Mammalian cell response and bacterial adhesion on titanium healing abutments: effect of multiple implantation and sterilization cycles.

OBJECTIVE: Multiple implantations of the implant healing abutment (IHA) could adversely impact its surface properties in vivo. Furthermore, the effect of sterilization and reuse of the IHA on soft tissue viability and bacterial contamination has not been extensively studied. The goal of this study was to perform an in vitro analysis of mammalian cell viability and bacterial adhesion on the surfaces of retrieved IHA after single and multiple implantations and repetitive cycles of sterilization. MATERIALS AND METHODS: IHA surface morphology was studied using optical microscopy. Cell viability of gingival fibroblasts (HGF-1) and oral keratinocytes (HOKg) in indirect contact with IHAs was assessed for 3 and 7 days. Immersion in bacterial culture was performed with a polyculture of Streptococcus species for 3 days and Streptococcus species with Fusobacterium nucleatum for 7 days. RESULTS: IHAs exhibited signs of surface damage even after a single exposure to the oral cavity. Fibroblasts did not show a significant preference towards control IHAs over used IHAs, whereas keratinocytes exhibited a significant decrease in viability when exposed to IHAs after multiple implantation cycles as compared with controls. Adherent bacterial count increased with increasing number of IHA implantations for both polycultures. CONCLUSIONS: Reusing of IHAs in vivo promoted surface degradation in addition to adversely impacting host cell viability and oral bacterial attachment in vitro. These findings show IHA reuse might potentially affect its clinical performance. CLINICAL RELEVANCE: Careful consideration should be taken when reusing IHAs in patients because this practice can result in permanent surface changes that might affect soft tissue integration during the healing period and promote bacterial colonization.

Effects of decontamination solutions on the surface of titanium: investigation of surface morphology, composition, and roughness.

AIM: To investigate the impact of treatments used to detoxify dental implants on the oxide layer morphology and to infer how changes in morphology created by these treatments may impact re-osseointegration of an implant. MATERIALS AND METHODS: Pure titanium (cpTi) and the alloy Ti6Al4V were subjected to a series of chemical treatments and mechanical abrasion simulating surface decontamination of dental implants. The morphology and roughness of the surface layer before and after treatment with these solutions were investigated with optical and atomic force microscopy (OM, AFM). The solutions employed are typically used for detoxification of dental implants. These included citric acid, 15% hydrogen peroxide, chlorhexidine gluconate, tetracycline, doxycycline, sodium fluoride, peroxyacetic acid, and treatment with carbon dioxide laser. The treatments consisted of both immersions of samples in solution and rubbing with cotton swabs soaked in solution for 1, 2, and 5 min. Cotton swabs used were analyzed with energy dispersive spectroscopy (EDS). RESULTS: The microscopy investigation showed that corrosion and pitting of the samples were present in both metal grades with immersion and rubbing methods when employing more acidic solutions, which had pH <3. Mildly acidic solutions caused surface discoloration when coupled with rubbing but did not cause corrosion with immersion. Neutral or basic treatments resulted in no signs of corrosion with both methods. EDS results revealed the presence of titanium particles on all rubbing samples. CONCLUSION: It was demonstrated in this study that acidic environments coupled with rubbing are able to introduce noticeable morphological changes and corrosion on the surface of both titanium grades.

Effects of Dicationic Imidazolium-Based Ionic Liquid Coatings on Oral Osseointegration of Titanium Implants: A Biocompatibility Study in Multiple Rat Demographics.

