Early-stage osseointegration capability of a submicrofeatured titanium surface created by microroughening and anodic oxidation.

OBJECTIVE: The role of nanoscale/submicron morphological features in the process of osseointegration is largely unknown. This study reports the creation of a unique submicrofeatured titanium surface by a combination of anodic oxidation and sandblasting and determines how the addition of this submicrofeature to a microroughened surface affects the early-stage process of osseointegration. MATERIALS AND METHODS: Nonmicroroughened implants were prepared by machining Ti-6Al-4V alloy in a cylindrical form (1 mm diameter and 2 mm long). Microroughened implants were prepared by sandblasting machined implants, while submicrofeatured implants were created by anodic oxidation of the sandblasted implants. Implants were placed into rat femurs and subjected to biomechanical, interfacial, and histological analyses at 1 and 2 weeks post-implantation (n = 6). RESULTS: The submicrotopography was characterized by 50-300 nm nodules and pits in addition to other submicron-level irregularities formed entirely within the sandblast-created microstructures. The biomechanical strength of osseointegration increased continuously from week 1 to 2 for the submicrofeatured implants but not for the microroughened implants. A significant increase in bone-implant contact and bone volume, as well as a reduction in soft tissue intervention, were commonly found for the microroughened surface and the submicrofeatured surface compared with the nonmicroroughened surface. However, there were no differences in these parameters between the microroughened surface and the submicrofeatured surface. An extensive area of bone tissue at the submicrofeatured implant interface was retained intact after biomechanical shear testing, while the microroughened implant-tissue interface showed a gap along the entire axis of the implant, leading to clear separation of the tissue during the shear procedure. CONCLUSIONS: This study demonstrates that a submicrofeatured titanium surface created by a combination of sandblasting and anodic oxidation enhances the strength of early-stage osseointegration, primarily because of the increased resistance of peri-implant bone tissue against external force rather than modulation of bone morphogenesis.

Improvement in the osteoblastic cellular response to a commercial collagen membrane and demineralized freeze-dried bone by an amino acid derivative: an in vitro study.

PURPOSE: The objectives of this in vitro study were (1) to determine whether a commercially available collagen membrane (CM) or human demineralized freeze-dried bone (DFDB) particles adversely affected viability or function in cultured osteoblasts through oxidative stress, and, if so, (2) to determine whether N-acetyl cysteine (NAC) successfully prevented loss of viability and dysfunction in osteoblasts. MATERIALS AND METHODS: Rat calvaria-derived osteoblasts were seeded onto polystyrene and commercially available CM (Cytoplast ®) or DFDB (DynaGraft ™) with or without pretreatment with NAC solution. The osteoblastic response was evaluated using a flow cytometric cell viability assay, measurement of attached viable cell number, quantification of reactive oxygen species (ROS) and alkaline phosphatase (ALP) staining. RESULTS: The percentage of viable cells on CM was <50% at 24 h after seeding. However, this increased to 70% by pretreatment with NAC. The numbers of attached osteoblasts on DFDB remained at 60% the level of that on polystyrene at 24 h after seeding, but increased to up to 90% the level of that on polystyrene with NAC pretreatment. Although collagen materials increased intracellular ROS generation 1.5-5 times that with polystyrene, this was significantly reduced by NAC pretreatment. The percentage of the ALP-positive area was consistently 7% or less on CM and DFDB at days 7 and 14, which was restored by NAC pretreatment up to 60% or more. CONCLUSIONS: Commercially available CM and DFDB impaired osteoblastic viability and function and markedly increased intracellular ROS, indicating an oxidative stress-mediated negative impact on osteoblasts. Pretreatment with NAC substantially alleviated these cytotoxic effects.

N-acetylcysteine attenuates PGE2 and ROS production stimulated by 4-META/MMA-based resin in murine osteoblastic cells.

This study examined the effects of N-acetylcysteine (NAC) on the inflammatory reactions of murine osteoblastic cells cultured on the 4-methacryloxyethyl trimellitate anhydride/methyl methacrylate (4-META/MMA)-based resin. Superbond C&B (SB) was used as the 4-META/MMA-based resin and placed in a 48-well cell culture plate. The cells were cultured in αMEM (control) as well as on SB and SB in αMEM with NAC (SB+NAC). They were examined using the WST-1 proliferation assay, real-time PCR, enzyme-linked immunosorbent assay (ELISA), intracellular reactive oxygen species (ROS) measurements, and cellular glutathione (GSH) detection. COX-2 and IL-6 gene expressions were upregulated in SB; however, they were suppressed by NAC. Furthermore, PGE2 production in the culture medium was increased in SB, whereas NAC decreased the PGE2 production. NAC lowered the ROS level in the culture medium and significantly increased the intracellular GSH level. The present in vitro study demonstrated that NAC might be effective for dental material detoxification.

