Influence of osteoporosis and mechanical loading on bone around osseointegrated dental implants: A rodent study.

This study aimed to evaluate the influence of estrogen deficiency and mechanical loading on bone around osseointegrated dental implants in a rat jaw model. The maxillary right first molars of 36 rats were extracted. One week later, the rats were divided into an unloaded group and a loaded group; short head implants and long head implants were inserted respectively. Nine weeks after implantation, the rats were further subjected to ovariectomy (OVX) or sham surgery. All animals were euthanized 21 weeks after OVX. Micro-computed tomography, histological and histomorphometrical evaluation were undertaken. Systemic bone mineral density and bone volume fraction decreased in OVX groups compared with the sham controls. Histomorphometrical observation indicated that unloaded OVX group showed significantly damaged osseointegration and bone loss versus the loaded OVX group. Both the bone density (BD) inside the peri-implant grooves and the percentage of bone-to-implant contact (BIC) were lower in the OVX groups than in the sham-surgery groups, although mechanical loading increased the BIC and BD in the loaded OVX group compared with the unloaded OVX group. An increased number of positive cells for tartrate-resistant acid phosphatase was observed in the OVX groups versus the sham controls. The percentage of sclerostin-positive osteocytes was lower under loaded compared with unloaded conditions in both the OVX groups and the sham controls. In conclusion, estrogen deficiency could be a risk factor for the long-term stability of osseointegrated implants, while mechanical loading could attenuate the negative influence of estrogen deficiency on bone formation and osseointegration.

Localization of Integrin Beta-4 Subunit at Soft Tissue-Titanium or Zirconia Interface.

Currently, along with titanium (Ti), zirconia is widely used as an abutment material for dental implants because it makes it possible to avoid gingival discoloration; however, the epithelial sealing capability of zirconia remains unknown. The purpose of the present study is to elucidate the localization of integrin ß4 subunit (Inß4), one of the main proteins in the attachment structure between gingival junctional epithelial (JE) cells and substrata. Maxillary first molars were extracted from rats, and implants were placed with Ti or zirconia transgingival parts; then, the localization of Inß4 was observed. Morphological and functional changes in rat oral epithelial cells (OECs) cultured on a culture dish (Dish) and Ti and zirconia plates were also evaluated with Inß4 immunofluorescence histochemistry and Western blotting. After four weeks of implant placement, the morphology of the peri-implant epithelium (PIE) and the localization of Inß4 around the Ti and zirconia transgingival parts were similar. However, both exhibited markedly shorter Inß4-positive bands in the PIE than in the JE around natural teeth. Decreased expression levels of Inß4 were observed in OECs cultured on Ti and zirconia plates compared with those cultured on Dish. In conclusion, although inferior to natural teeth, zirconia implants are thought to have epithelial sealing properties comparable to those of titanium.

Effect of a Single Injection of Benidipine-Impregnated Biodegradable Microcarriers on Bone and Gingival Healing at the Tooth Extraction Socket.

Objective: A dihydropyridine-type calcium channel blocker, benidipine (BD), is extensively used in hypertension therapy. In vitro study reported BD promoting bone metabolism. We evaluated the effect of sustained release of BD-loaded poly(lactic-co-glycolic acid) (PLGA) microcarriers on the promotion of bone and gingival healing at an extraction socket in vivo. In addition, the effect of BD on osteoblasts, osteocytes, fibroblasts, and epithelial cells was evaluated in vitro. Approach: The maxillary first molar of rats was extracted. Next, PLGA microcarriers containing BD were directly injected into the gingivobuccal fold as a single dose. After injection, bone and soft-tissue healing was histologically evaluated. Effect of BD on proliferation, migration, and gene expression of gingival and bone cell was also examined in vitro. Results: After tooth extraction, BD significantly augmented bone volume and density, and also epithelial wound healing. During in vitro studies, BD promoted significant proliferation and migration of fibroblasts and epithelial cells. Real-time RT-PCR revealed that BD upregulated messenger RNA expression of Ahsg (alpha 2-HS glycoprotein) and Csf2 (colony-stimulating factor 2) in osteoblasts. Innovation: The prevention of bone and soft-tissue reduction associated with tooth extraction has been eagerly anticipated in the field of dentistry. This study first reported the effect of BD on extraction socket healing. Conclusion: A single dose of topically administered BD-loaded PLGA microcarriers promoted bone and soft-tissue healing at the extraction site of tooth.

