The aim of this study was to evaluate bone healing at the bone–implant interface in rats with induced osteoporosis. The rats underwent a bilateral ovariectomy (OVX) and were fed a low calcium and phosphate diet. The OVX rats were divided into three groups: one was treated with raloxifene (OVX-RAL), one with alendronate (OVX-ALE), and one received no medication (OVX-NT). The control group rats (SHAM-DN) underwent sham surgery and were fed a normal diet. Each animal received one implant in each tibia: a machined surface implant in the right tibia and an implant with surface etching in the left tibia. All animals were euthanized after 42 days. Analysis of variance (ANOVA) and Tukey post hoc tests were applied to the biomechanics (reverse torque) and bone–implant contact (BIC) data ( P < 0.05). The RAL and ALE groups showed improved peri-implant bone healing. However, the ALE group showed no significant difference from the OVX-NT group. Surface treatment promoted higher corticalization at the bone–implant interface, but showed the same characteristics of mature bone and bone neoformation in concentric laminations as the machined implant. There were no statistically significant differences in reverse torque ( P = 0.861) or BIC ( P = 0.745) between the OVX-RAL and SHAM-DN groups. Therefore, the use of raloxifene resulted in good biomechanical, BIC, and histological findings in the treatment of induced osteoporosis in rats.
One of the determining factors for the proper osseointegration of dental implants is the quality of bone tissue, since the characteristics of the bone microarchitecture influence the ability of the bone to transmit and distribute physiological forces. Therefore, when the cortical or trabecular structure of the bone has low density, the bone–implant interface is compromised.
Decreased bone density is observed in two-thirds of women because of oestrogen deficiency after the menopause. Thus, oestrogen deficiency associated with ageing can cause osteoporosis. The age-related bone loss is due to decreased intestinal absorption of dietary calcium, which results mainly in cortical bone loss.
There are numerous published reports on rehabilitative treatment using dental implants. Yamazaki et al. , Ozawa et al. , and Dvorak et al. analyzed the contact between bone tissue and implants placed in the tibia of ovariectomized (OVX) female rats. These authors observed that the reduction in bone mass of osteoporotic rats led to a smaller contact area between the bone and the implant, which compromised the ability of the bone to support the prosthesis.
Among the treatments for osteoporosis, hormone replacement is the most used, but this therapy has several contraindications and side effects. Several other drugs, including bisphosphonates and selective oestrogen receptor modulators (SERMs), are promising alternative therapies for the treatment of postmenopausal osteoporosis.
Changes in the surface topography of dental implants through the addition and subtraction techniques have improved biological responses in peri-implant osteogenesis, especially in areas of lower density, as seen in osteoporosis. The surface treatment of dental implants increases porosity and surface roughness, which causes osteoblastic lineage cells to reach the peri-implant region more quickly and more efficiently.
The aim of this study was to evaluate the biomechanical behaviour of implants, with and without changes to their surfaces, in OVX rats fed on a low calcium and phosphate diet to induce osteoporosis. In addition, we sought to evaluate the healing at the bone–implant interface in the tibias of the same rats. Furthermore, we aimed to determine whether treatment with the SERM alendronate (ALE) or raloxifene (RAL) improves peri-implant bone healing and could, therefore, be beneficial in rehabilitation with dental implants.
It was hypothesized that drug treatment with RAL or ALE would improve the peri-implant healing process as well as increase the reverse torque and the bone–implant contact (BIC) values in OVX rats, compared with an OVX group that received no drug. Furthermore, it was hypothesized that surface treatment would promote better biomechanical, BIC, and histological results.
Materials and methods
This research project was approved by the Ethics Committee on the Use of Animals (CEUA). Female Wistar rats ( Rattus norvegicus albinus , n = 72) weighing approximately 200 g were divided into three groups according to the proposed analysis: group I, histological study; group II, histometric study; and group III, reverse torque biomechanical analysis. Within these groups, the rats were further divided into four subgroups ( n = 6) as follows: ‘SHAM’ – rats subjected to sham surgery only, with exposure of the ovaries, and fed a balanced diet; OVX-NT – OVX rats fed a low calcium (Ca 2+ ) and phosphate (PO 4− ) diet, without drug treatment; OVX-ALE – rats fed a low Ca 2+ and PO 4− diet and treated with sodium alendronate; and OVX-RAL – rats fed a low Ca 2+ and PO 4− diet and treated with raloxifene. Euthanasia was performed 42 days after implant placement.
Initially, all animals were kept in cages, fed a normal laboratory diet (NUVILAB, Curitiba PR, Brazil) containing 1.4% Ca 2+ and 0.8% PO 4− , and allowed access to water ad libitum . The animals were separated into treatment groups (SHAM-DN, OVX-NT, OVX-ALE, or OVX-RAL) prior to the induction of osteoporosis and drug treatment. After surgery, the animals in the SHAM group continued on the normal diet with water provided ad libitum , while all the OVX rats were fed a diet containing 0.1% Ca 2+ and 0.5% PO 4− (Rhoster Ind., Vargem Grande Paulista, SP, Brazil) and had access to water ad libitum .
