired particular site within the medullary cavity for support of biomedical devices. Local intramedullary injury was performed in the tibiae of rats and parathyroid hormone (PTH), alendronate, or saline was administered. Newly generated bone in the medulla was assessed by micro-CT and histology. To evaluate the function of newly generated bone, animals received intramedullary injury in tibiae followed by daily PTH. At day-14, implants were placed in the endocortical bone and the bone response to the implants was assessed. The fate of newly generated bone was compared with and without implants. We found that neither intramedullary injury nor medication alone resulted in bone formation. However, when combined, substantial bone was generated locally inside the diaphyseal medulla. Newly formed bone disappeared without implant placement but was retained with implants. Bone was especially retained around and between the implants. This study found that local bone marrow disruption followed by PTH or alendronate generated substantial cancellous bone locally in the diaphyseal medulla. This approach offers promise as a tissue engineering tool in medicine and dentistry.

Third molar transplantation combined with an osteotome sinus lift - two case reports.

We report here on our application of the socket lift technique to create a transplant socket for the transplanted maxillary molar. These outcomes suggest that this technique is likely to be effective for tooth transplantation when the maxillary sinus bottom is close to the alveolar crest.

Accelerated Osteogenic Differentiation and Bone Formation on Zirconia with Surface Grooves Created with Fiber Laser Irradiation.

BACKGROUND: Modification of the surface topography of biomaterials is a critical factor for the proliferation and differentiation of osteoblasts. Elucidating the biological response to surface roughening is necessary for clinical application of zirconia implants. PURPOSE: To investigate the effects of fiber laser-induced zirconia surface roughening on cultured osteoblast-like cell morphology, proliferation, differentiation, and calcification, and on in vivo bone formation. MATERIALS AND METHODS: Sixty-six machine-surfaced yttria-tetragonal zirconia polycrystal plates (S-Zr) and 16 machine-surfaced implants were used as controls. We prepared 66 rough plates (R-Zr) and 16 rough implants by surface treatment using a fiber laser. RESULTS: MC3T3-E1 cells spread well in all directions on S-Zr, whereas elongated cells with poorly organized actin stress fibers were observed on R-Zr. Cell proliferation was significantly greater on R-Zr than on S-Zr. The Runx2 mRNA level increased time dependently in osteogenic culture condition. Alkaline phosphatase activity and osteocalcin mRNA levels were higher on R-Zr compared with S-Zr. Alizarin red S staining revealed greater calcification on R-Zr than on S-Zr. Laser treatment of zirconia implant bodies placed in rat tibiae increased the bone-implant contact ratio and removal torque considerably. CONCLUSIONS: Our results suggest that fiber laser irradiation produces adequate surface roughening of zirconia ceramics to support osseointegration.

Human mandibular trabecular bone density correlation with mechanical strength: implications for implant dentistry.

PURPOSE: The prognosis of a dental implant depends on the mechanical properties of the bone. The preoperative diagnosis of bone quality has become more important, because the immediate loading procedure is now widely used, and a firm initial stability is required. Quantitative computed tomography (QCT) is an effective method of determining selectively the bone mineral density (BMD) of the trabecular bone mostly responsible for the survival of the implant. In this project, we investigated the relationship between the BMD measured by QCT and the mechanical strength of the mandible. MATERIALS AND METHODS: The BMD (mg/mL) of 58 trabecular bone specimens from 6 embalmed human cadaver mandibles were obtained using QCT. The compressive strength to break point was performed with a mechanical device. Each specimen was washed and weighed. RESULTS: The mean values of BMD for the anterior, premolar, and molar region were 633.3, 571.0, and 518.3 mg/mL, respectively. The mean values of compressive strength were 0.237, 0.216, and 0.196 kN, and the ash weights were 0.047, 0.044, and 0.039 g, respectively. There was a positive correlation between the BMD and the mechanical strength (R = 0.77) and between the ash weight and the mechanical strength (R = 0.84). CONCLUSION: There was strong correlation of bone strength, bone mineral, and bone density by QCT. QCT is an excellent preoperative diagnostic tool to select the most mechanically appropriate implant for initial stability and improve the survival prognosis.

Students' opinion of a predoctoral implant training program.

Implant treatment today is highly predictable and provides valid restorative options for the completely or partially edentulous patient. In Japan, many dental care facilities have incorporated implant treatment, and such treatment is no longer rare. For predoctoral students, the educational environment related to implants is not always applicable in present clinical settings. In this article, we describe the implant training program developed at our university for predoctoral education, and we report the changes in student opinions regarding implant treatment by comparing pre- and post-training opinions. The newly developed models for implant training were effective in increasing student understanding that implant treatment is one prosthetic option for restoring missing teeth. In a survey of predoctoral students, responses indicating negative opinions toward implant treatment decreased after training, and responses indicating positive opinions increased. These findings indicated that this training was effective in deepening student understanding of implant treatment.