Fibroblast response to initial attachment and proliferation on titanium and zirconium surfaces

ISSN Online 0719-2479 ©2016 Official publication of the Facultad de Odontología, Universidad de Concepción www.joralres.com 194 1. Laboratorio de Investigación Interdisciplinaria, Escuela Nacional de Estudios Superiores, Universidad Nacional de México, León, México. 2. Universidad Politécnica de Guanajuato, Cortázar, México. 3. Área de Salud Pública Bucal, Escuela Nacional de Estudios Superiores, Universidad Nacional de México, León, México.


INTRODUCTION.
In recent decades, dental implants have become one of the best options for comprehensive dental restoration in patients with partial or total prosthesis. Dental implants may help patients obtain a complete, healthy, functional and aesthetic dentition 1 . Implant placement is a complex, multidisciplinary task involving various disciplines and requiring a deep knowledge of periodontics, surgery and prosthetics 2 .
The successful placement of a dental implant is determined by the environment of the oral cavity and the response of adjacent tissues. The success of an implant is directly related to bone formation around it (osseointegration), in order to direct the forces of mastication to the bone structure 1 ; soft tissue integration to provide a biological seal between the oral cavity and the implant 2 and the appropriate biocompatibility of the tissue at systemic and local level 3 . As soon as an implant is placed in the appropriate site, there is a molecular interaction covering the implant in just nanoseconds. This is primarily influenced by the implant surface 3 , which also determines the type of coating and rehabilitation procedures 4 .
Anderson defined the term biocompatibility as the ability of a biomaterial, prosthesis or medical device to perform a specific function, with an appropriate host response. The evaluation of biological responses is measured by the magnitude and duration of adverse alterations in homeostatic mechanisms that determine the host response 5 .
Biomaterials commonly used for dental implants are manufactured from metal (titanium and its variants) and ceramic materials covered by porcelain (aluminum and zirconium oxide) 6 . Ti is widely used because it has proven to be a biocompatible and bio-inert material, stable and very well tolerated by soft tissues 7 . Although Ti implants have been used as the gold standard in the past 40 years, cell adhesion to this type of material is not always strong, and new formulations and modifications of the surfaces are developed to enhance cell attachment to the implant and accelerate osseointegration 8 .
In response to the above, the use of dental implants based on Zr represents a new frontier in implantology. Ceramic implants have been successfully used in orthopedic surgeries for many years because their biocompatibility tests have yielded positive results, while carcinogenicity and mutagenicity tests have shown negative results. Zr can provide an aesthetic advantage and result in less biofilm accumulation on the surface of the implant 9 . However, due to the lack of information on their performance in the short and long term 10 , Zr implants have not been widely used as Ti ones.
The hypothesis proposed in this study is based on the fact that a Zr surface can provide adhesion and proliferation for human gingival fibroblasts (HGF) equal to or greater than a Ti surface 11 , and therefore show more scientific evidence on the adhesion of fibroblast to Zr surfaces.
The aim of this study was to quantify in vitro the adhesion and proliferation of HGF response on Ti and Zr surfaces with a fast and reproducible colorimetric method using MTT bromide salt. Preparation of Ti samples Type 1 Ti plates of 10x10x0.5mm (n=3) were prepared. Samples were placed in epoxy resin and polished automatically at 160-200 rpm (Buehler, Lake Bluff, IL, USA) with water sandpaper of different roughness, # 400, 800, 1000, 1500 and 2000 (Fujistar, Sankyo, Rikagaku , Okegawa, Japan) and diamond suspension from 0.05 to 1μm with a cloth (Chemomet, Buehler, Lake Bluff, IL, USA). Samples were removed from the epoxy resin and washed in ultrasound with distilled water, 99.5% ethanol and 99.5% acetone for 10 minutes and dried at room temperature.

Preparation of Zr samples
Zr plates of 10x10x10mm (n=3) were prepared. Samples were sintered in a conventional manner in a furnace at 2822°F for 6 hours and then the temperature was gradually decreased for 3 hours. Then plates were cut, polished and sandblasted. All Ti and Zr plates were reused for each experiment after being re-polished, ultrasonic washed and sterilized.
Observation of samples by AFM Ti and Zr surfaces were evaluated using atomic force microscopy (NANOSURF FlexAFM, Liestal, Switzerland). The surface roughness was estimated based on Ra (arithmetic mean of the absolute values of the coordinates of the points of the roughness profile in relation to the midline within the length measurement) and Rms (the largest partial roughness present on the measuring Fibroblast response to initial attachment and proliferation on titanium and zirconium surfaces. Cell culture The human gingival fibroblasts (HGF) were obtained from gingival tissue biopsy of a third molar of an 18-yearold patient, with prior approval and after signing informed consent. The project was authorized by the Committee on Bioethics at ENES, Unit Leon, National Autonomous University of Mexico. The tissue was stored in PBS and 2% antibiotic. The sample was washed twice with PBS and 2% chlorhexidine. The primary cell culture was performed using explants of 1x1mm approximately. The tissue was suspended in α-MEM medium supplemented with 20% FBS, heat inactivated, 100IU/ml penicillin G and 100mg/ml streptomycin sulfate. Cells were incubated at 37°C with an atmosphere of 5% CO 2 and 95% humidity for two weeks changing the culture medium every third day until exponential growth was observed. HGFs have an in vitro life expectancy of approximately 40 PDL (population doubling level).

