Abstract
The aim of this study was to perform a comparative analysis of aspects of the osseointegration of titanium implants placed with and without the local application of a bisphosphonate agent, after 28 days in vivo. The study involved the placement of 50 commercially pure titanium implants in the middle third of the tibia of 10 rabbits, with the right tibia used as the control and the left as the test site. Sodium alendronate gel was applied locally in the test group and sterile saline solution in the control group. After euthanasia, 10 implants from each group were analyzed for maximum removal torque. The remainder of the sample was processed to obtain non-decalcified slides, approximately 30 μm thick, for histomorphological and histomorphometric analyses, including bone–implant contact (%BIC). Data were analyzed at the 5% level of significance. The removal torque values of the test group were, on average, half those obtained in the control group. The test group showed a lower %BIC and notable changes in bone quality. It is concluded that the initial events in the osseointegration of titanium implants are not favoured by the local application of sodium alendronate gel in rabbits.
Over the last few decades, there has been considerable growth in the demand for the replacement of lost teeth by means of therapy with dental implants and implant-supported dentures. Among other reasons, this is due to the fact that partial or complete edentulism affects a large proportion of persons of more advanced age, a population group that is growing steadily worldwide. The bone quality and quantity in these patients is frequently affected by systemic diseases, such as diabetes and osteoporosis, and they may also present lower potential bone regeneration, all of which may contribute to lower success rates in dental implant therapy. On the other hand, the desire to attain faster osseointegration is common for both health professionals and patients. This has motivated research into the development of materials and techniques to optimize the process of bone remodelling around dental implants.
During implant placement surgery, the primary stability of the implant in bone tissue is one of the aspects used to determine whether or not to apply an immediate load. In cases in which there is no primary stability during dental implant placement, it is recommended that the professional follows the protocol of two surgical stages, delaying functional loading for the period of osseointegration. In this scenario it becomes important to accelerate the process of osseointegration, so that the delay between implant placement surgery and re-opening for the connection of the prosthetic abutment can be reduced.
Interest in the use of bisphosphonates as bone biomodulators in implant dentistry was aroused because of the known ability of these drugs to inhibit the activity of osteoclasts; this inhibition activity is why the drugs are used widely in the treatment of diseases characterized by excessive bone resorption, such as osteoporosis, hypercalcemia, and bone metastases. Furthermore, it is known that an effect on bone formation may be expected, promoting a considerable reduction in bone turnover. Studies have suggested that bisphosphonates may have a positive influence on bone formation and remodelling, and may consequently improve the fixation of titanium implants in humans.
Because of the severe side effects caused by the systemic use of these drugs, researchers have turned their attention to developing methods for the local delivery of these drugs to the site of interest. The intention is that the bisphosphonate will positively influence the remodelling of bone adjacent to the implant, without causing undesirable systemic side effects. In this regard, immobilization of the bisphosphonate on the implant surface has been proposed as a way of delivering the drug locally. However, the methodology for this immobilization is complex and requires sophisticated equipment. The direct application of bisphosphonate to the surgical alveolus immediately before implant insertion would appear to be a simpler and more practical procedure; however this has not yet been tested extensively.
Therefore, the aim of this study was to propose the local application of a bisphosphonate drug (sodium alendronate) in gel form, directly to the surgical site, and to perform a comparative evaluation of aspects related to osseointegration of titanium implants inserted immediately after this application, in vivo. The hypothesis was that topically applied bisphosphonates would increase primary implant stability after 28 days.
Materials and methods
Animals and experimental groups
Ten adult male rabbits of the Oryctolagus cuniculus species, New Zealand lineage, with a mean body weight of 4.0 kg, were used in this research. The study was approved by the ethics committee of the university, and the animals received all the care stipulated by the institution.
A total of 50 implants were inserted in the sample. The right tibia was used as the control site and the left tibia as the test site. In the control group, sterile saline solution was applied to the surgical alveoli made in the right tibia of each animal. In the test group, a topical application of a 1 ml of sodium alendronate gel (10 mg/g) was administered to the surgical alveoli. The sodium alendronate gel was formulated specially for use in this study and the formulation was produced exactly as recommended by Reddy and Kumar.
Surgical procedure
After being weighed, the animals received pre-anaesthesia medication, comprising acepromazine maleate (0.2 mg/kg) and morphine sulphate (2 mg/kg), both administered intramuscularly. After approximately 10 min, the marginal ear vein of the animal was cannulated for the administration of fluid therapy with lactated Ringer solution and enrofloxacin (10 mg/kg) 20 min before surgery. Anaesthesia was induced by means of intravenous injection of ketamine chloride (10 mg/kg) and 1 mg midazolam (1 mg/kg). Epidural anaesthesia was administered with 2% lidocaine (0.25 ml/kg). After the induction of anaesthesia, the animals were shaved and antisepsis of the region was performed, including the skin adjacent to the shaved area.
Surgery began with a linear incision, measuring approximately 2 cm in extension, on the medial diaphyseal surface of the tibia. The sites where the surgical alveoli were to be cut were demarcated, with the perforations positioned 10 mm below the tibial condyle and a distance of 10 mm between perforations. Cutting for implant insertion was performed with appropriate burs, under irrigation, to a depth of 4 mm, using first a 2-mm lance-shaped bur and then a helical bur.
After the cavities had been prepared, sterile gauze was introduced and kept in the surgical alveolus by compression for 1–2 min in order to absorb and stop the bleeding. This process guaranteed that the bisphosphonate gel would come into direct contact with the entire wall of the alveolus, without the interposition of blood.