Assessment of the inhibitory effects of fissure sealants on the demineralization of primary teeth using an automatic pH-cycling system.

In this study, we examined the effects of fissure sealants on inhibition of demineralization of primary teeth using an automatic pHcycling system. Three fissure sealants were used: Teethmate F-1 2.0 (TM), BeautiSealant (BS), and Fuji III LC (IIILC). Using an automatic pH-cycling system, the specimens (n=12) were repeatedly demineralized and remineralized. Specimens were subjected to transverse microradiography (TMR), and changes in integrated mineral loss (IML) and lesion depth (Ld), indicated as ΔIML and ΔLd, respectively, were calculated. In addition, fluoride levels in the enamel were assessed using microparticle-induced gamma-ray emission/particle-induced X-ray emission (n=3). IIILC showed the lowest values for ΔIML and ΔLd, followed by BS and then TM. The highest amount of fluorine in the enamel was observed for IIILC, followed by TM and BS. All fissure sealants inhibited demineralization in primary teeth.

Release from optimal compressive force suppresses osteoclast differentiation.

Bone remodeling is an important factor in orthodontic tooth movement. During orthodontic treatment, osteoclasts are subjected to various mechanical stimuli, and this promotes or inhibits osteoclast differentiation and fusion. It has been previously reported that the release from tensile force induces osteoclast differentiation. However, little is known about how release from compressive force affects osteoclasts. The present study investigated the effects of release from compressive force on osteoclasts. The number of tartrate­resistant acid phosphatase (TRAP)­positive multinucleated osteoclasts derived from RAW264.7 cells was counted, and gene expression associated with osteoclast differentiation and fusion in response to release from compressive force was evaluated by reverse transcription­quantitative polymerase chain reaction. Osteoclast number was increased by optimal compressive force application. On release from this force, osteoclast differentiation and fusion were suppressed. mRNA expression of NFATc1 was inhibited for 6 h subsequent to release from compressive force. mRNA expression of the other osteoclast­specific genes, TRAP, RANK, matrix metalloproteinase­9, cathepsin­K, chloride channel 7, ATPase H+ transporting vacuolar proton pump member I, dendritic cell­specific transmembrane protein and osteoclast stimulatory transmembrane protein (OC­STAMP) was significantly inhibited at 3 h following release from compressive force compared with control cells. These findings suggest that release from optimal compressive force suppresses osteoclast differentiation and fusion, which may be important for developing orthodontic treatments.

Photofunctionalization increases the bioactivity and osteoconductivity of the titanium alloy Ti6Al4V.

This study examined the effect of photofunctionalization on bioactivity and osteoconductivity of titanium alloy Ti6Al4V. We also tested a hypothesis that the effect of photofunctionalization is as substantial as the one of surface roughening. Two different surface morphology, a roughened surface (sandblasted and acid-etched surface) and relatively smooth surface (machined surface), was tested. Ti6Al4V samples were photofunctionalized with UV light for 15 min using a photo device. Photofunctionalization converted Ti6Al4V surfaces from hydrophobic to superhydrophilic. The attachment, spread, proliferation, and the expression of functional phenotype of bone marrow-derived osteoblasts were promoted on photofunctionalized Ti6Al4V surfaces. The strength of bone-implant integration examined using a biomechanical push-in test in a rat femur model was at least 100% greater for photofunctionalized implants than for untreated implants. These effects were seen on both surface types. The strength of bone-implant integration for photofunctionalized machined implants was greater than that for untreated roughened implants, indicating that the impact of photofunctionalization may be greater than that of surface roughening. Newly prepared Ti alloy was hydrophilic, whereas the hydrophilic status degraded with time and was converted to hydrophobic in 4 weeks. This finding uncovered biological aging of Ti alloy and allowed us to consider photofunctionalization as a countermeasure for aging. These results suggest that photofunctionalization accelerates and enhances bone-implant integration of Ti6Al4V regardless of smooth and roughened surface features, supporting photofunctionalization as an effective and viable measure for improving efficacy of a wide range of Ti6Al4V-based materials used in dental and orthopedic medicine.

N-acetyl cysteine improves affinity of beta-tricalcium phosphate granules for cultured osteoblast-like cells.