Dicationic imidazolium-based ionic liquids with amino acid anions, such as IonL-phenylalanine (IonL-Phe), have been proposed as a multifunctional coating for titanium (Ti) dental implants. However, there has been no evaluation of the biocompatibility of these Ti coatings in the oral environment. This study aims to evaluate the effects of IonL-Phe on early healing and osseointegration of Ti in multiple rat demographics. IonL-Phe-coated and uncoated Ti screws were implanted into four demographic groups of rats to represent biological variations that could affect healing: young males (YMs) and females (YFs), ovariectomized (OVXFs) females, and old males (OMs). Samples underwent histopathological and histomorphometric analysis to evaluate healing at 7 and 30 days around IonL-coated and uncoated Ti. The real-time quantitative polymerase chain reaction was also conducted at the 2- and 7-day YM groups to evaluate molecular dynamics of healing while the IonL-Phe was present on the surface. IonL-coated and uncoated implants demonstrated similar histological signs of healing, while coated samples' differential gene expression of immunological and bone markers was compared with uncoated implants at 2 and 7 days in YMs. While YMs presented suitable osseointegration for both uncoated and IonL-Phe-coated groups, decreased success rate in other demographics resulted from lack of supporting bone in YFs and poor bone quality in OVXFs and OMs. Overall, it was found that IonL-coated samples had increased bone-to-implant contact across all demographic groups. IonL-Phe coating led to successful osseointegration across all animal demographics and presented the potential to prevent failures in scenarios known to be challenged by bacteria.

Cellular and Molecular Dynamics during Early Oral Osseointegration: A Comprehensive Characterization in the Lewis Rat.

OBJECTIVE: There is a need to improve the predictability of osseointegration in implant dentistry. Current literature uses a variety of in vivo titanium (Ti) implantation models to investigate failure modes and test new materials and surfaces. However, these models produce a variety of results, making comparison across studies difficult. The purpose of this study is to validate an oral osseointegration in the Lewis rat to provide a reproducible baseline to track the inflammatory response and healing of Ti implants. METHODS: Ti screws (0.76 mm Ø × 2 mm length) were implanted into the maxillary diastema of 52 adult male Lewis rats. Peri-implant tissues were evaluated 2, 7, 14, and 30 days after implantation (n = 13). Seven of the 13 samples underwent microtomographic analysis, histology, histomorphometry, and immunohistochemistry to track healing parameters. The remaining six samples underwent quantitative polymerase chain reaction (qPCR) to evaluate gene expression of inflammation and bone remodeling markers over time. RESULTS: This model achieved a 78.5% success rate. Successful implants had a bone to implant contact (BIC)% of 68.86 ± 3.15 at 30 days on average. Histologically, healing was similar to other rodent models: hematoma and acute inflammation at 2 days, initial bone formation at 7, advanced bone formation and remodeling at 14, and bone maturation at 30. qPCR indicated the highest expression of bone remodeling and inflammatory markers 2-7 days, before slowly declining to nonsurgery control levels at 14-30 days. CONCLUSION: This model combines cost-effectiveness and simplicity of a rodent model, while maximizing BIC, making it an excellent candidate for evaluation of new surfaces.

Investigation of the early healing response to dicationic imidazolium-based ionic liquids: a biocompatible coating for titanium implants.

Dicationic Imidazolum-based ionic liquids with amino acid anions (IonL) have been proposed as a multifunctional coating for titanium dental implants, as their properties have been shown to address multiple early complicating factors while maintaining host cell compatibility. This study aims to evaluate effects of this coating on host response in the absence of complicating oral factors during the early healing period using a subcutaneous implantation model in the rat. IonLs with the best cytocompatibility and antimicrobial properties (IonL-Phe, IonL-Met) were chosen as coatings. Three different doses were applied to cpTi disks and subcutaneously implanted into 36 male Lewis rats. Rats received 2 implants: 1 coated implant on one side and an uncoated implant on the contralateral sides (n=3 per formulation, per dose). Peri-implant tissue was evaluated 2 and 14 days after implantation with H&E staining and IHC markers associated with macrophage polarization as well as molecular analysis (qPCR) for inflammatory and healing markers. H&E stains revealed the presence of the coating, blood clots and inflammatory infiltrate at 2 days around all implants. At 14 days, inflammation had receded with more developed connective tissue with fibroblasts, blood vessels in certain doses of coated and uncoated samples with no foreign body giant cells. This study demonstrated that IonL at the appropriate concentration does not significantly interfere with and healing and Ti foreign body response. Results regarding optimal dose and formulation from this study will be applied in future studies using an oral osseointegration model.

Detection of apoptotic and live pre-osteoblast cell line using impedance-based biosensors with variable electrode design.

