Abstract
Objectives
The aim of this study was to evaluate the bone tissue response to fiber-reinforced composite (FRC) in comparison with titanium (Ti) implants after 12 weeks of implantation in cancellous bone using histomorphometric and ultrastructural analysis.
Materials and methods
Thirty grit-blasted cylindrical FRC implants with BisGMA–TEGDMA polymer matrix were fabricated and divided into three groups: (1) 60 s light-cured FRC (FRC-L group), (2) 24 h polymerized FRC (FRC group), and (3) bioactive glass FRC (FRC–BAG group). Titanium implants were used as a control group. The surface analyses were performed with scanning electron microscopy and 3D SEM. The bone–implant contact (BIC) and bone area (BA) were determined using histomorphometry and SEM. Transmission electron microscopy (TEM) was performed on Focused Ion Beam prepared samples of the intact bone–implant interface.
Results
The FRC, FRC–BAG and Ti implants were integrated into host bone. In contrast, FRC-L implants had a consistent fibrous capsule around the circumference of the entire implant separating the implant from direct bone contact. The highest values of BIC were obtained with FRC–BAG (58 ± 11%) and Ti implants (54 ± 13%), followed by FRC implants (48 ± 10%), but no significant differences in BIC or BA were observed ( p = 0.07, p = 0.06, respectively). TEM images showed a direct contact between nanocrystalline hydroxyapatite of bone and both FRC and FRC–BAG surfaces.
Conclusion
Fiber-reinforced composite implants are capable of establishing a close bone contact comparable with the osseointegration of titanium implants having similar surface roughness.
1
Introduction
The long-term clinical success of oral implants is based on the presence and maintenance of a proper bone response. Implant materials have been classified into three categories based on the biological response: (1) biotolerant type, characterized by distance osteogenesis where the implant is surrounded by a fibrous connective tissue capsule; (2) bioinert type, characterized by contact osteogenesis and formation of direct bone-to-implant contact without an intervening connective tissue layer; and (3) bioreactive type, where the implant allows new bone formation around itself, thereby exchanging ions to create a chemical bond with the bone .
Sufficient strength and stiffness, biocompatibility and long-term stability are important criteria that ceramic and polymeric composites have to fulfill for their successful use as non-metallic implants. In 1969, Hodosh placed custom-made polymer implants directly into the fresh extraction sockets of teeth for the first time . Since then most studies have been performed in experimental animals but some work has been published with selected human patients . It has been reported that the attachment of polymer implants can be achieved using connective tissue capsule resembling the periodontal ligament . However, due to the high failure rate of 60% after the seven years follow-up of 10 patients, this acrylic resin implant was not recommended for clinical use . At the same time, Brånemark et al. demonstrated good results with osseointegrated titanium implants , and defined the osseointegration as a “direct structural and functional connection between ordered living bone and the surface of a load-carrying implant” . Although osseointegration was meant originally to describe a biologic fixation of the titanium dental implants, it is now used to describe the attachment of other materials used for dental and orthopedic applications as well .
Currently, a majority of implants are made of high modulus metals and their alloys . The problem of stress-shielding, which results from an elastic modulus mismatch between these metallic materials and natural bone , has stimulated new research for the development of polymer composite materials that can more closely match the modulus of bone. Furthermore, bone can be considered as an anisotropic natural fiber-reinforced composite (FRC) material composed of collagen fibers and inorganic hydroxyapatite matrix. Therefore, non-metallic FRC implants have been developed for head-and-neck, maxillofacial and orthopedic applications , which also make them an interesting material for oral implants. Several surface modification or coating methods can be used in order to improve implant bioactivity and enhance the osseointegration process. Acid etching, grit blasting and various CaP coatings are frequently applied on titanium implants. FRC materials allow modifications by embedding bioactive ceramics such as bioactive glass (BAG) directly on the implant surface.
The bulk and surface properties of FRC implant materials have been characterized and evaluated in biological environments . FRCs have been found to be durable materials, whose strength and elasticity are well adapted to the physiological requirements of bone . FRC implants have also shown good mechanical performance in the laboratory environment . Furthermore, FRC materials have been found to be cytocompatible in cell cultures and have demonstrated a similar cellular response to titanium .