For the test group, a 1-ml quantity of sodium alendronate was injected into the surgical alveolus immediately before implant placement ( Fig. 1 a) . After this, the implants were inserted ( Fig. 1 b). Commercially pure titanium implants with an acid-treated surface were used (Porous Nano; Conexão Sistemas de Prótese, Arujá, São Paulo, Brazil); these were 2.2 mm in diameter and 4.0 mm long, and fabricated specifically for this study. The implants were inserted at a speed of 35 rpm, until they reached bone level.
To complete the surgery, the muscle and subcutaneous tissues were approximated with continuous sutures and the skin was approximated with simple, interrupted sutures, using resorbable suture thread (catgut 4–0; Johnson & Johnson/Ethicon, USA). The region was cleaned with gauze dampened with physiological solution to remove the residues of blood clots, and the wound was covered with an occlusive dressing and a gauze bandage.
During the postoperative period, all animals received analgesic medication consisting of tramadol hydrochloride (2 mg/kg) delivered subcutaneously, every 8 h, for 3 days. Antibiotic therapy consisting of enrofloxacin (10 mg/kg) was administered via intramuscular injection every 24 h for 7 days.
Euthanasia of animals
At 28 days postoperative, the rabbits were euthanized. Each animal received pre-anaesthetic medication comprising acepromazine maleate (l mg/kg), ketamine hydrochloride (15 mg/kg), and xylazine hydrochloride (2 mg/kg), all administered intramuscularly. After around 10 min, the palpebral, corneal, and pain reflexes were absent. With the animal in a plane of deep anaesthesia, 10% potassium chloride solution was administered intravenously until cardiorespiratory function ceased.
Removal torque measurement
The specimens were processed immediately after removal of the tibia for the measurement of the maximum removal torque of each implant. The tibias were first placed in 10% buffered neutral formalin solution; after 1 h, they were submitted to the torque removal test, thus they did not become dehydrated. The anatomical sample was carefully placed on the torque test equipment (CME; Técnica Industrial Oswaldo Filizola, Guarulhos, Brazil), which was controlled completely by the software programme DynaView Torque Standard/Pro M (Dyna Pro Dynamometers Ltd, United Kingdom), generating values automatically at a speed of 1 rpm and angular measurement of the system with a resolution of 0.002°. The maximum torque measurement to begin the inverse rotation was recorded, and the mean torque values were calculated for each group.
Histomorphological and histomorphometric analysis
Bone blocks containing the implants were dehydrated gradually in successive concentrations of alcohol and the samples embedded in methacrylate-based resin using an EMBed-812 embedding kit (Electron Microscopy Sciences, Hatfield, PA, USA) in accordance with the manufacturer’s instructions. The blocks were then cut into slices approximately 300 μm thick, with the centre of the implant in the direction of its long axis, using a diamond-coated disc in a metallographic cutter (Model DTQ5; Pantec, São Paulo, Brazil). After this, the samples were bonded to an acrylic plate with acrylate-based cement and left to dry for 24 h before the stripping and finishing processes. The sections were reduced to a final thickness of approximately 30 μm with the use of a series of water abrasive papers (400, 600, 800, 1200, and 2400 grit; 3 M do Brasil, São Paulo, Brazil) in a polishing machine (Polipan 2; Pantec) under irrigation with water. Finally the sample was stained with fuchsin and analyzed under an optical microscope (Nikon Eclipse E200; Nikon Corporation, Tokyo, Japan).
All the bone–implant histological sections were analyzed histomorphologically in order to establish the general tissue characteristics of the osseointegration process in each group, by means of observing the neoformed bone tissue and its typical cell elements. An endeavour was made to record the regions of the implants with the strongest evidence of osseointegration and to evaluate the bone tissue covering the implant threads.
In the histomorphometric analysis, the bone–implant contact (%BIC) was determined at 50–200× magnification by means of a software programme (Image Tool for Windows, version 5.02, Department of Dental Diagnostic Science of the University of Texas Health Science Center, Texas, USA). The regions of bone–implant contact along the perimeter of the implant were subtracted from the total implant perimeter, and calculations were done to determine the %BIC.
Statistical analysis
The maximum removal torque values and the %BIC were compared between the groups by means of the paired Student’s t -test. The tests were considered at the 5% level of significance. IBM SPSS Statistics for Windows, version 20.0 software (IBM Corp., Armonk, NY, USA) was used for the data analysis.
Results
Maximum removal torque
Table 1 shows the maximum removal torque values in the control and test groups. The values in the test group were, on average, half those in the control group ( P < 0.001).
| Group | Mean | SD | Median | Min | Max | N | P -value |
|---|---|---|---|---|---|---|---|
| Control | 17.86 | 5.15 | 17.9 | 10.1 | 25.3 | 10 | <0.001 |
| Test | 8.73 | 2.86 | 7.9 | 5.9 | 14.2 | 10 |
Histomorphological analysis
Qualitative evaluation of the histological slides demonstrated that the most cervical portion of all of the implants passed through the tibial cortical bone, and the apical portion was in contact with medullary bone ( Fig. 2 ).
In the control group, histological analysis showed bone neoformation in the areas adjacent to the implant surfaces, with regions of bone remodelling, showing evidence of a structural arrangement similar to that of the lamellar region. Close to the implant, a large quantity of voluminous osteocytes was observed located within wide gaps. Closer to the implant, immature bone trabeculae with innumerable large and voluminous osteoblasts were observed. The difference in staining – more intensely stained areas – revealed more recently formed bone tissue, which was found particularly in the regions between the implant threads ( Fig. 3 ).
In the majority of specimens in the test group, the histological analysis showed an absence of bone neoformation in the areas adjacent to the implant surfaces, with sites of bone remodelling close to the tops of the spirals and in the more cervical portion of the implant. In the majority of samples, granular tissue was observed filling the spaces between the spirals ( Fig. 4 ).
