In this study, dental cores were fabricated with materials that are used for implant superstructures, through the dental restorations fabrication process, Cell adhesion, morphology, proliferation, and differentiation of MC3T3-E1 pre-osteoblast cells involved in implant osseointegration, on each core were analyzed, and implant cytocompatibility was investigated. In most previous studies on implants, cytocompatibility has been studied through in-vitro cell experiments using osteoblast cells and titanium or titanium alloys [22].However, there have been few studies on osteoblast cells and the core, which is the superstructure of the implant prepared through the actual process of dental restoration fabrication.
The fabrication process of the dental restorations is an important process requiring professional technique and proficiency, and appropriate dental materials as the process is for the restoration of lost teeth while restoring the aesthetic aspect desired by the patient and the functional intraoral and dental aspects [23].In this experiment, the cores that form the superstructure of the implant were fabricated using materials including P. F.G, which is a gold alloy, Co-Cr alloy and Ni-Cr alloy, which are non-precious metal alloys, and Lithium disilicate glass ceramic and zirconia, which are ceramic materials. Other than precious metals, non-precious metals, ceramic, various biocompatible poly-substances such as polymers and hybrid-resin ceramic are available [24].Use of such biocompatible dental materials can enhance the patient’s satisfaction on the restorations in the aesthetic and functional aspects [25].
During dental restorations fabrication, sandblasting surface treatment plays an important role for metal materials in removing the impurities and enhancing the bonding strength between the core and metal materials [26].In case of ceramic materials, sintering plays an important role of enhancing the intermolecular bonding strength [27,28].
In this study, the cytocompatibility of the specimen fabricated through dental restorations fabrication procedure was investigated through in-vitro experiments using MC3T3-E1 cells, which are pre-osteoblast cells involved in implant osseointegration. Cell culture was performed under aseptic conditions. The experimental procedure using CLSM includes a post-treatment process and has disadvantages of difficulties in cell adhesion due to errors arising during the experimental procedure or environmental factors when proficient skills are not mastered. In this experiment, P. F.G was used for CLSM measurement and the surface and component analysis. CLSM measurement of P. F.G showed proper cell adhesion and an active spread of MC3T3-E1 cells (Fig. 2A). This is consistent with previous studies suggesting sufficient biocompatibility, corrosiveness, and corrosion resistance of the dental gold alloys; hence, the prostheses fabricated of the dental gold alloys among other metal alloys were biocompatible [29,30].However, this experiment showed more cell adhesive distribution on Co-Cr among the materials used for dental cores compared to P. F.G (Fig. 2E). Cell morphology and adhesion observed by CLSM measurement showed that the cells could not proliferate on the core made of Ni-Cr, and the non-precious metal alloys in particular (Fig. 2D). In general, studies on the intraoral tissue irritation caused by Ni-Cr alloys have been of the interest and nickel cytotoxicity including allergic reactions have been a real problem [31].
In the experiment on cell proliferation and differentiation, Co-Cr, Ni-Cr, zirconia, and lithium disilicate glass ceramic underwent the dental restoration fabrication procedure (Fig. 1) to form of 10 × 10 × 3 mm and 20 × 20 × 3 mm, according to the sizes of the cell culture plate. From the MTS assay for cell proliferation and ALP assay for cell differentiation, a color change of the indicator to blue was observed due to reaction with the ions released during cell culture (Fig. 3B). According to previous studies, Lithium disilicate and zirconia are known to have no pH change after immersion [32], and Co-Cr is also known to be not dissolution in the oral cavity. Therefore, it is considered that it does not cause internal environmental changes [18,33]. On the other hand, it seems that there is a change in pH of the Ni-Cr alloy [34], and the change in pH affects cytotoxicity. Also, the restorations fabricated from Ni-Cr alloys influenced the tissue cells surrounding the implanted teeth, causing side effects that may result in cytotoxicity and allergy, and interrupted the metabolism of cytokines and cells that play pivotal roles in the inflammatory process due to the release of metal ions [35].On the other hand, zirconia has been known for its advantage of lowering the risk of inflammatory reaction in tissues adjacent to the implant by reducing the adhesive strength and biofilm accumulation of the bacteria [36].
Therefore, the in-vitro experiment performed in this study is valuable as understanding and recognition of the characteristics and related cytocompatibility of materials used for dental restorations is essential for the appropriate choice and use of dental materials during actual treatment procedures [37].The success or failure of various dental restoration treatments using dental materials is determined by the appropriate choice and accurate handling of the dental materials possessing suitable characteristics [38].This is ultimately linked to cytocompatibility that the dental materials react with the patient’s intraoral tissues [39].After placement of the implant, micro-leakage of ions can occur from the marginal region of the core due to friction inside the mouth or long-term use [40,41].Hence, in-vitro study through clinical experiments and animal experiments to confirm the biocompatibility of dental materials are thought to be required. With the recent development of 3D printing technology, various experiments using MC3T3-E1 pre-osteoblast cells involved in osseointegration, are expected by studying novel materials and novel fabrication methods to seek for biocompatible dental materials.
In this study, MC3T3-E1 pre-osteoblast cells involved in implant osseointegration were used to analyze the cytocompatibility characteristics including cell adhesion, morphology, proliferation, and differentiation of gold alloy, non-precious metal alloy, and ceramic cores fabricated through the CAD/CAM milling method and the dental restorations fabrication process. MTS assay conducted for the comparison of cell proliferation revealed reduced cell proliferation in the Ni-Cr specimen at 5 days after cell culture. ALP assay for cell differentiation showed that the Ni-Cr specimen had the lowest cell activity. All other specimens presented more uniform cell adhesive distribution and more active cell proliferation and differentiation compared with Ni-Cr. Additionally, cell adhesion, proliferation, and differentiation were more active in dental ceramic materials than in metal materials. However, Co-Cr was found to be similar to the ceramic material because there was no significant difference from the ceramic material.