Injectable Porous Bioresorbable Composite Containing Fluvastatin for Bone Augmentation.

The purpose of this study was to evaluate the effects of an injectable composite made up of calcium sulfate (CAS), fluvastatin (FS), and atelocollagen on bone augmentation in rats. Porous structures and compressive strength of composites were evaluated. The cumulative release kinetics of FS were determined in vitro by a spectrophotometer. To observe bone regeneration in vivo, five different materials (normal saline; atelocollagen gel only; composite of CAS and atelocollagen; composite containing 0.5% FS; and composite containing 1.0% FS) were injected in extraction sockets and in the crania of rats. Microcomputed tomography and histological evaluation were performed after 2, 4, and 8 weeks of healing time. The composites had high porosity (greater than 55%). FS kept a slow and stable release for >30 days. In vivo results demonstrated that more new bone was formed in the FS groups compared with the other groups, and both bone mass and bone density had prominent increase in maxillae and crania. Resorption of the composite was also observed for cranial tissues. In conclusion, this composite can be applied percutaneously, without any incision. It has excellent properties with replaceability into bone and anabolic effects for bone formation, as well as a drug delivery system for bone formation.

Generation and histomorphometric evaluation of a novel fluvastatin-containing poly(lactic-co-glycolic acid) membrane for guided bone regeneration.

The purpose of this study was to evaluate the effects of a poly(lactic-co-glycolic acid) (PLGA) membrane containing fluvastatin on bone regeneration at bone defects in rat calvaria and tibia for possible use as a guided bone regeneration (GBR) membrane. PLGA and fluvastatin-containing PLGA (PLGA-fluvastatin) membranes were prepared and mechanical properties were evaluated. Standardized bony defects were created in rat calvaria and the right tibia, and covered with a PLGA or PLGA-fluvastatin membrane. Bone regeneration was evaluated using image analysis based on histologic examination. At 4 and 8 weeks after membrane implantation, the PLGA-fluvastatin group displayed enhanced new bone formation around the edge of the defect compared with the PLGA membrane group in the calvarial model. Thick bone regeneration was observed in tibia-defect sites in the PLGA-fluvastatin membrane group. These results suggest that the PLGA-containing fluvastatin membrane prepared in this study may potentially be used as a GBR membrane.

Mechanical loading induced osteocyte apoptosis and connexin 43 expression in three-dimensional cell culture and dental implant model.

Osteocytes are thought to act as stress sensors, and are known to display a gap junction-mediated stress-transfer mechanism. To demonstrate the stress-related function of osteocytes, cells of an osteocyte-like cell line derived from murine long bone osteocyte Y4 (MLO-Y4) were cultivated in a three-dimensional culture and subjected to cyclic loading from a titanium plate. This application of physiological loading using a titanium plate significantly increased connexin 43 (Cx43) expression, the number of dead and apoptotic cells, and receptor activator of nuclear factor κB ligand expression. Furthermore, the conditioned medium from the loaded osteocytes induced alkaline phosphatase activity in bone marrow cell culture. In addition, we immunohistologically determined whether bone metabolism increased as a result of the occlusal force in the bone surrounding the titanium implants in a rat model. Increased Cx43 expression and apoptotic osteocytes were observed in the loading group as well as a significantly increased number of tartrate-resistant acid phosphatase-positive cells. These findings indicate that stress from the implant adversely affected the osteocytes, which may promote osteoclastic and osteoblastic cell formation around the implants. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 815-827, 2019.

A peptide that blocks the interaction of NF-κB p65 subunit with Smad4 enhances BMP2-induced osteogenesis.

