Using implants for dental applications are well-accepted procedures as one of the solutions for periodontal defect repair. Suitable design and materials, their reaction with the surrounding hard tissues and interfacial biomechanical properties are still considered to be the primary criteria which need to be addressed. The purpose of present study was to evaluate the bone repair around pure titanium implants and porous surface using anodic oxidation technique, after their insertion in tibiae of rats (n = 15). Five animals received pure titanium-surface implants in tibia, 5 rough-surface implants (TiO2/Ti) in tibia and last five acted as control group. The interfacial integrity and compositional variation along the interface were studied using scanning electron microscope (SEM) with energy dispersive analysis of X-ray (EDX) and histopathology after 2 months. The rats were sacrificed 8 weeks after surgery and fragments of the tibiae containing the implants were submitted to histological analyses to evaluate new bone formation at the implant-bone interface as well as the tibiae were radio graphed. The SEM-EDX results confirmed the initial stability for the Ti implant, but the regen eration of new bone formation was faster in the case of TiO2/Ti implant, and hence could be used for faster healing. The results of the histological analysis showed that osseointegration occurred for both types of implants with similar quality of bone tissue. In conclusion, the porous-surface implants contributed to the osseointegration because they provide a larger contact area with surface roughness at implant-bone interface can help into the formation of physico-chemical bondage with the surrounding hard tissues.
Pure titanium and titanium alloys are the most used biomaterials for fabrication of surgical implants due to their excellent mechanical properties, biocompatibility [
The high biocompatibility of titanium derives partially from the stable and protective oxide layer, which apparently aids in connecting extracellular matrix to the implant surface [
Regardless of their external shape of the implants, microscopically they can present smooth, porous or textured surfaces [10-15]. Several studies have shown that the success or failure of surgical implants can be related to chemical [13,16] and biological properties [
Porous implants have been developed to be stabilized by bone ingrowth into the pore [13,15]. Oliveira et al. [
Titanium foil (Sigma-Aldrich Chemie GmbH, Riedstr. 2D-89555 Steinheim 497329 970) with 0.25 mm thick, 99.7% metals basis was used as base material in this study. The exposed metal surface (area: 1 cm2) of each specimen was ground with silicon carbide paper to 2000 grit, washed in distilled water and then rinsed with alcohol before implantation [
The surface morphology and chemical composition of the untreated and treated titanium samples, Ti, TiO2/Ti-plate, before and after implantation process were studied by scanning electron microscopy (SEM) with electron diffraction X-ray (EDX) system by JEOL-840 Electron prop micro analyzer.
Fifteen male, adult Sprague-Dawley rats were used in this study and were purchased from King Fahed Medical Research Centre in Jeddah (Kingdom of Saudi Arabia). The average weight of the animals at surgery was 224 g; after 8 wk of osseointegration the average weight was 375 g. This weight gain is normal in healthy male rats. As described below, titanium implants were implanted in tibia of each animal. The un-operated tibia was used as histological control. For the entire experimental period two or three animals were kept in each cage with an unlimited supply of fresh water and rodent pellets. The European Community Directive (86/609/EEC) and National rules on animal care have been followed.
Experimental implants were manufactured from pure titanium. The implants had an overall length of 3 mm. A 1.0 mm thickness; smooth middle section had a diameter of 1.0 mm. The implants were cleaned using oscillating ultrasound equipment after placing them in n-butanol within a glass container. They were processed two times for 10 min each time, with a change of liquid. The objects were then rinsed three times and processed another 10 min in 70 percent ethanol. From this stage, in order not to contaminate the titanium surface, the implants were kept in a dry glass container. Finally, the implants, together with all necessary instrumentation, were moist-sterilized at 134˚C for 40 min.
