In Vitro Microstructural Analysis of Dental Implants Subjected to Insertion Torque and Pullout Test

surface treatment on the osteoinduction and, therefore, on the osseointegration. The aim of this study was to evaluate by energy-dispersive X-ray spectroscopy (EDX) the effect of insertion torque and pullout test on the microstructure of dental implants with different shapes. Four shapes of implants (n=8) were selected: conical with surface treatment (COTS), cylindrical with surface treatment (CTS), cylindrical with double surface treatment (CTSD) and cylindrical with machined surface (CSU). Before and after performing the mechanical tests, the screw surfaces were subjected to analysis of chemical composition by EDX. The results obtained by the microstructural analysis showed presence of three main chemical elements: Ti, C and O. There was a significant change in the concentration of Ti and C. The implant with double surface treatment (CTSD) showed the greatest Ti reduction and the greatest C increase. It may be concluded that the mechanical manipulation may alter the implant surfaces as regards their microstructure. Therefore, surgical planning should take into consideration the choice of surface treatment because the characteristics of the implants may be modified as they are inserted and removed from the bone site. In Vitro Microstructural Analysis of Dental Implants Subjected to Insertion Torque and Pullout Test


Introduction
The contact between bone and endosseous implant is established and maintained after installation by the osseointegration process, in which the main element of the whole system is the implant itself, considering its macroscopic and microscopic characteristics (1)(2)(3).Morphological and chemical surface changes may alter the response of the bone tissue adjacent to the screw by inducing specific responses in the osteoblastic cells (4).
Modifications made by the manufacturers in the macro and microstructure of the implant, such as changes in shape and surface treatments, aim at increasing the success of oral rehabilitation by promoting greater surface area and bone/implant contact, induction of bone growth and load distribution (5)(6)(7) in order to obtain maximum surface anchoring, insertion with less bone trauma and greater resistance to insertion and removal torques (8).Titanium (Ti) and its alloys are widely used as biomaterials, either in the medical or dental field, due to its good mechanical strength, chemical stability and biocompatibility that are fundamental characteristics to the success of osseointegration, as stated by Branemark (9,10).Among the characteristics of Ti, biocompatibility is the one most related to its microstructure, i.e., chemical composition, roughness and surface energy (11).
The intimate implant/bone tissue contact originates on the surface.Therefore, it is important to keep it free from impurities to allow bone formation in this region.The Ti on the screw surface reacts spontaneously with the oxygen in the air to form a stable oxide layer (TiO 2 ) (1).This layer responds for the biocompatibility of this material due to its low electrical conductivity, high corrosion resistance and thermodynamic stability at physiological pH.However, the natural oxide layer can be easily broken, impairing the bone formation at the implant/ bone interface (12,13).
Incidents during surgery or absence of initial planning may require the replacement of the previously inserted implant in the bone site.In these cases, the action of inserting or removing the screw may alter its surface chemistry, especially as regards to breaking the Ti oxide layer, which may lead to damage in the bone tissue responses.
The purpose of the present study was to evaluate the effect of insertion torque and pullout test on the microstructure of dental implants of different shapes using energy-dispersive X-ray spectroscopy (EDX).

Material and methods
Four different shapes (n=8) of dental implants (Conexão ® , Arujá, SP, Brazil) were selected: conical with surface treatment (COTS); cylindrical with surface treatment (CTS); cylindrical with double surface treatment (CTSD) and cylindrical with machined surface (CSU).All implants had the same dimensions (Ø 3.75 mm x 11.5 mm), except for the conical one (COTS), which had a slightly smaller diameter (3.5 mm) (Table 1).The receptor site of choice for implant insertion was the femur head of a synthetic polyurethane bone (Synbone®, Malans, Switzerland), since this material is indicated as bone substitute due to its close characteristics to the natural bone (14)(15).

Implant Microstructure Analysis
An EDX device (IXRF Systems, Houston, TX, USA) was used to quantify the elements on the surface of the implants, before and after the insertion torque and pullout test.This device emits a thermal ionic beam of primary electrons from a tungsten filament and, after path correction by condenser lenses and focus adjustment by the objective lens, the beam hits the sample to be analyzed.This interaction generates X-ray photons, yielding the energy spectra (16).