Determination of the oestrous cycle
The rats were placed in individual cages for the oestrous cycle evaluation, which was conducted daily according to the method of Evans and Long. After two or three regular cycles, the animals were used for the experiments.
Induction of osteoporosis
The induction of osteoporosis was performed according to the model described by Teófilo et al. Briefly, this involved combining a bilateral OVX with the administration of a low Ca 2+ and PO 4− diet for a period of at least 4 weeks. To confirm the development of osteoporosis, the tibias of the SHAM-DN and osteoporotic animals (OVX-NT) were processed through quantitative computed microtomography (SkyScan 1176; Bruker MicroCT, Aartselaar, Belgium) and the bone mineral density (BMD) values of the cortical bones were obtained. Data from our laboratory showed that the BMD in the OVX-NT animals was 0.12525 g/cm 3 compared with 0.35255 g/cm 3 in the SHAM-DN animals. This result confirmed the presence of the osteopenia that is characteristically observed in the osteoporosis model in rats. The OVX-ALE and OVX-RAL groups showed BMD values of 0.33302 and 0.51231 g/cm 3 , respectively.
Bilateral ovariectomy (OVX)
The rats in the OVX-NT, OVX-ALE, and OVX-RAL groups were anaesthetized with xylazine (Coopazine; Coopers Brasil Ltda, Campinas, São Paulo, Brazil) and ketamine hydrochloride injection (Vetaset; Fort Dodge Saúde Animal Ltda, Campinas, São Paulo, Brazil), and incisions were made in both flanks to remove the ovaries. The rats in the SHAM-DN group underwent the same procedure, but without removal of the ovaries.
Drug treatment – sodium alendronate (ALE) and raloxifene (RAL)
Eight days after the OVX, rats in the OVX-ALE and OVX-RAL groups were treated with sodium alendronate (0.1 mg/kg/day) and raloxifene (1.0 mg/kg/day), respectively, for 30 days; the drugs were administered by gavage. Both drugs were dissolved in an aqueous solution. The drugs were administered for a total of 72 days of dosing, up to the end of the experiment (euthanasia).
Surgery for tibia implants
The animals were fasted for 8 h prior to surgery and anaesthetized with a combination of 50 mg/kg intramuscular ketamine and 5 mg/kg xylazine. The rats were also administered mepivacaine hydrochloride (0.3 ml/kg 2% Scandicaine, 1:100,000 epinephrine; Septodont, Saint-Maur-des-Fossés, France) for local anaesthesia and to provide haemostasis in the operative field.
Following the induction of anaesthesia, the surgical site on the medial portion of the right and left tibia of the animal was shaved, after disinfecting with topical polyvinylpyrrolidone iodine and degerming (10% PVP, riodeine degermante; Rioquímica, São José do Rio Preto, SP, Brazil). A 2.0-cm incision was made, followed by the separation of the soft tissue to the right and left of the exposed tibial metaphysis.
A grade 4 titanium implant with a machined surface (IMPLALIFE Biotechnology, Jales, São Paulo, Brazil) was installed in the right tibia of each rat. A grade 4 titanium double acid-etched surface implant (IMPLALIFE Biotechnology, Jales, São Paulo, Brazil) was installed in the left tibia. All implants were 2.0 mm in diameter and had a 4.0 mm long square-edge module. The milling was performed with a 1.4-mm diameter spiral cutter mounted on an electric motor (BLM 600; Driller, São Paulo, SP, Brazil) at a speed of 1000 rpm, with isotonic sodium chloride 0.9% irrigation (Physiological; Biosintética Laboratories Inc., Ribeirão Preto, SP, Brazil). The installation was done manually with a square digital key ( Fig. 1 ).
The tissues were sutured using an absorbable suture (Vycril (polyglactin 910) 4–0; Ethicon, Johnson & Johnson, São José dos Campos, SP, Brazil) in a deep plane and monofilament (nylon 5–0; Ethicon, Johnson & Johnson) with interrupted skin stitches. During the immediate postoperative period, the animals received intramuscular injections of Pentabiotic (0.1 ml/kg; Fort Dodge Saúde Animal Ltda) with sodium dipyrone (1 mg/kg; Ariston Indústrias Químicas E Farmacêuticas Ltda, São Paulo, Brazil).
On day 14 following implant placement, 24 animals were administered intramuscular fluorochrome calcein at a dose of 20 mg/kg. Fluorochrome Alizarin Red (20 mg/kg) was administered 20 days later. Following infiltration of the fluorochromes, the BIC assessment was performed using confocal microscopy. Biopsies from these animals were kept for hard tissue processing to obtain histometric data through the BIC analysis.
Group I – histological analysis
Laboratory procedures for obtaining histological slides
Animals were euthanized 42 days after implant placement with an overdose of the anaesthetic. The left and right tibial metaphyses of the group I rats were then removed and reduced with margins of approximately 1 cm, fixed in buffered 10% formalin (Analytical Reagents; Dynamic Dental-Hospital Ltd, Catanduva, SP, Brazil) for 48 h, and soaked in water for 24 h.