Meza-Rodríguez
The cells were detached enzymatically from the culture dish with 0.25% trypsin-EDTA 0.025% -2Na for each experiment. After the primary cell culture was established, the experiments were carried out using DMEM+10% FBS and antibiotics 12 .
Adhesion assay and cell proliferation.
Cells were inoculated at a density of 2x10 6 cells/ml in each of the Ti and Zr samples and incubated at room temperature (23°C) for 60 minutes 12 . Samples were washed twice with PBS to remove nonadherent cells. In the case of cell proliferation, cells were incubated for 24 hours more at 37°C with 5% CO 2 . The number of viable cells attached and proliferated on the surfaces was determined by MTT method. Subsequently, 0.2mg/ml of MTT reagent was mixed in DMEM +10% FBS and incubated for 3 hours at 37°C with 5% CO 2 . Formazan was completely dissolved with DMSO, transported to a 96-well plate and analyzed at 540nm in a microplate reader (Thermo Fisher Scientific, St. Louis, Missouri, USA). In cell adhesion and proliferation Ti plates were used as control value. Assays were performed in triplicate from three independent experiments.

Statistic analysis
The mean, standard deviation and percentage were calculated. All data were tested with Kolmogorov-Smirnov tests of normality (Lilliefors), Kruskal-Wallis test, and multiple comparisons using Mann-Whitney (SPSS, Chicago, IL, USA). Statistical significance was considered at p<0.05 and a 95% confidence interval.

RESULTS.
Topography of the Zr plates showed a higher roughness (Ra=0.39μm) (Fig. 1A and 1B) than Ti (Ra=0.049μm) ( Fig. 1C and 1D). Ti samples showed an almost flat surface with some sipes and micropores, in contrast, Zr plates showed a roughened surface with a high presence of micropores.
Quantification of HGF adhesion was significantly higher (p<0.05) in the Ti with 42% (±18.2%) more when compared to adherent cells in Zr samples ( Fig. 2A), while the proliferation revealed a greater number of cells on the surfaces of Ti with 37% (±27.4%) showing no statistical differences (Fig. 2B), having a similar biological response between both surfaces.

DISCUSSION.
The aim of modifying the surfaces of the implants is not only to adapt them to the demands to avoid the negative effects of implanted materials into the surrounding tissue, but to improve the interaction between the material and tissues 13 . The use of current technologies that modify the surface of the implants has become a trend in marketing and production of new implants, creating different morphologies and chemical treatments to improve and accelerate osseointegration 14 .
For a long time, osseointegration was identified as a local factor that could interfere with the success of dental implants. Now, it is known that not only the osseointegration of dental implants contributes to the integration of the adjacent tissues, but also of the soft tissues adjacent to the implant 11 .
Long-term stability of dental implants, the biological seal of soft tissues and implant interface are important features for the clinical success of oral rehabilitation. The transmucosal part of the dental implant requires sufficient attachment of connective tissue and inhibition of bacterial invasion 15 . Conventionally, surfaces in contact with soft tissues, particularly the abutment, are designed with smooth surfaces to prevent bacteria from adhering easily 16 .
The aim of this research was to quantify in vitro the HGF response on Ti and Zr surfaces by cell adhesion and proliferation. The results shown here indicate that initially Ti surfaces significantly increase HGF adhesion by 42%, while cell proliferation, comparing both surfaces showed Fibroblast response to initial attachment and proliferation on titanium and zirconium surfaces.
no difference in the number of proliferated cells, suggesting that the biological response in both surfaces after 24 hours can yield similar and comparable results. The type of material with which dental implants are manufactured is critical to their success, also the topography of the implant surface influences integration 14 qualitatively and quantitatively 14 .
However, the review of the current literature showed partly contradictory results. Yamano et al. 17 identified the topography of the surfaces as an important modulator of fibroblast behavior, both in vitro and in vivo, demonstrating that smooth Zr surfaces promote more adhesion and proliferation of fibroblasts. The difference between their results and this research could be caused by the polishing of the surfaces. Furthermore, Pae et al. 16 suggest that microporosity allows greater diffusion and alignment of fibroblasts, when compared to the smooth surface. The controversy lies not only in the topography of the Zr surfaces. Velasco-Ortega et. al. 8 found that osteoblastic cells cultured on rougher Ti surfaces differ faster than on smoother surfaces, however, Xiaohui Rausch-fan et al. 11 suggested that the smoothest surfaces are the ideal surfaces for better adhesion and cell proliferation. Furthermore, Esfahanizadeh et al. 18 showed that the difference between the adhesion on Zr and Ti surfaces is not significant, which is line with the findings of this study.
The method reported here is an easy and reproducible technique for the study of the interaction between Ti and Zr cells and surfaces. The method of rapid colorimetry by MTT is based on the identification of the metabolic activity of the cells adhered and proliferated on the surface. The results shown in this study are more representative than those previously published by direct counting with scanning electron microscopy 19 or enzymatic detachment with trypsin and ultrasonic vibration 20 . Such methods may underestimate the number of cells adhered and proliferated to surfaces. However, this study is preliminary and it is necessary to investigate the kinetics of the interaction, the specific morphological and functional relationship of the cells on surfaces, as well as controlling the expression of different genes associated with cell adhesion and proliferation, such as integrin expression on surfaces.

CONCLUSION.
Although initially Ti showed increased cell adhesion on the surface after 24h, Zr samples showed a similar proliferation. Therefore, both surfaces have comparable biological response.