Histomorphometric Analyses Of Bone Interface With Titanium-Aluminum-Vanadium and Hydroxyapatite-Coated Implants by Biomimetic Process

Abstract

Purpose: 

The surface properties of dental implants have been considered directly related to the success of the osseointegrated interface. Hydroxyapatite (HA)-coated implants promote a bioactive surface because they facilitate cellular migration and bone growth, resulting in a faster osseointegration.

Materials and Methods: 

This study analyzed and compared 2 differen implant surfaces: a group composed of titanium implants and another group coated with HA. The HA coating was produced by the biomimetic process to reduce costs.

Results and Conclusions: 

After histomorphometric analyses it was possible to demonstrate that there were no statistically significant differences between the groups.

Titanium has been used in orthopedic therapy for more than 40 years with no records of biological incompatibilities. It presents good mechanical properties, with tension strength close to the stainless steel. Titanium is utilized in surgical implants for many parts of human body subjected to physical loads after trauma reconstructions.¹

For dental implants, the desirable mechanical properties of metal titanium, as well as the implant’s surface characteristics and design, are considered important factors influencing the expected biological response.² In addiction to the implant’s composition, the method of production and the surface preparation should also be well controlled.³

The need for predictability and long term results of implant therapy has prompted dental research to focus on implant design as well as physical-chemical characteristics of implant surfaces, which are considered fundamental for treatment success.^4,5 Many studies have compared the performance of dental implants composed of commercial pure titanium, titanium alloys, or with some type of surface coating as hydroxyapatite (HA), with a focus on which type of implant presents a better long term bone reaction.⁶

It has been attributed to high dielectric properties of the titanium oxide - which exceed most of the other metallic oxides – part of the characteristics of the positive biological response to dental implants, since they are capable to be more reactive to biomolecules through the increasing electrostatic forces on their surfaces.⁷ Clinical success of commercially pure titanium machined screws could be affected by poor density bone.⁸ Consequently, many efforts to characterize and modify the implant surface aiming to increase clinical success have been performed. Some dental implant designs have a substructure of commercially pure titanium or titanium alloys with some surface coatings, mainly the traditional method of plasma thermal aspersion with a fine coat of calcium phosphate.^6,9 The reason for this type of implant coating is to allow that the calcium and phosphate compound induce a biological reaction that promote bone growth and increase the speed of direct contact process between bone and implant.¹⁰

The high technology required for such method is yet not available in our country, as it would increase the costs involved in large scale implant production. Other methods of implant surface coatings could be utilized with national technology and lower costs and, among them, the biomimetic methodology. This method of HA coating with the width of 50 nm, approximately 99% of pure HA and good stability at high temperatures.¹⁰ It utilizes heterogenic precipitation over a substrate previously attacked by sodium hydroxide, in such a way that the TiO₂ formed over the metallic surface of titanium becomes sodium titanate and, in the sequence, have a tendency to transform to calcium titanate when it makes contact to aqueous solution with 1M of calcium and ph 7.0. After this procedure, the new surface of calcium titanate is immersed in a simulating body fluid which leads to the formation of a titanate-apatite titanium coating, followed by the nucleation of HA over this coating, converting as an intermediary phase on the chemical integration between HA and titanium.¹¹

Nevertheless, well designed studies should be implemented to verify the applicability of alternative methods to those considered traditional and well known. After the evaluation of new surface coatings by in vitro studies, it is mandatory to perform experimental in vivo analyses by observing microscopically the percentage of bone-implant contact.¹² The aim of the present study is to compare the percentage of osseointegration of implants produced from a titanium alloy and those with the same material, but coated with HA applied by the biomimetic method.

Materials and Methods

Biomimetic Process and Implant Characterization

The sixteen screws implants of the present experiment were made form titanium alloy normally used in bone grafts reconstruction surgery, with 2 mm of length and 1.5 mm of diameter (Neodent, JJGC Indústria e Comércio de Materiais Dentários Ltda., Curitiba, Paraná, Brazil). Half of them were maintained as-received (Ti with 5.5% to 6.75% aluminum and 3.5% to 4.5% vanadium, namely Ti6Al4V), while the other half were coated by HA using the biomimetic process. The HA biomimetic coating was performed according to the protocol described by Kokubo et al⁶ and modified by Andrade.¹²

Eight samples of implants were immersed in NaOH 5M solution at 60°C for 24 hours and then, washed in bi-distillated water at room temperature. The aim of this procedure was to produce a sodium titanate layer on their surfaces, replacing the titanium oxide surface. At the next step, sodium titanate coating was stabilized trough a thermal treatment in a tubular furnace with N₂ flux, in a heating rate of 300°C h⁻¹. After reached the 600°C, the screws were maintained at this temperature for one hour, and cooled with a rate of 55°C/h⁻¹. Then, the screws were immersed in a human plasma simulating solution (SBF), at 37°C, during 21 days, with the objective to induce the formation of calcium titanate followed by the precipitation on hydroxyapatite on their surfaces. After the treatment in the SBF solution, some surfaces show agglomerates that were removed by de-ionized distilled water during 5 minutes in ultra-sonic bath. Finally, the samples were sterilized with a total dose of 25 KGy for 10 hours, using a Cesiun-137 source for irradiation.

