Yoichiro Ogino

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.

Simvastatin enhances bone formation around titanium implants in rat tibiae

Statins are cholesterol-lowering drugs that have been reported to promote bone formation. The purpose of this study was to investigate the effect of simvastatin on the enhancement of bone formation around titanium implants. Thirty-week-old female rats received pure titanium implants in both tibiae. The animals were intra-peritoneally administered 0, 0.125, 1, 5 or 10 mg kg(-1) of simvastatin daily. After 30 days, the animals were sacrificed, and specimens were prepared. The bone contact ratio of the implant, bone density in the medullary canal and percentage of cortical bone were obtained. Markers for bone turnover were also measured using sera collected at the time of euthanasia. In the medullary canal, a scanty amount of bone was observed in the 0, 0.125 and 1 mg kg(-1) groups. In contrast, in both the 5 and 10 mg kg(-1) groups, thicker bone trabeculae were abundant. Histometric observations showed that the bone contact ratio and the bone density of both groups were significantly greater than those of the other groups (anova, P < 0.01). However, no significant difference in the percentage of cortical bone was found between groups. Serum chemistry showed that statin increased bone formation markers and decreased bone resorption markers. In conclusion, although the dose equivalent to that used in human patients with hypercholesterolemia was not effective, a simvastatin dose of 5 mg kg(-1) or higher increased medullary bone formation around the titanium. In contrast, no effect of simvastatin on pre-existing cortical bone was indicated.

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.

Platelet-rich plasma suppresses osteoclastogenesis by promoting the secretion of osteoprotegerin

BACKGROUND AND OBJECTIVE Platelet-rich plasma is characterized by containing fundamental protein growth factors. Although many in vitro studies have documented the capability of platelet-rich plasma to induce the growth of osteoblasts or osteoblast-like cells, the effect of platelet-rich plasma on osteoclastogenesis has not yet been studied. The aim of the present study was to evaluate the effects of platelet-rich plasma and platelet-poor plasma on osteoclastogenesis with rat bone marrow cell culture. MATERIAL AND METHODS Platelet-rich plasma and platelet-poor plasma were produced from the whole blood of rat. For cell culture, rat bone marrow cells were isolated from rat tibiae and then treated with 1,25alpha dihydroxy vitamin D(3) and with different concentrations of platelet-rich plasma or platelet-poor plasma. After 4 d of culture, rat bone marrow cells were stained with tartrate-resistant acid phosphatase (TRAP), and TRAP-positive cells that had more than three nuclei (TRAP-positive multinucleated cells) were counted as osteoclast-like cells. Osteoprotegerin, known as an osteoclastogenesis-related factor, cells was quantified using an enzyme-linked immunosorbent assay (ELISA). RESULTS Although platelet-poor plasma had no effect on the formation of TRAP-positive multinucleated cells, platelet-rich plasma decreased the number of TRAP-positive multinucleated cells in a dose-dependent manner. The amount of osteoprotegerin produced from rat bone marrow cells and from MC3T3-E1 cells was enhanced in platelet-rich plasma-treated groups. CONCLUSION Under our experimental conditions, platelet-rich plasma decreased the formation of TRAP-positive multinucleated cells and increased the secretion of osteoprotegerin. This study suggests that platelet-rich plasma suppresses osteoclastogenesis, therefore inhibiting bone resorption. In addition we also demonstrated that platelet-rich plasma increased the secretion of osteoprotegerin, an inhibitor for osteoclast formation, thus suggesting that the enhancement of osteoprotegerin secretion induces this inhibitory effect.

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.

RhoA‐Mediated Functions in C3H10T1/2 Osteoprogenitors Are Substrate Topography‐Dependent

