Surface characteristics and in vitro biocompatibility of titanium preserved in a vitamin C-containing saline storage solution.

The purpose of this study is to explore a storage solution for titanium implants and investigate its osteogenic properties. The commercial pure titanium (cp-Ti) surface and double-etched (SLA) titanium surface specimens were preserved in air, saline, 10 mM Vitamin C (VitC)-containing saline and 100 mM VitC-containing saline storage solutions for 2 weeks. The surface microtopography of titanium was observed by scanning electron microscopy (SEM), the surface elemental compositions of the specimens were analyzed by Raman and X-ray photoelectron spectroscopy (XPS), and water contact angle and surface roughness of the specimens were tested. The protein adsorption capacity of two titanium surfaces after storage in different media was examined by BCA kit. The MC3T3-E1 osteoblasts were cultured on two titanium surfaces after storage in different media, and the proliferation, adhesion and osteogenic differentiation activity of osteoblasts were detected by CCK-8, laser confocal microscope (CLSM) and Western blot. The SEM results indicated that the titanium surfaces of the air group were relatively clean while scattered sodium chloride or VitC crystals were seen on the titanium surfaces of the other three groups. There were no significant differences in the micromorphology of the titanium surfaces among the four groups. Raman spectroscopy detected VitC crystals on the titanium surfaces of two experimental groups. The XPS, water contact angle and surface roughness results suggested that cp-Ti and SLA-Ti stored in 0.9% NaCl and two VitC-containing saline storage solutions possessed less carbon contamination and higher surface hydrophilicity. Moreover, the protein adsorption potentials of cp-Ti and SLA-Ti surfaces were significantly improved under preservation in two VitC-containing saline storage solutions. The results of in vitro study showed that the preservation of two titanium surfaces in 100 mM VitC-containing saline storage solution upregulated the cell adhesion, proliferation, osteogenic related protein expressions of MC3T3-E1 osteoblasts. In conclusion, preservation of cp-Ti and SLA-Ti in 100 mM VitC-containing saline storage solution could effectively reduce carbon contamination and enhance surface hydrophilicity, which was conducive to osteogenic differentiation of osteoblasts.

Osseointegration of titanium dental implant under fluoride exposure in rabbits: Micro-CT and histomorphometry study.

OBJECTIVE: This study aims to investigate the influence of fluoride exposure on implant osseointegration. METHODS: A total of 24 male New Zealand white rabbits were randomly divided into the control group and the fluoride exposure group. Rabbits in the control group were fed with tap water, while those in the fluoride exposure group were given 200 mg/L sodium fluoride solution. After 2-month feeding, implants were inserted into the extraction socket immediately after extraction of rabbit mandibular anterior teeth. Four rabbits in each group were sacrificed to collect the implants samples at 1, 2, and 3 months post-implantation, respectively. Radiographic and histomorphometry examinations were performed to evaluate the condition of implant osseointegration. RESULTS: Bone volume around the implants increased in a time-dependent manner in both groups. Micro-CT images illustrated that the bone mineral density (BMD) in the fluoride exposure group was significantly lower than that in the control group after implantation for 2 and 3 months. The bone-implant contact ratio (BIC) in the fluoride exposure group was much lower than that of the control group at 3 months post-implantation according to histomorphometry examination. CONCLUSIONS: In rabbit animal model, high fluoride exposure affected the quality of bone surrounding the implant and significantly reduced bone integration of the implant, especially in the late stage of osseointegration.

Regulation of osteoblast behaviors via cross-talk between Hippo/YAP and MAPK signaling pathway under fluoride exposure.