Only a few in vivo studies about the tissue response to FRC implants have been reported . These studies have shown that highly polymerized FRC is biocompatible and induces neither toxic nor inflammatory reactions. FRC surfaces induce a bone response similar to titanium after 4 and 12 weeks of healing in the cortical bone of pig tibia . Neither grit-blasted FRC implants nor FRC–BAG implants revealed toxicity in the pig bone tissue during the 12-week healing period. The establishment of strong bone contact with the FRC implant surface indicates that the material is biocompatible in the bone environment . However, the quality and quantity of new bone formation on the FRC implant compared to titanium is not known.
The present study set out to compare the quantity and quality of bone formation between surface modified FRC and titanium implants.
2
Materials and methods
2.1
Experimental implants
Cylinder designed FRC implants with a length of 6 mm and a diameter of 4 mm were fabricated for this study. The preparation of FRC implants has been described in detail in the previous study .
The FRC implants were made by combining five fiber rovings each consisting of 4000 continuous unidirectional E-glass fibers (diameter of one fiber ca. 15 μm). Fiber rovings were impregnated manually with light-polymerizable monomer system of BisGMA–TEGDMA (50 wt%:50 wt%) (Stick, Stick Tech Ltd., Turku, Finland) resin with a camphorquinone-amine initiator system. The nominal composition of the E-glass fibers (wt%) was SiO 2 (53–55%), Al 2 O 3 (14–16%), CaO (20–24%), MgO (20–24%), B 2 O 3 (6–9%), K 2 O (<1%), Na 2 O (<1%), and Fe 2 O 3 (<1%). Commercially available particles of BAG (S53P4, Vivoxid Ltd., Turku, Finland) (particle size ≤45 μm) were used in the preparation of FRC implants with a BAG surface layer.
Three FRC implant groups were prepared: (I) light polymerized implants (FRC-L group); (II) light- and heat-polymerized implants (FRC group); and (III) light- and heat-polymerized FRC implants with BAG granules embedded on the implant surface (FRC–BAG group) .
The FRC-L group was polymerized for 60 s with an Optilux 501 dental hand light-curing unit (Kerr Mfg., Orange, CA, USA). The FRC and FRC–BAG groups were polymerized starting from initial light polymerization for 60 s using an Optilux 501 hand light-curing unit, and further polymerized for 1 h in a light curing oven at 80 °C (LicuLite, Dentsply De Trey GmbH, Dreieich, Germany) and 23 h post-cured at 120 °C which was above the temperature of glass transition ( T g ) of the BisGMA–TEGDMA copolymer . Cylindrical titanium implants, with similar shape manufactured of pure titanium bars (grade 2) served as the control group. All tested implants were grit blasted with Al 2 O 3 particles (size 50–60 μm; Danville Engineering, San Ramon, CA, USA).
2.2
Surface characterization
Scanning electron microscopy (SEM) and energy-dispersive X-ray fluorescence (EDX) analysis were used for surface characterization of the implants. The composite implants were coated with 15 nm of AuPd (50/50) prior to the SEM/EDX analysis using Gatan Precision Etching Coating System, Model 682.
The SEM (Supra 40 VP, Zeiss, Germany) was used to observe the surface morphology of the implants at different magnifications. The EDX (Oxford Instruments Analytical Ltd.) was used to acquire elemental spectra at selected locations on the implants for determining their elemental compositions.
For SEM imaging and EDX analysis, acceleration potentials of 2 and 20 kV, respectively, were used. The regular secondary electron (SE) detector was used for ordinary SEM imaging, whereas the in-lens detector was used for three-dimensional (3D) SEM imaging (see below).
The acquired images were imported into MeX 4.15 (Alicona Imaging GmbH, Germany) to generate 3D representations and to calculate roughness from the tilted SEM images. The spectra were imported into INCA Suite version 4.15 for EDX spectra and mapping.
To enable 3D visualization with the SEM technique, stereo image pairs were collected by tilting the specimen around the same point on the surface with a tilting SmartSEM for SEM angle of −4° and +4° (3000× to 5000× enlargement), and −4° and +12° (2000× enlargement).
The surface chemistry of the specimens was analyzed by time-of-flight secondary ion mass spectroscopy (TOF-SIMS; TOF-SIMS IV, ION-TOF GmbH, Münster, Germany). TOF-SIMS analysis was done on at least two areas (500 μm × 500 μm) on each sample, using 25 kV Bi 3+ primary ions at a target current of 0.1 pA. The SurfaceLab 6.3 (ION-TOF GmbH) software was used for data analysis.