Bone morphogenetic protein (BMP) potentiates bone formation through the Smad signaling pathway in vitro and in vivo. The transcription factor nuclear factor κB (NF-κB) suppresses BMP-induced osteoblast differentiation. Recently, we identified that the transactivation (TA) 2 domain of p65, a main subunit of NF-κB, interacts with the mad homology (MH) 1 domain of Smad4 to inhibit BMP signaling. Therefore, we further attempted to identify the interacting regions of these two molecules at the amino acid level. We identified a region that we term the Smad4-binding domain (SBD), an amino-terminal region of TA2 that associates with the MH1 domain of Smad4. Cell-permeable SBD peptide blocked the association of p65 with Smad4 and enhanced BMP2-induced osteoblast differentiation and mineralization without affecting the phosphorylation of Smad1/5 or the activation of NF-κB signaling. SBD peptide enhanced the binding of the BMP2-inudced phosphorylated Smad1/5 on the promoter region of inhibitor of DNA binding 1 (Id-1) compared with control peptide. Although SBD peptide did not affect BMP2-induced chondrogenesis during ectopic bone formation, the peptide enhanced BMP2-induced ectopic bone formation in subcortical bone. Thus, the SBD peptide is useful for enabling BMP2-induced bone regeneration without inhibiting NF-κB activity.

Acceleration of hard and soft tissue healing in the oral cavity by a single transmucosal injection of fluvastatin-impregnated poly (lactic-co-glycolic acid) microspheres. An in vitro and rodent in vivo study.

Antihyperlipidemic drug statins reportedly promote both bone formation and soft tissue healing. We examined the effect of sustained-release, fluvastatin-impregnated poly(lactic-co-glycolic acid) (PLGA) microspheres on the promotion of bone and gingival healing at an extraction socket in vivo, and the effect of fluvastatin on epithelial cells and fibroblasts in vitro. The maxillary right first molar was extracted in rats, then one of the following was immediately injected, as a single dose, into the gingivobuccal fold: control (no administration), PLGA microspheres without a statin (active control), or PLGA microspheres containing 20 or 40 µg kg(-1) of fluvastatin. At days 1, 3, 7, 14, and 28 after injection, bone and soft tissue healing were histologically evaluated. Cell proliferation was measured under the effect of fluvastatin at dosages of 0, 0.01, 0.1, 1.0, 10, and 50 µM. Cell migration and morphology were observed at dosages of 0 and 0.1 µM. Following tooth extraction, the statin significantly enhanced bone volume and density, connective tissue volume, and epithelial wound healing. In the in vitro study, it promoted significant proliferation and migration of epithelial cells and fibroblasts. A single dose of topically administered fluvastatin-impregnated PLGA microspheres promoted bone and soft tissue healing at the extraction site.

Bending of protonema cells in a plastid glycolate/glycerate transporter knockout line of Physcomitrella patens.

Arabidopsis LrgB (synonym PLGG1) is a plastid glycolate/glycerate transporter associated with recycling of 2-phosphoglycolate generated via the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). We isolated two homologous genes (PpLrgB1 and B2) from the moss Physcomitrella patens. Phylogenetic tree analysis showed that PpLrgB1 was monophyletic with LrgB proteins of land plants, whereas PpLrgB2 was divergent from the green plant lineage. Experiments with PpLrgB-GFP fusion proteins suggested that both PpLrgB1 and B2 proteins were located in chloroplasts. We generated PpLrgB single (∆B1 and ∆B2) and double (∆B1/∆B2)-knockout lines using gene targeting of P. patens. The ∆B1 plants showed decreases in growth and photosynthetic activity, and their protonema cells were bent and accumulated glycolate. However, because ∆B2 and ∆B1/∆B2 plants showed no obvious phenotypic change relative to the wild-type or ∆B1 plants, respectively, the function of PpLrgB2 remains unclear. Arabidopsis LrgB could complement the ∆B1 phenotype, suggesting that the function of PpLrgB1 is the same as that of AtLrgB. When ∆B1 was grown under high-CO2 conditions, all novel phenotypes were suppressed. Moreover, protonema cells of wild-type plants exhibited a bending phenotype when cultured on media containing glycolate or glycerate, suggesting that accumulation of photorespiratory metabolites caused P. patens cells to bend.

In vivo and in vitro studies of epithelial cell behavior around titanium implants with machined and rough surfaces.