Titanium is widely used in implant materials, while excessive fluoride may have negative effects on the osseointegration between the titanium and osteoblasts. Although the underlying mechanisms are still not clear, the mitogen-activated protein kinase (MAPK) or Yes-associated protein (YAP) signaling pathways are thought to be involved. This study evaluated the role of Hippo/YAP and MAPK signaling pathway in osteoblast behaviors under excessive fluoride exposure in vitro and in vivo. Commercially pure Ti (cp-Ti) samples were exposed to fluoride (0, 0.1, and 1.0 mM NaF) for 7 days. Cell adhesion was observed using a laser scanning confocal microscope. Cell viability and apoptosis were evaluated by CCK-8 assay and flow cytometry, respectively. The expressions of osteoblast markers and key molecules in MAPK and YAP pathway were detected by Western blot. In vivo studies were evaluated by histology methods in C57/BL6 mice model. Our results showed that 1.0 mM NaF destroyed the passivation film on cp-Ti surface, which further inhibited the osteoblast adhesion and spreading. Meanwhile, compared to other groups, 1.0 mM NaF led to a remarkable reduction in cell viability (P < 0.05), as well as increased apoptosis (P < 0.05) and downregulation of osteogenesis protein expression (P < 0.05). MAPK and YAP signaling pathways were also activated under 1.0 mM NaF exposure, and JNK seemed to regulate YAP phosphorylation in response to NaF impacts on osteoblasts. In vivo fluorosis mouse model further indicated that 100 ppm NaF group (high fluoride group) increased bone resorption and inhibited the nuclear translocation of YAP. The osteoblast behaviors were negatively altered under excessive fluoride, and MAPK/JNK axis contributed to YAP signaling activation in regulating NaF-induced osteoblast behaviors. KEY MESSAGES: • Excessive fluoride inhibited osteoblast behaviors and bone formation. • YAP and MAPK signaling pathways were activated in osteoblasts under fluoride exposure. • Fluoride regulated osteoblast behaviors via the cross-talk between YAP and MAPK.

Cigarette Smoke Extract Exposure: Effects on the Interactions between Titanium Surface and Osteoblasts.

The aim of this study was to explore the changes in the characteristics of titanium surface and the osteoblast-titanium interactions under cigarette smoke extract (CSE) exposure. In this study, CSE was used to simulate the oral liquid environment around the implant under cigarette smoke exposure. Titanium samples were immersed in CSE to explore the changes in the characteristics of titanium surface. The physical properties of titanium surface were measured, including surface micromorphology, surface elemental composition, roughness, and surface hydrophilicity. MC3T3-E1 cells were cultured on the titanium surface in vitro under different concentrations of CSE exposure, and cell adhesion, cell proliferation, and osteogenic differentiation were observed. The surface micromorphology and elemental composition of titanium surface changed under CSE exposure. No obvious changes were found in the surface roughness and the hydrophilicity of titanium samples. Moreover, the results of in vitro study showed that CSE exposure downregulated the cell spreading, proliferation, and osteogenic differentiation of MC3T3-E1 cells on the titanium surface. It could be speculated that some carbon-containing compounds from CSE adsorbed on the titanium surface and the osteoblast-titanium interactions were influenced under CSE exposure. It is hoped that these results could provide valuable information for further studies on smoking-mediated inhibition of implants osseointegration.

Enhanced corrosion resistance of zinc-containing nanowires-modified titanium surface under exposure to oxidizing microenvironment.

Titanium (Ti) and its alloys as bio-implants have excellent biocompatibilities and osteogenic properties after modification of chemical composition and topography via various methods. The corrosion resistance of these modified materials is of great importance for changing oral system, while few researches have reported this point. Recently, oxidative corrosion induced by cellular metabolites has been well concerned. In this study, we explored the corrosion behaviors of four common materials (commercially pure Ti, cp-Ti; Sandblasting and acid etching-modified Ti, Ti-SLA; nanowires-modified Ti, Ti-NW; and zinc-containing nanowires-modified Ti, Ti-NW-Zn) with excellent biocompatibilities and osteogenic capacities under the macrophages induced-oxidizing microenvironment. The results showed that the materials immersed into a high oxidizing environment were more vulnerable to corrode. Meanwhile, different surfaces also showed various corrosion susceptibilities under oxidizing condition. Samples embed with zinc element exhibited more excellent corrosion resistance compared with other three surfaces exposure to excessive H2O2. Besides, we found that zinc-decorated Ti surfaces inhibited the adhesion and proliferation of macrophages on its surface and induced the M2 states of macrophages to better healing and tissue reconstruction. Most importantly, zinc-decorated Ti surfaces markedly increased the expressions of antioxidant enzyme relative genes in macrophages. It improved the oxidation microenvironment around the materials and further protected their properties. In summary, our results demonstrated that Ti-NW-Zn surfaces not only provided excellent corrosion resistance properties, but also inhibited the adhesion of macrophages. These aspects were necessary for maintaining osseointegration capacity and enhancing the corrosion resistance of Ti in numerous medical applications, particularly in dentistry.