Mechanical Tests
A single operator performed all assays for standardization purposes.The surgical insertions were performed with the sequence of bits specified by the manufacturer of an electric motor (MC 101, Omega; Dentscler®, Ribeirão Preto, SP, Brazil).The implants were inserted in the polyurethane femoral head (Synbone, BaySystems ® ) with a standardized torque of 32 N.cm.Four implants (n=4) were inserted into each femoral head; one of each type (COTS, CTS, CTSD, CSU).
For the pullout test, a universal testing machine (Emic DL-10000; São José dos Pinhais, PR, Brazil) was used with a 200 kgf load cell.The screw head was attached to the testing machine by connectors that allow multidirectional movements and applying axial tensile load without torque application.Pre-load of 5 N and settling time of 10 s were used.The axial tensile load was applied at constant 0.2 mm/ min speed until pullout of the implant.These parameters were defined according to the need to adapt the implant to the machine (2).

Data Analysis
Quantification of Ti and carbon (C) in the samples before the insertion torque and after the pullout test yielded the percentage of each ion analyzed by EDX and conducted a descriptive analysis.

Results
The microstructural EDX analysis showed that the main components of the tested implants were Ti and C. Comparing the concentration of each chemical element (Table 2), before and after the test pullout, there was a significant decrease in the concentration of Ti and increase of C, especially in the implants with treated surface (COTS, CTS, CTSD).The CTSD implant showed greater reduction of Ti, while the CSU presented an increase of this element.

Discussion
Since the discovery of osseointegration, different models of implants have been developed.Modifications occurred in both shape and surface, with the evolution of smooth machined to microscopically rough surfaces, which enhance bone healing around implants (4,17).
The titanium alloy used for manufacturing of implants has in its composition a wide variety of elements and chemical compounds, beyond the pure metal, as this would only be only possible under auto vacuum or equivalent conditions (18).In addition to the elements present in the material, the impurities on the screw surface, both due to poor handling or by contamination during surgery, can lead to the destruction of titanium dioxide (TiO 2 ) layer and result in the failure of osseointegration, by the development of pathological processes such as periimplantitis (19).
The surface treatment modifies the implant insofar as it promotes micro-roughness (porosity), responsible for increasing the mechanical imbrication at the implant/ bone interface (17,20,21).This modification on the implant surface leads to a thicker TiO 2 layer, favoring the process of bone neoformation (9).
In the present study, implants with treated surface (COTS, CTS and CTSD) showed the greatest reduction in the concentration of Ti, which indicates a greater reduction of the TiO 2 layer when compared with implants presenting smooth/machined surface (CSU).This result suggests that inserting and removing the implant at the surgical site can break the surface TiO 2 layer , especially in surface-treated implants.Comparing the surface-treated screws, it was found that the CTSD model (cylindrical) showed the highest reduction of Ti and the COTS model (conical) presented the smallest reduction.
The presence of C in the sample is related to the presence of organic material particles.In the present study, it is represented by the synthetic bone.The machinedsurface implants (CSU) had a lower aggregation of organic particles and, therefore, showed lower C concentration.On the other hand, the surface-treated implants (COTS, CTS and CTDS) showed higher C concentration after the mechanical test, demonstrating the influence of the surface roughness produced by the treatment, on the mechanical imbrication (22).
Despite the limitations of this study, the obtained results confirm the importance of conducting a surgical planning prior to implant placement and contraindicate the reuse of previously inserted screws due to the alteration of the superficial TiO 2 layer.Given the benefits of the TiO 2 layer to increase the biological responses and subsequent osseointegration success (23,24), it was found that the surface-treated implants exhibited the greatest modifications in the TiO 2 layer, demonstrating the need for greater care when handling these implants.

Table 2 .
Chemical elements percentage

Table 1 .
Shape, surface treatment and size of the tested implants