The animals were anesthetized intraperitoneally with a solution of 8 mg ketamine chlorlhydrate and 1.28 mg xylazine per 100 g body weight. The skin of right tibiae was shaved before a 1.5 cm incision was made along the tibial crest. The region of surgery surface was cleaned with antiseptic. The subcutaneous tissue, muscles and ligaments were dissected to expose the lateral external surface of the diaphyseal bone. An end-cutting bur was used to drill make a crack 1.5 mm in diameter with manual rotating movements to avoid overheating and necrosis of the bone tissue [
In vivo biocompatibility of TiO2/Ti coating was determined by implanting in the plates in rat tibia. The animals were anesthetized with the same procedure used for implant surgery. The rats were sacrificed after eight weeks of healing and the bone specimens with uncoated Ti and coated Ti implants were retrieved. The Tibiae were removed and all specimens were X-rayed using dental equipment. A hydrated aluminum chloride solution (7 percent, w/v) with formic acid (5 percent, v/v), HCl (8.5 percent, v/v), and distilled water was used to decalcify the bone specimens. The bone became sufficiently soft after 2 - 3 d in this solution at 4˚C. Phosphate buffer rinse stopped the decalcification process. The specimens were fixed in 10% phosphate-buffered formalin (pH 7.25) for 10 days and dehydrated in graduated ethanol (70% for 30 min, 95% for 30 min, and 100% for 2 × 1 h) series. After embedding samples in Spurr’s resin, each undecalcified implant block was sectioned perpendicular to the implant surface using a low speed diamond saw [
The results of SEM micrographs, Figures 1(a) and (b), of Ti samples before and after anodization, respectively show that: a) the surface appearance of the mechanically polished pure Ti sample (uncoated) represented the typical morphology of native oxide film, with thin and nonporous structure; b) the anodic oxide film, TiO2, showed that the surface of Ti specimen (coated) appear as the network forms with nano porous slots. The EDX spectrum of untreated and treated titanium spacemen’s were presented in Figures 2(a) and (b), which indicated that the chemical composition of both samples oxide layer is Ti in addition to oxygen and small percentage from fluoride for only anodized Ti sample. The surface analysis results of untreated and anodized Ti samples confirmed that the major element present on the surface is Ti.
All implants had characteristic signs of bone ingrowth in various regions along their length. There were apparent differences in the amount or distribution of bone in
growth between the uncoated and coated Ti evaluations (Figures 3(a) and (b)). Densification of bone immediately adjacent to the porous fiber metal, suggestive of bone ingrowth and load transfer, was observed locally at coated TiO2/Ti (
The results of SEM micrographs of different examined samples after implantation process (
and O, the essential ingredients of bone cells on anodized titanium implant sample, TiO2/Ti. It is clear that an array of TiO2 nano-porous structure well adherent on Ti implant surface can be useful for accelerated bone growth in orthopedic/dental applications. We noticed that the Ca peaks are detected in two implantation process this pointed to the bone healing occur either with Ti or TiO2 /Ti but with a good proliferation with TiO2/Ti as the arrows indicate in
All animals presented satisfactory postoperative results, without any evidence of inflammation or infection of the
surgical site. No adverse reaction was observed during the procedure. After implant insertion, slight initial limping was noticed in some animals, but no pronounced motion disorders were seen; neither were there signs of infection, failure to thrive, or other complications. The structure of bone surrounding the titanium implants appeared normal after 8 wk of intramedullary osseointegration. There were obvious signs of bone remodeling adjacent to the proximal implant plate including changes in the size and shape of the bone and osteoclast activity resulting in new bone lamellae.
Histological analysis of the cross sections from all tibiae confirmed the presence of bone ingrowth after 4 and 8 weeks of implantation of Ti-plate (
the osteotomy site of bone two months after implantation of TiO2/Ti-plate. Enlarging the haversian canals (HC), but several canals show no evidence of repair. The vast majority of the chondrocytes appear viable, with only small patches of a cellular matrix. There is no evidence of an inflammatory response.