Low density lipoprotein adsorption on a titanium surface and its effect on osteoblast behaviors.

Objective: This study aims to investigate the adsorption of low density lipoprotein (LDL) on a titanium surface and to explore its effect on osteoblast behaviors. Materials and methods: LDL adsorption on a titanium surface was analyzed using LDL assay and X-ray photoelectron spectroscopy (XPS). Physical properties, including topography, surface roughness and wettability of a control smooth titanium surface and a LDL pre-adsorbed titanium surface, were assessed. Subsequently, the adhesion, proliferation and differentiation abilities of MC3T3-E1 cells (an osteoblast-like cell line) on the surfaces of control titanium and LDL pre-adsorbed titanium were investigated. Results: LDL assay and XPS confirmed LDL adsorption on the titanium surface. The maximum adsorption of LDL on the titanium surfaces was observed after 150 minutes of incubation. In comparison with the control smooth titanium surface, the roughness and hydrophilicity of the LDL pre-adsorbed titanium surface were significantly altered. Furthermore, in vitro studies demonstrated that LDL adsorption obviously attenuated the adhesion, proliferation and differentiation of MC3T3-E1 cells on the titanium surface. Conclusion: LDL could adsorb on a titanium surface. Meanwhile, LDL adsorption changed the characteristics of the titanium surface, which, in turn, negatively regulated osteoblast behaviors.

Effect of titanium ions on the Hippo/YAP signaling pathway in regulating biological behaviors of MC3T3-E1 osteoblasts.

Titanium (Ti) and its corresponding alloys have been widely applied in dental and orthopedic implants. Owing to abrasion and corrosion of implants in the unfavorable electrolytic aqueous environment of the host body, Ti ions could be released from implants and accumulated in local tissues. Recent studies have found that excessive Ti ions were toxic to osteoblasts in adjacent bone tissues and subsequently influenced long-term effects on implant prostheses. However, the potential molecular mechanisms underlying the damage to osteoblasts induced by Ti ions remained unclear. Hippo signaling has been confirmed to be involved in organ size and tissue regeneration in many organs, while its roles in osteoblasts differentiation and bone repair remained elusive. Therefore, we hypothesize that YAP, a regulator of Hippo pathway, inhibited osteoblast growth, skeletal development and bone repair, as well as excessive Ti ions promoted the progression of YAP activation. This study aimed to explore the role of Hippo/YAP signaling pathway in the biotoxicity effect of Ti ions on osteoblast behaviors. Here, we confirmed that 10 ppm Ti ions, a minimum concentration gradient previously reported that was capable of suppressing osteoblasts growth, induced nuclear expression of YAP in osteoblasts in our study. Furthermore, 10 ppm Ti ion-induced YAP activation was found to downregulate osteogenic differentiation of MC3T3-E1 cells. Most importantly, the hypothesis we proposed that knockdown of YAP did reverse the inhibitory effect of 10 ppm Ti ions on osteogenesis has been verified. Taken together, our work provides insights into the mechanism of which YAP is involved in regulating osteoblast behaviors under the effect of Ti ions, which may help to develop therapeutic applications for Ti implant failures and peri-implantitis.