2.3
Experimental design and implantation procedure
Ten adult female New Zealand white rabbits (mean weight of 4 kg) were used for this study.
The animals were obtained from the Laboratory Animal Breeding and Experimental Studies Center of Gazi University. The experimental protocol was reviewed and approved by the Animal Ethics Committee of Gazi University (G.Ü.ET-07.052). The animals were kept in cages under standardized conditions and fed with standard hard diet pellets. Gazi University guidelines for the care and use of laboratory animals were followed.
Surgery was performed under general anesthesia, induced by intravenous injection of a mixture consisting of ketamine hydrochloride (50 mg/kg; Ketalars, Parke-Davis, France), xylazine hydrochloride (3.9 mg/kg; Rompun, Bayer AG, Leverkusen, Germany) in normal saline (0.9% NaC1, Baxter nv, Lessine, Belgium). To reduce the perioperative infection risk, prophylactic antibiotic (Terramycin) at a dose of 1 cm 3 /10 kg (oxytetracycline hydrochloride; Pfizer, Egypt) was administered postoperatively by subcutaneous injection.
During the operation, the animals were immobilized, and the bilateral knees were shaved, washed and disinfected with povidone–iodine. A longitudinal incision was made on the medial side of the left and right knees. Subsequently, the skin, the subcutaneous tissue and the muscle were retracted in order to gain access to the bone, and the medial and lateral femoral condyles of both legs were exposed. Thereafter, two (medial and lateral) holes were carefully prepared in the femoral condyles using gradually increasing drill sizes (final drill diameter and depth: 3.8 mm and 6 mm, respectively). The bone preparation was done using low rotational drill speed (450 rpm) with continuous internal cooling. The bone cavities were washed with saline during and after drilling. The total number of inserted implants in each group was 10.
2.4
Histological procedure
At the end of implantation periods, all animals were sacrificed by injecting an overdose of pentobarbitalsodium (Nembutal; Abbott Laboratories, North Chicago, IL). After sacrificing the animals, the femoral condyles were removed and fixed in 10% buffered formalin solution. Subsequently, the formalin-fixed tissue–implant block was dehydrated in a graded series of ethanol and embedded in LR White (London Resin Company Ltd., Berkshire, UK) prior to cutting along the long axis of the implant using a diamond saw. The bone–implant block was divided into two. One half was used to prepare a ground section for histological and histomorphometric examination, whereas the other half was used for SEM and TEM analysis. The ground sections for histological examination were prepared using sawing and grinding machines (Exakt Apparatebau GmbH & Co, Norderstedt, Germany), until a final thickness of 10–20 μm was obtained. The sections were subsequently stained with 1% toluidine-blue in 1% borax solution, mixed in a 4:1 proportion with 1% pyronin-G solution. The quantitative histomorphometric analysis was performed blind by one investigator, whereas the qualitative histological analysis was performed by two investigators.
The most center section was analyzed for each implant. The length along the implant surface that was directly contacting the bone was determined for all implants. Each implant image was divided in half longitudinally, and the actual surface length along both sides was measured. Computer-based histomorphometrical analyses on ground sections were performed in a Leitz Aristoplan light microscope with a Leitz Microvid unite, connected to a PC enabling the observer to perform quantifications directly in the eye-piece of the microscope, using occuars of 10× magnification. A higher magnification objective with 40× magnification was used for qualitative analysis.
The degree of bone–implant contact (BIC), expressed as percentage of the implant surface in direct contact with bone, was calculated . The area of bone around the implants was assessed by calculating the percentage of the surface area occupied by bone (BA) in the rectangular area extending 200 μm, and 400 μm from the implant surface into bone ( Fig. 1 ), thereby defining the area around the implant .
2.5
Scanning and transmission electron microscopy
The counterpart of the embedded tissue–implant block was used for scanning electron microscopy including observations using the back-scattered mode (SEM-BS). The blocks were glued upwards on a regular SEM stub using a carbon adhesive tape and sputter coated by a thin layer of gold (10 nm) prior to mounting in the vacuum chamber. The microscopy used was a Leo 440 SEM (Leo Electron Microscopy Ltd, Cambridge, UK) equipped with a LaB 6 electron emitter, operating at 20 kV. Imaging was performed with a quadrupole detector using back-scattered electrons in composition mode for high Z-contrast. Magnifications between 100× and 10.000× were used for the evaluation of the implant–tissue interface. Further, chemical evaluation of the blocks was performed using EDX. Generated element maps of areas of interest were made using a personal computer.