Electrical impedance-based sensing of cell activity has become a powerful analytical tool that allows the monitoring of several relevant biological processes associated with cell evolution and morphology. In these types of biosensors, the electrode design has a direct impact on the sensitivity because it defines the capability of the biosensor to measure small changes in the impedance resulting from cell activities. Herein, impedance-based biosensors arrays with several configurations were successfully developed and used to study the impact of the electrode layout on the dynamics of cultured pre-osteoblast cells. The biosensor design was initially validated by measuring the effect of electrode design on the capacitance of a dielectric polymer (parylene) that mimics the dielectric characteristics of cell populations, results are shown in the Supplementary information section. Results from in vitro cell growth indicate that the optimized design of single electrodes with a diameter of 50 µm, are the most sensitive to cell motion whereas increasing the number of electrodes allows clear differentiation between living and dead cells after 3 h of inducing apoptosis. Apoptosis death was induced with Staurosporine, a chemical mediator of apoptosis in osteoblasts. These impedance results have been validated with optical imaging and flow cytometry analysis that were performed on parallel cultures. Frequency and electrolyte concentration effects are also discussed.

Effects of titanium oxide surface properties on bone-forming and soft tissue-forming cells.

BACKGROUND: Previous studies have concluded that certain titanium oxide (TiO2 ) surface properties promote bone-forming cell attachment. However, no comprehensive studies have investigated the effects of TiO2 surface and film morphology on hard and soft tissues. PURPOSE: The aim of this study is to understand the effects of TiO2 morphology on the proliferation and differentiation of murine preosteoblasts (MC3T3-E1) and proliferation of human gingival fibroblasts (HGF-1) using in vitro experiments. MATERIALS AND METHODS: Samples were fabricated with several TiO2 thickness and crystalline structure to mimic various dental implant surfaces. in vitro analysis was performed for 1, 3, and 7 days on these samples to assess the viability of MC3T3-E1 and HGF-1 cells in contact with the modified oxide surfaces. RESULTS: Results showed that HGF-1 cells exhibited no significant difference in viability on modified oxide surfaces versus a titanium control across experiments. MC3T3-E1 cells exhibited a significantly higher viability for the modified oxide surface in 1 day experiments, but not in 3 or 7 day experiments. Alkaline phosphatase expression in MC3T3-E1 was not significantly different on modified oxide surfaces versus the control across all experiments. A slight positive trend in viability was observed for cells in contact with rougher modified oxide surfaces versus a titanium control in both cell types. CONCLUSIONS: These observations suggest that crystallinity and thickness do not affect the long-term viability of hard or soft tissue cells when compared to a cpTi surface. Therefore, treatments like anodization on implant components may not directly affect the attachment of hard or soft tissue cells in vivo.

Surface characterization of titanium implant healing abutments before and after placement.

BACKGROUND: Implant healing abutments (IHA) have a vital role in soft tissue healing after implant placement. Although there is thorough investigation on the implant surface, little is known about the effects potentially damaging oral conditions impose on healing abutments. PURPOSE: To characterize the surface of titanium healing abutments before and after clinical placement to understand the effects of the oral environment and time on the device surface. MATERIALS AND METHODS: Ten regular Straumann IHA were subjected to characterization pre and postplacement to elucidate the effects of the oral environment on device surfaces. Changes in surface crystallinity, morphology, and elemental composition were monitored with Raman spectroscopy, scanning electron microscopy, optical microscopy, and x-ray photoelectron spectroscopy, respectively. In addition, corrosion rate and polarization resistance were obtained to assess electrochemical device stability after placement. RESULTS: Control analysis indicated the titanium oxide of IHAs was thicker than natural commercially pure titanium and had the structure of crystalline anatase. After removal, the abutments possessed large amounts of biological debris, visible scratches, and discoloration sparsely on the surface. Spectroscopic analysis revealed the titanium oxide on the surface of IHAs was structurally unchanged, with crystalline titanium dioxide still present on the surface. Electrochemical results revealed that implanted healing abutments possessed a significantly higher corrosion rate than controls (change in corrosion rate = 2.34 ± 0.58 nm/year). CONCLUSIONS: Healing abutments were stable in the oral environment due to the chemical stability of the oxide, and were likely subjected to abrasions from unintentional loading and oral hygiene techniques.