Abstract
Objective
Studies observing early wound healing periods around dental implants demonstrate an implants ability to enhance osseointegration, the bone–implant interactions for extended healing periods though have not been thoroughly studied.
Methods
Twenty threaded titanium alloy (Ti6Al4V, Grade 5) implants were inserted bilaterally, half prepared to impart stable hydroxyapatite nanoparticles onto a sand blasted and acid etched surface (HA) and half with a non-coated control surface with only heat treatment (HT), into eighteen rabbit femurs. At 12 weeks, the bone–implant blocks were retrieved for micro computed tomography (μCT), histologic processing and histomorphometric evaluation.
Results
The bone-to-implant contact for the entire threaded portion of the implant revealed 57.1% (21.0) for the HT group and 38.8% (17.7) for the HA group with a total bone area within the threads 72.5% (13.9) (HT) and 59.7% (12.5) (HA). The 3D reconstructed μCT image corresponded to the histomorphometric results.
Significance
It is suggested that multiple factors such as the change in topography and chemistry may have influenced the outcomes.
1
Introduction
The primary goal of oral implant treatment is to achieve a stable and harmonious anchoring in bone, which is able to support a dental prosthesis. It is a well-known fact that the bone-to-implant interfacial interaction is one of the important factors in mediating this success . Although a successful treatment option, the high implant success rates that are seen in the parasymphaseal mandible have yet to be achieved in more clinically challenging situations . To overcome these shortcomings, implant surface modifications and designs are being constantly developed in the hopes of generating a stable bone to implant interface.
Calcium–phosphate (CaP) derived coatings, such as hydroxyapatite (HA), have been researched extensively . Several methods have been documented in the literature, such as plasma sprayed, electrochemical deposition, biomimetic deposition, and nanospray deposition, each resulting in different bone responses . In a similar fashion, nanoscale modification of implant surfaces have been shown to improve the biomimicry of dental implants, since extracellular matrix proteins, growth factors, and numerous osteogenic potential cells interact at this level .
Intriguingly, the application of nanostructured CaP to implant surfaces has been explored as a possible means to enhance osseointegration. It has been previously reported that nanometer sized HA coatings on electro-polished surfaces can result in approximately 300% more bone-to-implant contact when compared to non-coated electro-polished surfaces . It has suggested that nanotopography is one of the decisive factors influencing osseointegration, since significant bone apposition was seen without the presence of moderately rough microstructures . What is interesting about nanostructured HA surfaces is that they appear to impart benefits from both the nanometer scale topography and the inherent calcium phosphate chemistry . Studies comparing turned to nano HA surfaces have demonstrated that nano HA surfaces have significantly enhanced osteogenic gene expression as well as similarly enhanced osteoclastic activities, this has lead to the theory that nano HA is slowly bioresorbed from the implant surface, demonstrating nano HA potential to enhance bone remodeling .
It should be noted, however, that in clinical situations, the presence of microtopography may be important for the success of implant treatment. It is known to contribute to the initial stability by mechanically interlocking the bone and the implant, and without it, implants present with lower success regardless of the nanotopography of the surface . Thus, studying the effect of nanostructures applied on roughened implant surfaces is of great interest and relevance. In fact, studies observing the effect of fluoride treated nanostructured surfaces proved that the bone apposition is enhanced significantly as compared to controls without nanostructures . However, it is also a fact that the effect of nanostructures may be difficult to discern from the effects of the microroughened substrate surface topography . Thus, it is of great importance to observe the long-term responses of such nanostructured surfaces, since most studies have focused on the early time points to discover their bone forming properties. This may be especially of interest for nanostructured CaP surfaces, since it has been proven that the coating substances will biodegrade or detach from the implant surface , and little is known about the impact of the coating after it has been altered by biology. Hence in this study, nanostructured HA were coated onto sandblasted and acid etched implants, and were placed in rabbits for 12 weeks. The objective of this study was to histologically observe the long-term effect of the coating to surrounding bone and to clarify whether there would be differences in bone forming properties between the non-coated and coated moderately roughened implants.
2
Materials and methods
2.1
Implant surface preparation
Twenty threaded titanium alloy (Ti6Al4V) implants (3.3, length 6 mm) were sand blasted and acid etched by the manufacturer (Aadva surface, GC Dental, Tokyo, Japan). Ten experimental implants were coated with a nano sized HA as described previously (HA; test) . In brief, the HA coating was achieved by dipping the implant into an HA nanoparticle dispersion, followed by heat treatment at 550 °C for 5 min in air. To stabilize the HA surface and prevent precipitation of the particles the implants were subjected to electrostatic nanoparticle stabilization in a basic environment (pH = 9) and steric stabilization by addition of surfactants. The remaining ten implants were left untreated but were subjected to the same heat treatment as experimental implants (HT, control).
2.2
Topographical and morphometrical evaluation
Surface topography in the nano level was characterized by means an atomic force microscope (AFM). Three randomly selected implants were used, and three regions on each implant of bottoms were measured (XE-100, Park Systems, Suwon, Korea) using a non-contact mode setup in air and at room temperature (scan size 1 μm × 1 μm). The parametric calculation was performed after the removal of errors of form and waviness by the use of a Gaussian filter (0.25 μm × 0.25 μm).
