Eizaburo Kobayashi

Hard and soft tissue responses to three different implant materials in a dog model.

The aim of this study was to assess hard and soft tissue responses using three dental implants made of different materials. Implants made of titanium (Ti), yttria-stabilized tetragonal zirconia polycrystals (Y-TZP) and ceria partially stabilized zirconia/alumina nanocomposite (Ce-TZP/Al2O3) were used in a dog model. Five male beagles were sacrificed at three months after implantation, and harvested mandible were observed and analyzed. Histological observations were similar in all groups. There were no significant differences in any histomorphometric parameters. Our results suggested the possibility of Ce-TZP/Al2O3 as a dental implant material, similar to Ti and Y-TZP.

Effects of Antiseptic Solutions Commonly Used in Dentistry on Bone Viability, Bone Morphology, and Release of Growth Factors

PURPOSE Antiseptic solutions are commonly used in dentistry for a number of sterilization procedures, including harvesting of bone chips, irrigation of extraction sockets, and sterilization of osteonecrotic bone. Despite its widespread use, little information is available regarding the effects of various antiseptic solutions on bone cell viability, morphology, and the release of growth factors. MATERIALS AND METHODS The antiseptic solutions included 1) 0.5% povidone iodine (PI), 2) 0.2% chlorhexidine diguluconate (CHX), 3) 1% hydrogen peroxide (H2O2), and 4) 0.25% sodium hypochlorite (HYP). Bone samples collected from porcine mandibular cortical bone were rinsed in the antiseptic solutions for 10 minutes and assessed for cell viability using an MTS assay and protein release of transforming growth factor (TGF-β1), bone morphogenetic protein 2 (BMP2), vascular endothelial growth factor (VEGF), interleukin (IL)-1β, and receptor activator of nuclear factor κB ligand (RANKL) using an enzyme-linked immunosorbent assay at 15 minutes and 4 hours after rinsing. RESULTS After antiseptic rinsing, changes to the surface protein content showed marked alterations, with an abundant protein layer remaining on CHX-rinsed bone samples. The amount of surface protein content gradually decreased in the following order: CHX, H2O2, PI, and HYP. A similar trend was also observed for the relative cell viability from within bone samples after rinsing, with up to 6 times more viable cells found in the CHX-rinsed bone samples than in the HYP- and PI-rinsed samples. An analysis of the growth factors found that both HYP and PI had significantly lower VEGF and TGF-β1 protein release from bone samples at 15 minutes and 4 hours after rinsing compared with CHX and H2O2. A similar trend was observed for RANKL and IL-1β protein release, although no change was observed for BMP2. CONCLUSIONS The results from the present study have demonstrated that antiseptic solutions present with very different effects on bone samples after 10 minutes of rinsing. Rinsing with CHX maintained significantly higher cell viability and protein release of growth factors potent to the bone remodeling cycle.

Guided bone regeneration with recombinant human bone morphogenetic protein 9 loaded on either deproteinized bovine bone mineral or a collagen barrier membrane

BACKGROUND Recombinant human bone morphogenetic protein 9 (rhBMP9) has been considered the most osteoinductive growth factor of the BMP-family and has much translation potential for guided bone regeneration (GBR) procedures. PURPOSE The aim of this study was to compare bone formation using rhBMP9 loaded with different carrier systems including deprotenized bovine bone mineral (BioOss, BO) or collagen barrier membranes (BioGide, BG) in a rabbit GBR model. MATERIALS AND METHODS rhBMP9 was loaded either on BO; named BO/BMP9, or BG; named BG/BMP9 to investigate the better carrier system for rhBMP9. New bone formation was quantified in a rabbit calvarial defect model using four groups; (1) control (empty, n = 9), (2) BO + BG (n = 9), (3) BO/BMP9 + BG (n = 9; BMP9 loaded onto BO), and (4) BO + BG/BMP9 (n = 9; BMP9 loaded onto BG) by radiographically and histologically at 8 weeks post-surgery. RESULTS Both BO/BMP9 + BG and BO + BG/BMP9 samples significantly promoted new bone formation when compared to BO + BG samples based on parameters including mineralized tissue volume by microCT analysis, as well as new bone height and new bone area by histomorphometry. Interestingly, BO + BG/BMP9 samples but not BO/BMP9 + BG achieved near perfect horizontal bone defect closure, while demonstrating new bone layers in the defect areas implanted with BG materials and bone formation around BO materials. CONCLUSION Both BO and BG positively induced bone formation with rhBMP9 in an experimental rabbit GBR model when compared to BO + BG alone. This study revealed that BG-loaded with rhBMP9 promoted better wound closure when compared to BO-loaded with rhBMP9. GBR procedures with growth factors may thus benefit from loading rhBMP9 onto BG-collagen barrier membranes when compared to BO-bone grafting particles. Future large animal studies with different types of bone grafts and barrier membranes are needed to further investigate these trends.