Osseointegration is fundamental process in orthopedic. Several literatures explained about the integration of the implant with adjacent bone and tissue [27-29]. Osseointegration defined as the process of formation of new bone and bone healing. The incapability of an implant surface was improved to join with the adjacent bone and other tissues through the formation of a fibrous tissue around the implant and promote loosening of the prostheses. Thus, materials with a proper surface are extremely essential for the implant to integrate well with the sur-
rounding bone. Surface chemistry, roughness and topography are all parameters that influence both the osseointegration and biocompatibility [
Since Ti and its alloys has won as a good metallic biomaterial, researchers were keen to further improve the osseointegration of Ti by applied different surface modification method by altering the nature of the surface [30- 33]. Recently, TiO2 has been suggested as a bioactive surface to improve the osseointegration process. The advantage of using TiO2 is that it can be grown directly on the Ti surface, by cost-effective techniques such as anodic oxidation [34-36]. Also, it is well known that one problem with bone healing is poor adhesion strength at the Ti/bone interface [37-40]. By using anodic oxidation, TiO2 is formed with a chemical bond between the oxide and Ti substrate that likely results in enhanced adhesion strength of the bone.
Porous TiO2 films with controlled nanostructures were prepared reproducibly and conveniently by potentiostatic anodic oxidation in different electrolyte [41-43]. Our previous research has shown that it is possible to increase the range of titanium in medical application by depositing a porous layer of TiO2 on the metal surface [44-46]. The objectives of the present work are to assess the effect of TiO2 coat prepared 1 M H2SO4 + 0.5 wt% NaF to obtain a new anodized titania to evaluate histological effect of Ti/bone and TiO2/Ti/bone interfaces and to contribute clinically relevant data on the permanence of titanium metal structures used in osteosynthesis in the body.
The oxide film formed in 1 M H2SO4 and 0.5 wt% NaF has higher nano porous structure compared with our previous work that formed in 0.5 M H2SO4, and that formed in 1.4 M H3PO4 [
By comparing the tibiae of implanted uncoated Ti with that of coated anodized Ti, note that there is a different in the size of two implanted tibiae. The coated implanted tibiae are larger than the uncoated implanted tibiae (Figures 3(a) and (b)). This result is considered as a good observation and may be attributed to the excellent bone healing process on TiO2/Ti/bone interface and perhaps draws the authors to further future study. The detection of both implanted tibiae by X-ray investigation indicated that the high bone regeneration on coated implanted tibiae need more time to get a normal bone shape without any apparent defect (
Electrochemical formation and characterization of porous titanium (TiO2) films [
Microscopic observation of the implant/bone interface at this time-point indicated successful osseointegration with normal remodeled bone adjacent to the fixture [
Brånemark has studied the processes of osseointegration for endosseous titanium implants in long bones under various conditions [
Bone growth is also dependent on factors such as percentage of surface porosity and the presence of gaps between the implant and the bone at the time of placement [
Some previous studies used a 4-week healing period to evaluate the biocompatibility of metal materials [15,59]. Healing periods were longer than 4 weeks added no benefits to increase the quantity of bone tissue ingrowth into porous-surface implants, and observed that only bone tissue maturation took place after this period [
Our results show improvement in cell attachment and spreading on TiO2/Ti coated as compared to uncoated Ti, which is in line with previous studies [6,62]. During the initial period of bone healing, the mesenchymal cells move into the inflamated site and differentiate into osteoblasts, which allow the osteoid formation. The presence of mesenchymal cells in abundance at the healing zone between the TiO2-coated implant and the mature bone indicates the commencement of bone regeneration [
According to the methodology employed in this study, it was possible to conclude that, the anodic oxidation technique improved the surface of titanium by forming a film of nano-porous oxide layer. This porous-surface improved the osseointegration process because it encourages the bone healing at TiO2/Ti/bone interface. Therefore, the results showed that the roughness TiO2/Ti implant surface is better than the smooth Ti surface and well tolerated when placed in rat tibiae, thus corroborating the findings of previous studies that indicated modified titanium plate as the best biomaterial for bone surgical implants.
This research was supported by funding source from Center of Research Excellence in Corrosion, King Fahd University of Petroleum and Minerals, Al-Read, KSA. This study was parts of the Grant No. CR-12-2010.