Micro level surface topography was evaluated by an optical interferometer (MicroXam; ADE Phase Shift, Inc., Tucson, AZ, USA). Three implants from each group were randomly selected and each of them measured on 9 regions (3 tops, 3 thread valleys, and 3 flanks).
The parametric calculation was performed after the removal of errors of form and waviness by the use of a Gaussian filter (50 μm × 50 μm).
For both evaluations, the following 3D parameters were selected: Sa (i.e. arithmetic average height deviation from a mean plane), Sds (i.e. density of summits), and Sdr (i.e. developed surface ratio). Descriptive 3D images were reconstructed with imaging software MountainsMap 6.2 (Digital Surf, Paris, France).
Surface morphology of the implants was examined by scanning electron microscopy (SEM) with LEO Ultra 55 FEG (Zeiss, Oberkochen, Germany) at an accelerating voltage of 6 kV. Three randomly selected implants from each group were investigated.
2.3
Implantation in vivo and sample preparation
This animal study was approved by the Malmö/Lund, Sweden region animal ethics committee (approval number: M282-09). Ten Swedish lop-eared rabbits (mean body weight, 4.3 kg) were used for the study. The animals were anesthetized with intramuscular injection of a 0.15 mL/kg medetomidine (1 mg/mL Dormitor; Orion Pharma, Sollentuna, Sweden) and 0.35 mL/kg ketamine hydrochloride (50 mg/mL Ketalar; Pfizer AB, Sollentuna, Sweden) mixture. Thereafter, the fur above the left and right proximal tibiae were shaved and standard preoperative disinfection procedures were conducted on the skin with 70% ethanol and 70% chlorohexidine, and local anesthesia was administered with lidocaine hydrochloride (Xylocaine; AstraZeneca AB, Göteborg, Sweden). Incision was made with a no. 15 blade and the periosteum was carefully raised, and the osteotomy was prepared. One HA and HT implants were randomly placed in the left and right proximal tibia. After insertion, the muscle and the skin were sutured layer by layer with a 4-0 bioresorbable suture (Ethicon Johnson, Miami, FL, USA). Post-operatively, the animals were given antibiotics (buprenorphine hydrochloride, 0.5 mL Temgesic; Reckitt Benckiser, Slough, UK) for 3 days.
After 12 weeks postoperatively, all animals were euthanized by an overdose of pentobarbital (Mebumal, ACO AB, Solna, Sweden). No signs of inflammation or lose implants were present suggesting uneventful healing. After removal of the muscle layer around the implant, the implant–bone samples were carefully retrieved en bloc by using a hand saw.
2.4
Micro CT imaging
The samples were fixed in 4% phosphate buffered formalin for 24 h, thereafter, were gradually dehydrated in a series of ethanol concentrations. After dehydration, the samples were infiltrated and embedded in light-curing resin (Technovit 7200 VLC; Heraeus Kulzer Wehrheim, Germany). The three-dimensional bone formation around the implant was examined using micro computed tomography (μCT, TOSCANER-30000μ, Toshiba IT Control System, Tokyo, Japan) with a slice resolution of 30 μm. Four hundred and fifty μCT slices were imaged at an X-ray energy level of 100 kV, and a current of 80 μA. All data were exported in DICOM-format and imported in Amira software (Visage Imaging GmbH, Berlin, Germany) for evaluation. The data were cropped along the implant axis to where the cortical bone started to exclude unnecessary information above the cortical bone. Before segmentation, threshold levels for bone and implant were determined, based on the complete slices. This was done by determining the upper and lower threshold levels for bone and implant, and finally, descriptive 3D images were generated.
2.5
Ground section preparation and histological analysis
After the CT scanning, all samples were processed for undecalcified ground sectioning. Embedded samples were sectioned in the middle of the long axis of the implant. One central undecalcified cut was prepared utilizing Exakt sawing and grinding equipment. The sections were ground to a final thickness of approximately 30 μm and stained with toluidine blue and pyronin.
Histological evaluations were performed with a light microscope (Eclipse ME600; Nikon, Japan) and histomorphometrical data were analyzed by image analysis software (Image J v. 1.43u; National Institutes of Health). The bone–implant contact (BIC) percentage along the entire implant was calculated at 10× objective magnification.
2.6
Statistical analysis
Statistical analyses were performed using SPSS (SPSS Inc., Chicago, IL, USA) software. The non-parametric Wilcoxon signed-rank test was used for bilaterally inserted implants with a statistical significance level of 0.05.
2
Materials and methods
2.1
Implant surface preparation
Twenty threaded titanium alloy (Ti6Al4V) implants (3.3, length 6 mm) were sand blasted and acid etched by the manufacturer (Aadva surface, GC Dental, Tokyo, Japan). Ten experimental implants were coated with a nano sized HA as described previously (HA; test) . In brief, the HA coating was achieved by dipping the implant into an HA nanoparticle dispersion, followed by heat treatment at 550 °C for 5 min in air. To stabilize the HA surface and prevent precipitation of the particles the implants were subjected to electrostatic nanoparticle stabilization in a basic environment (pH = 9) and steric stabilization by addition of surfactants. The remaining ten implants were left untreated but were subjected to the same heat treatment as experimental implants (HT, control).