Animals and Surgical Technique

Eight adult female albino New Zealand rabbits, weighting between 3 and 4 kilograms were used in the experiment. The surgical procedures were performed under general anesthesia, using 2-(2,6-xylidine)-5,6 dihydro-4H-1,3 tyazine chlorohydrate (Rompur^R 2% solution, Bayer do Brasil S.A., Área Veterinária, Belo Horizonte, MG, Brazil) at the following dose: 3 mg/kg, im, associated with 20 mg/kg, im, cetamine chlorohydrate (Ketalar, Aché Laboratórios Farmacêuticos S.A., Guarulhos, São Paulo, Brazil). After local anesthesia with 2% lidocaine solution with vasoconstrictor, the surgical procedures begun with a incision on skin and a mucoperiosteal flap, with the exposition of the tibiae bone. Low speed surgical burs under copious saline irrigation were used to prepare the implant site. Following, the screws were inserted with a hand wrench.² Each animal received two different types of implants. After surgery, the animals were medicated with Decadron (0.5 mg/Kg weight), (Promode Química e Farmacêutica Ltda, Campinas, São Paulo, Brazil) im, and Novalgina (40 mg/kg weight) (Hoechst Marion Rossel S/A, São Paulo, São Paulo, Brazil) orally for 10 days.¹³

Samples Preparation

Three months after the surgery the animals were sacrificed with a 200 mg iv dose of sodium Phenobarbital (Cristália Produtos Químicos Farmacêuticos Ltda., São Paulo, São Paulo, Brazil). All the implants were removed together with neighbor tissue, preserving 3 mm of bone structure around it. All the assembly was fixed by immersion in 2.5 glutaraldehyde solution, in a buffer 0.05M cacodylate, ph 7.2, for 48 hours. Following, they were dehydrated by an increasing-concentration series of ethanol. It was also applied a clorophormium solution to remove any fat tissue and to facilitate the penetration of the resin in which the samples would be inside. Then, the samples were embedded to a Spurr resin infiltration at 20°C in vacuum and polymerized at 70°C. The blocks were abrasion trimmed to 50% of their volume, along the long axis of the implants. They were sequenced ground with a sequence of silica carbide papers, from 200, to 600 and cleaned with acetone solution in ultra-sonic bath for 10 minutes. Finally, the resin blocks with the samples were coated with a gold film to improve the electron conductibility in the scanning electron microscopy (SEM).⁴ After metallization, the blocks were positioned in the vacuum chamber for SEM analyses.

Histomorphometric Analyses

All the samples were visualized by SEM (DSM 940A, Zeiss, Germany), with a magnification of 100X, using backscattered electrons to evaluate the direct contact between bone and implant surfaces and thus interpreted as the percentage of osseointegration. Figure 1A shows the region corresponding to the sixth thread of one screw, before the histomorphometric analyses. Dark zones represent areas with no bone contact; gray zones show the bone area, while the implant is represented as white color. (Fig. 1B).

Fig. 1.: SEM images of implant samples. (A) Region of the threads analyzed. (B) Bone-implant interface sample.

All digital images were processed using image-analyses software (Global Lab Image, Data Translation, Marlboro, Ma, USA) to calculate bone-implant interface perimeter as showed in Figure 2.

Fig. 2.: Demarcation of osseointegrated perimeter (red line) with image-analyses software (Global Lab Image).

Statistics

The results obtained from the histomorphometric analyses, which determined the percentage of osseointegration, were compared with the non-parametric Wilcoxon test.

Results

At the end of the experiment all implants were considered osseointegrated. The results of the percentage of bone-implant contact are presented on Table 1.

Table 1: Percentage of Osseointegration Referred to Implant Surface and Animal Sample

Statistic analyses indicated that different amounts of contact between bone and hydroxyapatite-coated implant was not significantly different, when compared with the titanium alloy screws (P > 0,05).

Discussion

Gottlander et al⁴ and Vidigal Jr. et al¹⁴ observed more bone formation around hydroxyapatite-coated implants, when compared to titanium surface samples. The results of the present study show no statistically significant differences between HA-coated surfaces and titanium alloy implants, even with the utilization of the biomimetic methodology.

The lack of differences could be related to some sort of particular characteristic of the present coating process such as: purity level, crystallinity, calcium/phosphate ratio, and the presence of other calcium-phosphate phases. Less crystalline coatings, ie with a Ca/P rate lower than the stoichiometric values, and other Ca/P phases, besides hydroxyapatite, have a tendency to be more soluble, and, therefore, more bioactive.¹⁵ On the contrary, coatings of high grade degree of crystallinity could present similar behavior to a bioinert material, like titanium. Therefore, the comparison of the histological results from different studies related to hydroxyapatite-coated implants could indicate the effect of different physical-chemical properties of the variety of coating on the biological response. It could be considered that the term hydroxyapatite does not represent one single type of material, but a variety of substances that present different degrees of crystallinity, Ca/P rate, solubility and bioactivity. Besides, differences on the implant design, as well as the shape and distance between the implant threads, could also contribute to the results. It should be considered that direct transference of the results obtained from animal experiments could not be extrapolated to the human body.

Conclusions

It could be concluded, based on the results of the present study, that the percentage of contact of different implant surfaces and bone show no difference of osseointegration percentage in biomimetic HA-coated implants, when compared with titanium alloy samples inserted in rabbits′ tibiae, after three months. The development of other studies related to a better predictability of osseointegration with alternative implant surface coating methods should be stimulated aiming better clinical results.

Disclosure

The authors claim to have no financial interest in any company or any of the products mentioned in this article.