Enhancement of bone substitute's biocompatibility may accelerate healing of surrounding bone. Although widely used as a biodegradable alloplastic bone substitute for alveolar bone augmentation, the osteocompatibility of beta-tricalcium phosphate (ß-TCP) remains to be proven. The adverse cellular response to biomaterials is associated with oxidative stress. We hypothesized that commercially available ß-TCP granules for clinical use, caused oxidative stress and was not optimal in osteocompatibility and that application of antioxidant amino acid derivative N-acetyl cysteine (NAC) would improve osteoblastic responses to the material. Only 20% of rat calvarial osteoblasts cultured on ß-TCP granules remained viable at 24 h after seeding as opposed to 90% on polystyrene. Cell death on ß-TCP granules was characterized by necrosis. However, the percentage of viable osteoblasts cultured on ß-TCP granules showed a 100% increase with pre-treatment with NAC. NAC restored suppressed alkaline phosphatase activity on ß-TCP granules at day 5. Intracellular ROS level on ß-TCP granules was 16-fold greater than that on polystyrene, but decreased by half with pre-treatment with NAC. Cell death and intracellular ROS elevation were also induced in polystyrene culture under ß-TCP granules even when the osteoblasts were not in direct contact with the ß-TCP granules. NAC, however, prevented induction of cell death and elevation of intracellular ROS under ß-TCP granules. These results indicate that commercially available ß-TCP granules negatively affect cultured osteoblastic viability and function via oxidative stress and that NAC improves these negative responses to the material. This implies enhanced bone regeneration around biodegradable calcium phosphate-based bone substitute by NAC.

Effect of N-acetylcysteine on rat dental pulp cells cultured on mineral trioxide aggregate.

INTRODUCTION: The purpose of this study was to evaluate the cytotoxicity of mineral trioxide aggregate (MTA) and its potential detoxification by an antioxidant amino acid, N-acetylcysteine (NAC). METHODS: Rat dental pulp cells extracted from rat maxillary incisors were directly cultured on MTA with or without NAC in culture medium. The number of cells and their spreading behavior were both assessed 24 hours after seeding. The intracellular levels of reactive oxygen species (ROS) and glutathione (GSH) were also assessed after 24 hours of culture. RESULTS: The number of cells attached to MTA was 60% greater when NAC was added to the culture medium. In addition, the area and perimeter of the cells were found to be 2-fold greater in the culture containing NAC. Cells cultured on MTA alone showed large ROS concentrations, which disappeared when the medium was supplemented with NAC. The intracellular GSH level, however, increased 3.5-fold with NAC addition. CONCLUSIONS: This study demonstrated that the presence of NAC in environments can substantially improve attachment and spreading behaviors of dental pulp cells on MTA. This biological effect was associated with an improvement in the cellular redox system by NAC and warrants further exploration of NAC for determining its therapeutic value in improving the biocompatibility of MTA.

Effects of pico-to-nanometer-thin TiO2 coating on the biological properties of microroughened titanium.

The independent, genuine role of surface chemistry in the biological properties of titanium is unknown. Although microtopography has been established as a standard surface feature in osseous titanium implants, unfavorable behavior and reactions of osteogenic cells are still observed on the surfaces. To further enhance the biological properties of microfeatured titanium surfaces, this study tested the hypotheses that (1) the surface chemistry of microroughened titanium surfaces can be controllably varied by coating with a very thin layer of TiO(2), without altering the existing topographical and roughness features; and (2) the change in the surface chemistry affects the biological properties of the titanium substrates. Using a slow-rate sputter deposition of molten TiO(2) nanoparticles, acid-etched microroughened titanium surfaces were coated with a TiO(2) layer of 300-pm to 6.3-nm thickness that increased the surface oxygen levels without altering the existing microtopography. The attachment, spreading behavior, and proliferation of osteoblasts, which are considered to be significantly impaired on microroughened surfaces compared with relatively smooth surfaces, were considerably increased on TiO(2)-coated microroughened surfaces. The rate of osteoblastic differentiation was represented by the increased levels of alkaline phosphatase activity and mineral deposition as well as by the upregulated expression of bone-related genes. These biological effects were exponentially correlated with the thickness of TiO(2) and surface oxygen percentage, implying that even a picometer-thin TiO(2) coating is effective in rapidly increasing the biological property of titanium followed by an additional mild increase or plateau induced by a nanometer-thick coating. These data suggest that a super-thin TiO(2) coating of pico-to-nanometer thickness enhances the biological properties of the proven microroughened titanium surfaces by controllably and exclusively modulating their surface chemistry while preserving the existing surface morphology. The improvements in proliferation and differentiation of osteoblasts attained by this chemical modification is of great significance, providing a new insight into how to develop new implant surfaces for better osseointegration, based on the established microtopographic surfaces.