2.2
Topographical and morphometrical evaluation
Surface topography in the nano level was characterized by means an atomic force microscope (AFM). Three randomly selected implants were used, and three regions on each implant of bottoms were measured (XE-100, Park Systems, Suwon, Korea) using a non-contact mode setup in air and at room temperature (scan size 1 μm × 1 μm). The parametric calculation was performed after the removal of errors of form and waviness by the use of a Gaussian filter (0.25 μm × 0.25 μm).
Micro level surface topography was evaluated by an optical interferometer (MicroXam; ADE Phase Shift, Inc., Tucson, AZ, USA). Three implants from each group were randomly selected and each of them measured on 9 regions (3 tops, 3 thread valleys, and 3 flanks).
The parametric calculation was performed after the removal of errors of form and waviness by the use of a Gaussian filter (50 μm × 50 μm).
For both evaluations, the following 3D parameters were selected: Sa (i.e. arithmetic average height deviation from a mean plane), Sds (i.e. density of summits), and Sdr (i.e. developed surface ratio). Descriptive 3D images were reconstructed with imaging software MountainsMap 6.2 (Digital Surf, Paris, France).
Surface morphology of the implants was examined by scanning electron microscopy (SEM) with LEO Ultra 55 FEG (Zeiss, Oberkochen, Germany) at an accelerating voltage of 6 kV. Three randomly selected implants from each group were investigated.
2.3
Implantation in vivo and sample preparation
This animal study was approved by the Malmö/Lund, Sweden region animal ethics committee (approval number: M282-09). Ten Swedish lop-eared rabbits (mean body weight, 4.3 kg) were used for the study. The animals were anesthetized with intramuscular injection of a 0.15 mL/kg medetomidine (1 mg/mL Dormitor; Orion Pharma, Sollentuna, Sweden) and 0.35 mL/kg ketamine hydrochloride (50 mg/mL Ketalar; Pfizer AB, Sollentuna, Sweden) mixture. Thereafter, the fur above the left and right proximal tibiae were shaved and standard preoperative disinfection procedures were conducted on the skin with 70% ethanol and 70% chlorohexidine, and local anesthesia was administered with lidocaine hydrochloride (Xylocaine; AstraZeneca AB, Göteborg, Sweden). Incision was made with a no. 15 blade and the periosteum was carefully raised, and the osteotomy was prepared. One HA and HT implants were randomly placed in the left and right proximal tibia. After insertion, the muscle and the skin were sutured layer by layer with a 4-0 bioresorbable suture (Ethicon Johnson, Miami, FL, USA). Post-operatively, the animals were given antibiotics (buprenorphine hydrochloride, 0.5 mL Temgesic; Reckitt Benckiser, Slough, UK) for 3 days.
After 12 weeks postoperatively, all animals were euthanized by an overdose of pentobarbital (Mebumal, ACO AB, Solna, Sweden). No signs of inflammation or lose implants were present suggesting uneventful healing. After removal of the muscle layer around the implant, the implant–bone samples were carefully retrieved en bloc by using a hand saw.
2.4
Micro CT imaging
The samples were fixed in 4% phosphate buffered formalin for 24 h, thereafter, were gradually dehydrated in a series of ethanol concentrations. After dehydration, the samples were infiltrated and embedded in light-curing resin (Technovit 7200 VLC; Heraeus Kulzer Wehrheim, Germany). The three-dimensional bone formation around the implant was examined using micro computed tomography (μCT, TOSCANER-30000μ, Toshiba IT Control System, Tokyo, Japan) with a slice resolution of 30 μm. Four hundred and fifty μCT slices were imaged at an X-ray energy level of 100 kV, and a current of 80 μA. All data were exported in DICOM-format and imported in Amira software (Visage Imaging GmbH, Berlin, Germany) for evaluation. The data were cropped along the implant axis to where the cortical bone started to exclude unnecessary information above the cortical bone. Before segmentation, threshold levels for bone and implant were determined, based on the complete slices. This was done by determining the upper and lower threshold levels for bone and implant, and finally, descriptive 3D images were generated.
2.5
Ground section preparation and histological analysis
After the CT scanning, all samples were processed for undecalcified ground sectioning. Embedded samples were sectioned in the middle of the long axis of the implant. One central undecalcified cut was prepared utilizing Exakt sawing and grinding equipment. The sections were ground to a final thickness of approximately 30 μm and stained with toluidine blue and pyronin.
Histological evaluations were performed with a light microscope (Eclipse ME600; Nikon, Japan) and histomorphometrical data were analyzed by image analysis software (Image J v. 1.43u; National Institutes of Health). The bone–implant contact (BIC) percentage along the entire implant was calculated at 10× objective magnification.
2.6
Statistical analysis
Statistical analyses were performed using SPSS (SPSS Inc., Chicago, IL, USA) software. The non-parametric Wilcoxon signed-rank test was used for bilaterally inserted implants with a statistical significance level of 0.05.
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