Preparation of specimens for histological analysis
Samples from this group were fixed in formalin, decalcified in ethylenediaminetetraacetic acid (EDTA, 18%), and then dehydrated using a series of ethanol concentrations. After these steps, the samples were cleared with xylol, embedded in paraffin, and sectioned to obtain 5-mm slices. These slices were mounted on slides and the slides subsequently stained with haematoxylin and eosin (H&E).
Images were captured using a conventional optical microscope (Aristoplan Leitz; Leica Microsystems, Bensheim, Germany) coupled to a camera (Leica DFC 300FX; Leica Microsystems, Heerbrugg, Switzerland) and connected to a computer.
Group II – histometric analysis
Specimen preparation for histometric analysis
Following fixation in formaldehyde, the other rat tibias were also washed in water, dehydrated in an ascending series of ethanol concentrations, and then submerged in photopolymerizable resin (Technovit 7200 VLC; Exakt Advanced Technologies GmbH, Norderstedt, Germany). After resin polymerization, the specimens were semi-precision cut using a saw (Exakt Advanced Technologies) and subjected to an automatic polisher (Exakt Advanced Technologies) with sandpaper of different weights. The cut pieces were polished to a thickness of 80 μm, mounted on slides, cover-slipped, and stabilized with mineral oil.
Confocal laser scanning microscopy
Longitudinal sections of the bone–implant interface corresponding to the third, fourth, and fifth turns of the implants placed in the right and left tibias, were captured using a Leica CTR 4000 CS SPE camera (Leica Microsystems, Heidelberg, Germany) with a 10 × objective (original increase 100; Fig. 2 ). The confocal microscopy images were reconstructed, and thereby the overlap of the two fluorochromes (calcein and Alizarin) in the peri-implant bone was evident.
Histometric analysis – BIC
To calculate the linear length of the BIC interface, the perimeter (in micrometres) of the peri-implant interface in the regions of the third and fourth threads of the implants were standardized for all groups. The ImageJ analysis program (Processing Software and Image Analysis, Ontario, ON, Canada) was used for this purpose. Only the green (calcein) and red (Alizarin) fluorescent lines, which were precipitated in the peri-implant interface, were measured.
Group III – biomechanical test
Prior to euthanizing the animals on day 42 after implant placement, they were sedated with a combination of 50 mg/kg intramuscular ketamine and 5 mg/kg xylazine. The right and left tibial metaphyses were reopened to expose the implants, and reverse torque testing was performed. A assemble implant (Conexao, Sistemas de Próteses, Sao Paulo, SP, Brazil) was adapted to the edge of the implant module and a digital torque wrench (Instrutherm, São Paulo, SP, Brazil) was coupled to the implant mounts. A counter-clockwise movement was applied by increasing the reverse rotation torque to the implant within the bone tissue until it broke loose from the bone. The torque wrench then recorded the peak torque in Newton centimetres (N cm).
Data obtained from the reverse torque test and BIC analysis were analyzed statistically using the test of homoscedasticity (Kolmogorov–Smirnov test, P < 0.05), which showed a normal distribution (Sigmaplot 12.5 software; Systat Software Inc., San Jose, CA, USA). A two-factor (drug treatment vs. surface) analysis of variance (ANOVA) was performed, followed by the Tukey post hoc test for significant results. For all tests, a confidence level of 5% ( P < 0.05) was considered significant.
The histological analysis was performed 42 days after installation of the machined surface implants. The results showed peri-implant bone healing in the SHAM-DN group, with corticalization at the bone–implant interface in all spaces corresponding to the implant threads. There was also mature bone and bone formation in concentric laminations ( Fig. 3 A). The OVX-NT group showed new bone formation at the bone–implant interface, with corticalization only in the first implant threads in the cervical region. In the medullary portion, minor bone formation was observed with a large amount of adipose tissue ( Fig. 3 B).
The OVX-ALE group showed an improvement in bone repair ( Fig. 3 C). However, there was no significant difference compared with the OVX-NT group, indicating a delay in the repair process, especially in the bone marrow. Treatment with raloxifene (OVX-RAL) showed an improvement in peri-implant bone maturation ( Fig. 3 D), which was similar to that observed in the histological analysis of the SHAM-DN group.
For implants with a treated surface, the bone–implant interface was repaired to a greater extent in the OVX-NT and OVX-ALE groups compared with the corresponding groups with machined implants. However, this process was still delayed, particularly in the medullary bone, which possessed large amounts of adipose tissue. Results for the SHAM-DN and OVX-RAL groups were similar, showing the formation of new mature bone across the bone–implant interface ( Fig. 4 A–D) .
The results of a two-factor (drug treatment vs. surface) ANOVA showed statistically significant differences between the effects of the surface variable ( P = 0.02) and drug treatment ( P < 0.001) compared separately. The analysis comparing the effects of surface treatment and medication used showed no statistical difference ( P = 0.560, Table 1 ).