BACKGROUND: The surface roughness of a dental implant affects the epithelial wound healing process and may significantly enhance implant prognosis. PURPOSE: We explored the influence of surface roughness on peri-implant epithelium (PIE) sealing and down-growth by comparing machine-surfaced (Ms) and rough-surfaced (Rs) implants. MATERIALS AND METHODS: (1) Maxillary first molars were extracted from rats and replaced with Ms or Rs implants. (2) We also compared changes in the morphology of cultured rat oral epithelial cells (OECs) grown on Ms or Rs titanium (Ti) plates. RESULTS: (1) After 4 weeks, the PIE around Ms and Rs implants showed a similar structure to junctional epithelium (JE). At 16 weeks, Rs implants appeared to form a weak epithelial seal at the tissue-implant interface and exhibited markedly less PIE down-growth than Ms implants but was deeper than that observed in natural teeth. (2) We observed less expression of adhesion proteins in OECs cultured on Rs plates than in cells grown on Ms plates. Additionally, cell adherence, migration, and proliferation on Rs plates were lower, whereas apoptosis was reduced on Ms plates. CONCLUSION: Ms implants are a better choice for integration with an epithelial wound healing process.

The effect of low-magnitude, high-frequency vibration stimuli on the bone healing of rat incisor extraction socket.

Effects of small vibration stimuli on bone formation have been reported. In the present study, we used morphological and morphometric procedures to elucidate whether low-magnitude, high-frequency (LMHF) vibration stimuli could enhance the bone healing of rat incisor extraction sockets. After extraction of incisors from six-week-old rats, animals were assigned into a control group and two experimental groups to receive 50 Hz stimuli at either 0.05 mm or 0.2 mm peak-to-peak for an hour/day. LMHF vibration stimuli were generated by placing the mandibles of the animals onto a vibration generator. All groups were subdivided into two, according to the study periods (1 and 3 weeks). After the study period, undecalcified ground sections were taken and morphological and morphometric analyses performed. At both 1 and 3 weeks, newly formed bone was observed mainly in the upper wall of the extraction socket in all groups. Morphometric analyses revealed that the trabecular thickness in both experimental groups at 1 week was significantly greater than that in the control. LMHF vibration stimuli had a positive effect on bone at the early stage of bone healing, particularly in trabecular thickness, at the incisor extraction socket.

Evaluations of epithelial sealing and peri-implant epithelial down-growth around "step-type" implants.

OBJECTIVE: Implant designs that can stimulate and integrate with an epithelial wound-healing process may significantly enhance the efficacy of dental implants. Here, we evaluated the potential of "step-type" implant systems to improve the sealing between the peri-implant epithelium (PIE) and the implant surface, and investigated the effect of implant structure on PIE down-growth. MATERIALS AND METHODS: Right maxillary first molars were extirpated from rats and implanted with either a straight-type or a step-type implant varying in step height and/or width (N(s): 0.8 mm height, 0.1 mm width; W(s): 0.8 mm height, 0.2 mm width; H(s): 0.4 mm height, 0.1 mm width). Maxillae were harvested at various time points over 16 weeks to evaluate laminin-5 distribution as an indicator of wound healing and PIE formation, horse-radish peroxidase (HRP) penetration as a measurement of epithelial sealing, and PIE down-growth formation. RESULTS: In all implant models, the PIE formed from the oral sulcular epithelium and spread apically along the implant surface. In the W(s) group, HRP penetration was detected only in the coronal region of the PIE at 4 weeks, whereas in the straight-type, it was observed in the apical region and the connective tissue. At 16 weeks, the W(s) implants exhibited markedly less PIE down-growth than the Con, N(s) or H(s) implants, and were equivalent to that observed in natural teeth. CONCLUSION: The step-type implant system may have the potential for improving epithelial sealing at the tissue-implant interface, as well as reducing apical PIE down-growth, thus enhancing dental implant efficacy.

The effect of a single remote injection of statin-impregnated poly (lactic-co-glycolic acid) microspheres on osteogenesis around titanium implants in rat tibia.

The aim of this study was to evaluate the effects of newly developed injectable poly (lactic-co-glycolic acid) (PLGA) microspheres containing fluvastatin on osteogenesis around titanium implants in the rat tibia. After confirmation of the sustained-release profile of fluvastatin from the microspheres by an in vitro assay, the microspheres were administered to the back skin of the rats by a single transdermal injection. At 2 and 4 weeks after the implant surgery, the fluvastatin groups showed enhanced new bone formation around the titanium implants without any influence on the serum biochemistry. In addition, the fluvastatin groups showed increased three-point bending strengths of their femurs. The results of this study indicate that a single remote injection of PLGA/fluvastatin microspheres safely and successfully stimulated bone formation around titanium implants and increased the mechanical properties of bone.