Surface topography broadly influences cellular responses. Adherent cell activities are regulated, in part, by RhoA, a member of the Rho‐family of GTPases. In this study, we evaluated the influence of surface topography on RhoA activity and associated cellular functions. The murine mesenchymal stem cell line C3H10T1/2 cells (osteoprogenitor cells) were cultured on titanium substrates with smooth topography (S), microtopography (M), and nanotopography (N) to evaluate the effect of surface topography on RhoA‐mediated functions (cell spreading, adhesion, migration, and osteogenic differentiation). The influence of RhoA activity in the context of surface topography was also elucidated using RhoA pharmacologic inhibitor. Following adhesion, M and N adherent cells developed multiple projections, while S adherent cells had flattened and widespread morphology. RhoA inhibitor induced remarkable longer and thinner cytoplasmic projections on all surfaces. Cell adhesion and osteogenic differentiation was topography dependent with S < M and N surfaces. RhoA inhibition increased adhesion on S and M surfaces, but not N surfaces. Cell migration in a wound healing assay was greater on S versus M versus N surfaces and RhoA inhibitor increased S adherent cell migration, but not N adherent cell migration. RhoA inhibitor enhanced osteogenic differentiation in S adherent cells, but not M or N adherent cells. RhoA activity was surface topography roughness dependent (S < M, N). RhoA activity and ‐mediated functions are influenced by surface topography. Smooth surface adherent cells appear highly sensitive to RhoA function, while nano‐scale topography adherent cell may utilize alternative cellular signaling pathway(s) to influence adherent cellular functions regardless of RhoA activity. J. Cell. Physiol. 231: 568–575, 2016.

Vertical bone augmentation with fluvastatin in an injectable delivery system: a rat study

OBJECTIVES HMG-CoA reductase inhibitors (statins) are widely used for hyperlipidemia. Previous studies demonstrate that statins stimulate bone morphogenetic protein-2 (BMP-2) expression and lead to bone formation. The aim of this study was to evaluate whether percutaneously injected statin with a novel statin delivery system achieved vertical bone augmentation. MATERIAL AND METHODS As experimental groups, atelocollagen-alpha-tricalcium phosphate (alphaTCP) composites containing 3.3 mg (low dose) or 6.7 mg (high dose) of fluvastatin were injected (one shot) subcutaneously over the calvarial periosteum of rats. The animals were then sacrificed 1, 2, and 4 weeks after injection. Vertically augmented bone was assessed by histomorphometric procedures, i.e., by measuring new bone thickness (NBT) and bone density (BD). RESULTS In control groups, no newly formed bone could be seen over the calvarial bone. In the experimental groups, in contrast, a large amount of newly formed bone could be seen over the preexisting calvarial bone. The newly formed bone was seen to be in direct contact with the preexisting bone. During the entire observation, significant NBT was observed in the experimental groups (P<0.05). At the final stage of observation (4 weeks), NBT was 66.7% (low-dose group) and 59.7% (high-dose group), while they were from 1% to 16.3% in the control groups. In the experimental groups, BD significantly increased in a time-dependent manner. CONCLUSION Percutaneously applied fluvastatin (one shot) with a composite of alphaTCP and collagen has great potential to augment the height of the bone.

Topography Influences Adherent Cell Regulation of Osteoclastogenesis.

The importance of osteoclast-mediated bone resorption in the process of osseointegration has not been widely considered. In this study, cell culture was used to investigate the hypothesis that the function of implant-adherent bone marrow stromal cells (BMSCs) in osteoclastogenesis is influenced by surface topography. BMSCs isolated from femur and tibia of Sprague-Dawley rats were seeded onto 3 types of titanium surfaces (smooth, micro, and nano) and a control surface (tissue culture plastic) with or without osteogenic supplements. After 3 to 14 d, conditioned medium (CM) was collected. Subsequently, rat bone marrow–derived macrophages (BMMs) were cultured in media supplemented with soluble receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) as well as BMSC CM from each of the 4 surfaces. Gene expression levels of soluble RANKL, osteoprotegerin, tumor necrosis factor α, and M-CSF in cultured BMSCs at different time points were measured by real-time polymerase chain reaction. The number of differentiated osteoclastic cells was determined after tartrate-resistant acid phosphatase staining. Analysis of variance and t test were used for statistical analysis. The expression of prominent osteoclast-promoting factors tumor necrosis factor α and M-CSF was increased by BMSCs cultured on both micro- and nanoscale titanium topographies (P < 0.01). BMSC CM contained a heat-labile factor that increased BMMs osteoclastogenesis. CM from both micro- and nanoscale surface-adherent BMSCs increased the osteoclast number (P < 0.01). Difference in surface topography altered BMSC phenotype and influenced BMM osteoclastogenesis. Local signaling by implant-adherent cells at the implant-bone interface may indirectly control osteoclastogenesis and bone accrual around endosseous implants.