Focused-ion beam (FIB) milling was used to prepare electron-transparent transmission electron microscopy (TEM) specimens for ultrastructural analysis of the intact interface between the bone and implants ( Fig. 2 ). The FIB device used in this study was an FEI Strata DB235 FIB/SEM (FEI Company, Eindhoven, the Netherlands) equipped both with an ion and an electron gun (dual beam) and an Omniprobe micromanipulator, allowing in situ lift-out of the TEM specimen. A 100-nm-thick electron-transparent specimen of the cross-section of the interface was made by using an in situ lift-out method ( Fig. 2 d), which is explained in detail elsewhere .
2.6
Statistical analyses
Statistical analysis was performed using Statistical Package for the Social Sciences (version 19.0; SPSS Inc., Chicago, IL, USA). The data were analyzed using one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test. Differences were considered significant at 95% confidence level.
2
Materials and methods
2.1
Experimental implants
Cylinder designed FRC implants with a length of 6 mm and a diameter of 4 mm were fabricated for this study. The preparation of FRC implants has been described in detail in the previous study .
The FRC implants were made by combining five fiber rovings each consisting of 4000 continuous unidirectional E-glass fibers (diameter of one fiber ca. 15 μm). Fiber rovings were impregnated manually with light-polymerizable monomer system of BisGMA–TEGDMA (50 wt%:50 wt%) (Stick, Stick Tech Ltd., Turku, Finland) resin with a camphorquinone-amine initiator system. The nominal composition of the E-glass fibers (wt%) was SiO 2 (53–55%), Al 2 O 3 (14–16%), CaO (20–24%), MgO (20–24%), B 2 O 3 (6–9%), K 2 O (<1%), Na 2 O (<1%), and Fe 2 O 3 (<1%). Commercially available particles of BAG (S53P4, Vivoxid Ltd., Turku, Finland) (particle size ≤45 μm) were used in the preparation of FRC implants with a BAG surface layer.
Three FRC implant groups were prepared: (I) light polymerized implants (FRC-L group); (II) light- and heat-polymerized implants (FRC group); and (III) light- and heat-polymerized FRC implants with BAG granules embedded on the implant surface (FRC–BAG group) .
The FRC-L group was polymerized for 60 s with an Optilux 501 dental hand light-curing unit (Kerr Mfg., Orange, CA, USA). The FRC and FRC–BAG groups were polymerized starting from initial light polymerization for 60 s using an Optilux 501 hand light-curing unit, and further polymerized for 1 h in a light curing oven at 80 °C (LicuLite, Dentsply De Trey GmbH, Dreieich, Germany) and 23 h post-cured at 120 °C which was above the temperature of glass transition ( T g ) of the BisGMA–TEGDMA copolymer . Cylindrical titanium implants, with similar shape manufactured of pure titanium bars (grade 2) served as the control group. All tested implants were grit blasted with Al 2 O 3 particles (size 50–60 μm; Danville Engineering, San Ramon, CA, USA).
2.2
Surface characterization
Scanning electron microscopy (SEM) and energy-dispersive X-ray fluorescence (EDX) analysis were used for surface characterization of the implants. The composite implants were coated with 15 nm of AuPd (50/50) prior to the SEM/EDX analysis using Gatan Precision Etching Coating System, Model 682.
The SEM (Supra 40 VP, Zeiss, Germany) was used to observe the surface morphology of the implants at different magnifications. The EDX (Oxford Instruments Analytical Ltd.) was used to acquire elemental spectra at selected locations on the implants for determining their elemental compositions.
For SEM imaging and EDX analysis, acceleration potentials of 2 and 20 kV, respectively, were used. The regular secondary electron (SE) detector was used for ordinary SEM imaging, whereas the in-lens detector was used for three-dimensional (3D) SEM imaging (see below).
The acquired images were imported into MeX 4.15 (Alicona Imaging GmbH, Germany) to generate 3D representations and to calculate roughness from the tilted SEM images. The spectra were imported into INCA Suite version 4.15 for EDX spectra and mapping.