Local application of fluvastatin improves peri-implant bone quantity and mechanical properties: a rodent study.

Statins are known to stimulate osteoblast activity and bone formation. This study examines whether local application of fluvastatin enhances osteogenesis around titanium implants in vivo. Ten-week-old rats received a vehicle gel (propylene glycol alginate (PGA)) or PGA containing fluvastatin (3, 15, 75 or 300 microg) in their tibiae just before insertion of the implants. For both histological and histomorphometric evaluations undecalcified ground sections were obtained and the bone-implant contact (BIC), peri-implant osteoid volume and mineralized bone volume (MBV) were calculated after 1, 2 and 4 weeks. Using the same models mechanical push-in tests were also performed to evaluate the implant fixation strength. After 1 week the MBV and push-in strength were significantly lower in the 300 microg fluvastatin-treated group than in the other groups (P<0.01). At 2 weeks, however, the BIC and MBV were both significantly higher in the 75 microg fluvastatin-treated group than in the non-fluvastatin-treated groups (P<0.01). Similar tendencies were observed at week 4. Furthermore, the data showed a good correlation between the MBV and the push-in strength. These results demonstrate positive effects of locally applied fluvastatin on the bone around titanium implants and suggest that this improvement in osseointegration may be attributed to calcification of the peri-implant bone.

Local application of statin promotes bone repair through the suppression of osteoclasts and the enhancement of osteoblasts at bone-healing sites in rats.

OBJECTIVE: We investigated whether the local administration of simvastatin affected both the cellular events and the bone formation at surgically created bone defects in rat. STUDY DESIGN: Simvastatin (or a vehicle) was injected into a rat bony defect for 3 consecutive days from the day of surgery. Five or ten days after the injection, new bone tissue was collected, and the gene expressions of bone-related proteins were examined. For the histomorphometry, new bone area was measured. RESULTS: At day 5, the statin group demonstrated significantly larger new bone area. The number of tartrate-resistant acid phosphatase-positive multinucleated cells in the statin group was less than in the control group. In the statin group, the expressions of both alkaline phosphatase and bone morphogenetic protein 2 mRNA significantly increased. In contrast, the expression of cathepsin K was significantly suppressed in the statin group. Although the levels of both RANK and osteoprotegerin were not affected by statin, the expression of RANKL was depressed. At day 10, there were no significant differences among the groups in either histomorphometric or reverse-transcription polymerase chain reaction analyses. CONCLUSION: New bone area increased under the influence of simvastatin; however, the effect did not continue when the administration was terminated. Osteoclast suppression may be the consequence of RANKL depression.

Topical application of statin affects bone healing around implants.

OBJECTIVES: 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) are widely used for hyperlipidemia. Recent studies demonstrate that statins stimulate bone morphogenetic protein-2 expression and lead to bone formation. The aim of this study was to evaluate whether the topical application of statin enhances the osteogenesis around a titanium implant. MATERIALS AND METHODS: Ten-week-old female rats received pure titanium rods in both tibiae with or without fluvastatin. Propylene glycol alginate (PGA) was used as a carrier. The rats were divided into five groups: implant-only group, implant with PGA group, low-dose group [implant+PGA containing 3 microg of fluvastatin (FS)], medium-dose group (15 microg of FS), and high-dose group (75 microg of FS). The animals were sacrificed at 1 and 2 weeks after implantation. Peri-implant bone formation was assessed by histomorphometric procedures, i.e., measuring the bone-implant contact (BIC) and peri-implant bone volume (BV). A mechanical push-out test was also performed to evaluate the implant fixation strength. Statistical differences among the groups were determined by ANOVA and P < 0.05 was considered significant. RESULTS: At week 1, there was no significant difference in BIC among the groups, however, BV and the push-out strength were significantly higher in the high-dose group than in the implant-only group. At week 2, BIC and BV had significantly increased in the high-dose group in comparison with the non-statin groups. The fluvastatin-treatment group showed a significant increase in push-out strength compared with the non-statin groups. CONCLUSION: Our histomorphometrical and mechanical evaluations revealed the positive effect of topically applied fluvastatin on the bone around the implant.