Hyaluronic Acid Gel-Based Scaffolds as Potential Carrier for Growth Factors: An In Vitro Bioassay on Its Osteogenic Potential

Hyaluronic acid (HA) has been utilized for a variety of regenerative medical procedures due to its widespread presence in connective tissue and perceived biocompatibility. The aim of the present study was to investigate HA in combination with recombinant human bone morphogenetic protein 9 (rhBMP9), one of the most osteogenic growth factors of the BMP family. HA was first combined with rhBMP9 and assessed for the adsorption and release of rhBMP9 over 10 days by ELISA. Thereafter, ST2 pre-osteoblasts were investigated by comparing (1) control tissue culture plastic, (2) HA alone, and (3) HA with rhBMP9 (100 ng/mL). Cellular proliferation was investigated by a MTS assay at one, three and five days and osteoblast differentiation was investigated by alkaline phosphatase (ALP) activity at seven days, alizarin red staining at 14 days and real-time PCR for osteoblast differentiation markers. The results demonstrated that rhBMP9 adsorbed within HA scaffolds and was released over a 10-day period in a controlled manner. While HA and rhBMP9 had little effect on cell proliferation, a marked and pronounced effect was observed for cell differentiation. rhBMP9 significantly induced ALP activity, mRNA levels of collagen1α2, and ALP and osteocalcin (OCN) at three or 14 days. HA also demonstrated some ability to induce osteoblast differentiation by increasing mRNA levels of OCN and increasing alizarin red staining at 14 days. In conclusion, the results from the present study demonstrate that (1) HA may serve as a potential carrier for various growth factors, and (2) rhBMP9 is a potent and promising inducer of osteoblast differentiation. Future animal studies are now necessary to investigate this combination approach in vivo.

Superior bone-inducing potential of rhBMP9 compared to rhBMP2: EFFECT OF RHBMP9 VS RHBMP2 ON NEW BONE FORMATION

Recombinant human bone morphogenic protein (rhBMP) 9 has recently been reported to have more osteopromotive potential in vitro when compared to rhBMP2. The aim of the present study was to investigate the bone-inducing potential of rhBMP2 and rhBMP9. We compared rhBMP2, rhBMP7, and rhBMP9 at five different concentrations and showed convincingly that rhBMP9 possesses much greater potential for osteoblast differentiation even at 20 times lower concentrations in vitro. We further show that Noggin, an inhibitor for rhBMP2-induced osteogenesis, did not alter rhBMP9-induced osteogenesis. Thereafter, we show for the first time that rhBMP9 loaded onto atelo-collagen membranes is osteoinductive and has greater potential to form ectopic bone formation when compared to rhBMP2 even at four times lower doses. Similarly new bone formation of rhBMP2 and 9 when loaded on deproteinized bovine bone mineral (DBBM) was investigated in a rabbit calvarial defect. At 8 weeks, both rhBMP2 and rhBMP9 induced significantly higher new bone formation when compared to DBBM alone samples. Interestingly, once again four times lower dose of rhBMP9 group induced comparable or even greater levels of new bone height and new bone area when compared to the rhBMP2 group. The present study revealed that (1) rhBMP9 is capable of inducing ectopic new bone formation in vivo and (2) up to four times lower doses of rhBMP9 may be utilized to regenerate same-size bone defects when compared to rhBMP2.

Effect of recombinant human bone morphogenic protein 9 (rhBMP9) loaded onto bone grafts versus barrier membranes on new bone formation in a rabbit calvarial defect model

Recent research has demonstrated that recombinant human bone morphogenetic protein 9 (rhBMP9) has been considered the most osteoinductive growth factor of the BMP-family. In the present study, rhBMP9 was investigated for its influence in combination with two biomaterials for bone regenerative medicine. Either porcine-derived collagen membrane (CM) or deproteinized bovine bone mineral (DBM) combined with 20 µg of rhBMP9 were implanted in 6 mm rabbit calvarial defects. Bone augmentation was evaluated by microCT and histomorphometry at 8 weeks post-surgery. Both CM + rhBMP9 and DBM + rhBMP9 groups significantly promoted mineralized tissue volume (microCT) and area, new bone height and area (histomorphometric measurements) when compared to CM and DBM alone groups or control (empty). All specimens in the CM + rhBMP9 group but not all in the DBM + rhBMP9 group induced a complete horizontal bone defect closure. Multinucleated giant cells (MNGCs) were observed directly in contact with DBM surfaces irrespective of rhBMP9, whereas CM was generally not associated to the presence of MNGCs. When combined with rhBMP9, DBM augmented a larger volume of mineralized tissue (including the mineralized bone graft), whereas CM induced greater volume of native host bone. While DBM in combination with rhBMP9 induced higher mineralized tissue mostly associated with the bone grafting material, CM may have presented preferable results based on a higher horizontal defect closure with a faster regeneration of host new bone. The effect of including collagen within the carrier system of rhBMP9 on bone regeneration justifies further evaluation of this combination procedure in larger animal models.

Hard and soft tissue responses to implant made of three different materials with microgrooved collar in a dog model

The objective of the present study was to assess hard and soft tissue around dental implants made of three different materials with microgrooves on the collar surface. Microgrooved implants were inserted in the mandibles of five male beagles. Implants were made of three kinds of material; titanium (Ti), yttria-stabilized tetragonal zirconia polycrystals (Y-TZP) and ceria partially stabilized zirconia/alumina nanocomposite (Ce-TZP/Al2O3). The animals were euthanatized at three months after implantation, and harvested tissue was analyzed by means of histology. All kinds of implant were osseointegrated, and there were no significant differences in any histomorphometric parameters among the three groups of microgrooved implants made of different materials. Within the limitations of this study, implants with microgrooves integrated into the surrounding bone tissue, without statistically significant differences among the three tested materials, Ti, Y-TZP, and Ce-TZP/Al2O3.