To enable 3D visualization with the SEM technique, stereo image pairs were collected by tilting the specimen around the same point on the surface with a tilting SmartSEM for SEM angle of −4° and +4° (3000× to 5000× enlargement), and −4° and +12° (2000× enlargement).
The surface chemistry of the specimens was analyzed by time-of-flight secondary ion mass spectroscopy (TOF-SIMS; TOF-SIMS IV, ION-TOF GmbH, Münster, Germany). TOF-SIMS analysis was done on at least two areas (500 μm × 500 μm) on each sample, using 25 kV Bi 3+ primary ions at a target current of 0.1 pA. The SurfaceLab 6.3 (ION-TOF GmbH) software was used for data analysis.
2.3
Experimental design and implantation procedure
Ten adult female New Zealand white rabbits (mean weight of 4 kg) were used for this study.
The animals were obtained from the Laboratory Animal Breeding and Experimental Studies Center of Gazi University. The experimental protocol was reviewed and approved by the Animal Ethics Committee of Gazi University (G.Ü.ET-07.052). The animals were kept in cages under standardized conditions and fed with standard hard diet pellets. Gazi University guidelines for the care and use of laboratory animals were followed.
Surgery was performed under general anesthesia, induced by intravenous injection of a mixture consisting of ketamine hydrochloride (50 mg/kg; Ketalars, Parke-Davis, France), xylazine hydrochloride (3.9 mg/kg; Rompun, Bayer AG, Leverkusen, Germany) in normal saline (0.9% NaC1, Baxter nv, Lessine, Belgium). To reduce the perioperative infection risk, prophylactic antibiotic (Terramycin) at a dose of 1 cm 3 /10 kg (oxytetracycline hydrochloride; Pfizer, Egypt) was administered postoperatively by subcutaneous injection.
During the operation, the animals were immobilized, and the bilateral knees were shaved, washed and disinfected with povidone–iodine. A longitudinal incision was made on the medial side of the left and right knees. Subsequently, the skin, the subcutaneous tissue and the muscle were retracted in order to gain access to the bone, and the medial and lateral femoral condyles of both legs were exposed. Thereafter, two (medial and lateral) holes were carefully prepared in the femoral condyles using gradually increasing drill sizes (final drill diameter and depth: 3.8 mm and 6 mm, respectively). The bone preparation was done using low rotational drill speed (450 rpm) with continuous internal cooling. The bone cavities were washed with saline during and after drilling. The total number of inserted implants in each group was 10.
2.4
Histological procedure
At the end of implantation periods, all animals were sacrificed by injecting an overdose of pentobarbitalsodium (Nembutal; Abbott Laboratories, North Chicago, IL). After sacrificing the animals, the femoral condyles were removed and fixed in 10% buffered formalin solution. Subsequently, the formalin-fixed tissue–implant block was dehydrated in a graded series of ethanol and embedded in LR White (London Resin Company Ltd., Berkshire, UK) prior to cutting along the long axis of the implant using a diamond saw. The bone–implant block was divided into two. One half was used to prepare a ground section for histological and histomorphometric examination, whereas the other half was used for SEM and TEM analysis. The ground sections for histological examination were prepared using sawing and grinding machines (Exakt Apparatebau GmbH & Co, Norderstedt, Germany), until a final thickness of 10–20 μm was obtained. The sections were subsequently stained with 1% toluidine-blue in 1% borax solution, mixed in a 4:1 proportion with 1% pyronin-G solution. The quantitative histomorphometric analysis was performed blind by one investigator, whereas the qualitative histological analysis was performed by two investigators.
The most center section was analyzed for each implant. The length along the implant surface that was directly contacting the bone was determined for all implants. Each implant image was divided in half longitudinally, and the actual surface length along both sides was measured. Computer-based histomorphometrical analyses on ground sections were performed in a Leitz Aristoplan light microscope with a Leitz Microvid unite, connected to a PC enabling the observer to perform quantifications directly in the eye-piece of the microscope, using occuars of 10× magnification. A higher magnification objective with 40× magnification was used for qualitative analysis.
The degree of bone–implant contact (BIC), expressed as percentage of the implant surface in direct contact with bone, was calculated . The area of bone around the implants was assessed by calculating the percentage of the surface area occupied by bone (BA) in the rectangular area extending 200 μm, and 400 μm from the implant surface into bone ( Fig. 1 ), thereby defining the area around the implant .