Electrostatic control of protein adsorption on UV-photofunctionalized titanium.

Ultraviolet (UV)-photofunctionalization of titanium to enable the establishment of a nearly complete bone-implant contact was reported recently. However, the underlying mechanism for this is unknown. We hypothesized that UV-treated titanium surfaces acquire distinct electrostatic properties that may play important roles in determining the bioactivity of these surfaces. The objective of this study was to determine the protein adsorption capability of UV-treated titanium surfaces under various electrostatic environments. The amount of albumin adsorbed on UV-treated and untreated titanium disks was evaluated under different pH conditions above and below the isoelectric points of albumin and titanium. The effects of additional treatment with various ionic solutions were also examined. Albumin adsorption on UV-treated surfaces at pH 7.0 was considerably greater (6-fold after 3h of incubation and 2.5-fold after 24h) than that to UV-untreated surfaces. UV-enhanced albumin adsorption was abrogated at pH 3.0 or when these titanium surfaces were treated with anions, while maintaining UV-induced superhydrophilicity. Albumin adsorption on UV-untreated titanium surfaces increased after treating these surfaces with divalent cations but not after treating them with monovalent cations. These results indicated that UV-treated titanium surfaces are electropositively charged as opposed to electronegatively charged UV-untreated titanium surfaces. This distinct UV-induced electrostatic property predominantly regulates the protein adsorption capability of titanium, superseding the effect of hydrophilic status, and converts titanium surfaces from bioinert to bioactive. As a result, direct titanium-protein interactions take place exclusively on UV-treated titanium surfaces without the aid of bridging ions.

Amino acid derivative-mediated detoxification and functionalization of dual cure dental restorative material for dental pulp cell mineralization.

Current dental restorative materials are only used to fill the defect of hard tissues, such as dentin and enamel, because of their cytotoxicity. Therefore, exposed dental pulp tissues in deep cavities must be first covered by a pulp capping material like calcium hydroxide to form a layer of mineralized tissue. However, this tissue mineralization is based on pathological reaction and triggers long-lasting inflammation, often causing clinical problems. This study tested the ability of N-acetyl cysteine (NAC), amino acid derivative, to reduce cytotoxicity and induce mineralized tissue conductivity in resin-modified glass ionomer (RMGI), a widely used dental restorative material having dual cure mechanism. Rat dental pulp cells were cultured on untreated or NAC-supplemented RMGI. NAC supplementation substantially increased the percentage of viable cells from 46.7 to 73.3% after 24-h incubation. Cell attachment, spreading, proliferative activity, and odontoblast-related gene and protein expressions increased significantly on NAC-supplemented RMGI. The mineralization capability of cells, which was nearly suppressed on untreated RMGI, was induced on NAC-supplemented RMGI. These improved behaviors and functions of dental pulp cells on NAC-supplemented RMGI were associated with a considerable reduction in the production of intracellular reactive oxygen species and with the increased level of intracellular glutathione reserves. These results demonstrated that NAC could detoxify and functionalize RMGIs via two different mechanisms involving in situ material detoxification and antioxidant cell protection. We believe that this study provides a new approach for developing dental restorative materials that enables mineralized tissue regeneration.

Effect of mineral trioxide aggregate on rat clonal dental pulp cells: expression of cyclooxygenase-2 mRNA and inflammation-related protein via nuclear factor kappa B signaling system.

INTRODUCTION: Recently, mineral trioxide aggregate (MTA) has been routinely used for endodontic treatment. It is well-known that MTA induced secondary dentin formation in pulp cavity when it was applied to dentin, whereas its cytotoxicities were unclear. The purpose of this study was to evaluate the effect of MTA on rat clonal dental pulp cells, RPC-C2A. METHODS: This study was conducted to observe the response of RPC-C2A cells on MTA with reverse-transcriptase polymerase chain reaction, Western blot analysis, and enzyme immunoassay. Data were compared by analysis of variance. Statistical significance was established at P <.01. RESULTS: MTA significantly caused an up-regulation of cyclooxygenase-2 (COX-2) and inducible form of nitric oxide synthase (iNOS) mRNA expression. Furthermore, MTA caused inhibitory kappa B (IkappaB) phosphorylation and translocation of nuclear factor-kappa B (NF-kappaB) subunits to nucleus. Curucumin, an inhibitor of NF-kappaB activation, suppressed MTA-induced COX-2 and iNOS mRNA expressions. In addition, MTA increased the production of prostaglandin E(2) in comparison with the controls. CONCLUSIONS: MTA induces inflammation via NF-kappaB signaling system.