Histologic and Biomechanical Evaluation of 2 Resorbable-Blasting Media Implant Surfaces at Early Implantation Times

The contact between bone and endosseous implants is usually well established and maintained after implant placement, resulting in a success rate often exceeding 90% over 10 years.^1,2  Past investigations have shown that surface modification methods have been successful in increasing the host response to the implants, resulting in better long-term bone morphology as well as the initial interaction between the host and the implant.³  As a result, surface modifications in texture and chemistry such as increasing the roughness and the addition of calcium- and phosphorus-based bioceramic coatings are among the most investigated aspects of the implant studies.^3–6 

In previous studies, surface texture modification has proven to be effective in increasing the host response in in vivo, in vitro, and ex vivo studies.⁷  More bone-to-implant interaction has been shown in surfaces with increased roughness as compared with as-machined or smooth surfaces (Sa < 0.5 μm). The best osseointegration measurable by bone-to-implant contact (BIC) has been shown in the moderately rough surfaces (Sa between 1 and 2 μm).^3–6  Surface roughness can be achieved in a variety of ways, including acid etching, anodization, and grit blasting with nonresorbable media such as alumina, silica, or titanium oxides or with resorbable-blasting media (RBM) such as hydroxyapatite, tricalcium phosphates, or a combination of Ca-P phases followed by acid etching or passivation treatment that leaves little to absent amount of Ca and P on the final product.³ 

Increasing the surface roughness has resulted in improved early host-to-implant response, but a surface chemistry alteration involving bioceramic coating of implants using the thick plasma-sprayed hydroxyapatite (PSHA) has shown to result in higher degrees of fixation at earlier implantation times compared with moderately rough uncoated implants.^8–10  However, PSHA-coated implants rely on mechanical interlocking between the grit-blasted or etched metallic surfaces and the ceramic-like PSHA coating, and this physical interaction among the bulk metallic, metallic oxide, and bioceramic surface has been considered a weak link based on frequent adhesive failures that have reportedly occurred on different implant configurations.^8–10 

In an attempt to take advantage of increased osseoconductivity of the Ca-P while avoiding the mechanical drawbacks of PSHA coatings, integration of Ca-P particles at a reduced amount through various techniques such as ion beam–assisted deposition,^11–13  sputtering,^14,15  discrete crystalline deposition,^16–19  and RBM^20,21  processes have shown promising results as compared with uncoated, roughened implants. Specific to RBM processing, various factors such as blasting media composition, blasting particle size, processing parameters such as blasting pressure and distance, and subsequent acid etching are shown to have effect on early host-to-implant surfaces.³  However, the amount and the form of Ca-P that demonstrate optimal host-bone interaction have not been determined conclusively. Consequently, the aim of this investigation was to evaluate the biomechanical fixation and to histomorphologically and histomorphometrically characterize 3 different implant surfaces: (1) RBM + acid etching (RBMa), (2) alumina blasting + acid-etching (AB/AE), and (3) AB/AE + RBMa surface (hybrid).

The endosseous Ti-6Al-4V implants used in this study were 3.75 mm in diameter by 10 mm in length. Three surface modifications were investigated: RBMa, AB/AE, and the hybrid.

The AB/AE surface was achieved by blasting the surface with large particles of Al₂O₃ with a size of ∼300 to 400 μm followed by acid etching with hydrochloric/sulfuric acid. The RBMa surface was achieved by blasting with HA/TCP (20%/80% ratio) particles with a size of ∼200 to 400 μm followed by cleaning with HNO₃ at room temperature for 10 minutes. The hybrid surface (AB/AE + RBMa) was obtained by first applying the AB/AE treatment and subsequently applying the RBMa surface treatment. All implant surfaces were sterilized under gamma radiation.

Nine implants (n = 3 each group) were used for surface topography assessment by scanning electron microscopy (SEM) and atomic force microscopy (AFM). SEM (Philips XL 30, Eindhoven, the Netherlands) was performed at various magnifications under an acceleration voltage of 15 kV. Surface 3-dimensional (3D) imaging was collected by AFM (Nanoscope IIIa Multimode system, Digital Instruments, Santa Barbara, Calif) in contact mode. A scanner with a maximum 125-μm horizontal and 5-μm vertical range and a 200-μm Si₃N₄ cantilever tip using a constant force of 0.12 N/m was used. The region analyzed was the flat part of the implant cutting edges, and 35- × 35-μm scan areas were used.^22,23 Three scans per implant were performed, and S_a (arithmetic mean of the absolute value of the height within the sampling area) and S_q (root mean square value of the surface departures within the sampling area) parameters determined. Statistical analysis at the 95% level of significance was performed by one-way analysis of variance.

Surface-specific chemical assessment was performed by x-ray photoelectron spectroscopy (XPS). The implants were inserted in a vacuum transfer chamber and degassed to 10⁻⁷ torr. The samples were then transferred under vacuum to a Kratos Axis 165 multitechnique XPS spectrometer (Kratos Analytical Inc, Chestnut Ridge, NY). Survey and high-resolution spectra were obtained using a 165-mm mean radius concentric hemispherical analyzer operated at constant pass energy of 160 eV for survey and 80 eV for high-resolution scans. The take-off angle was 90°, and a spot size of 150 μm × 150 μm was used. The implant surfaces were evaluated at various locations.

For the animal model, 16 implants of each of the 3 surfaces were used. The study was composed of 8 adult male beagle dogs ∼1.5 years of age. The protocol received the approval of the Ethics Committee for Animal Research at Pontifica Universidade Católica do Rio Grande do Sul, Brazil.

Prior to general anesthesia, intramuscular atropine sulfate (0.044 mg/kg) and xilazine chlorate (8 mg/kg) were administered. A 15-mg/kg ketamine chlorate dose was then used to achieve general anesthesia.

The proximal medial tibia on the right side was initially shaved with a razor blade and followed by the application of antiseptic iodine solution. An incision through the skin of ∼5 cm in length was used for access to the periosteum, which was elevated for bone exposure.

Standardized osteotomies were made with sequential drills (pilot drill, followed by 2 mm, 2.5 mm, 3.0 mm, 3.5 mm) at 1200 rpm under abundant saline irrigation. The first implant was inserted 2 cm below the joint capsule line at the central anteromedial position of the proximal tibiae (procedures were performed bilaterally). The other two devices were placed along the distal direction at distances of 1 cm from each other along the central region of the bone. The implants were screwed into the drilled sites with a torque wrench and remained for 2 weeks. It should be noted that because of the dimensional interplay between drilling and implant design (implant threads' 3.25-mm inner diameter and the osteotomy's 3.5-mm diameter), intimate contact was achieved between bone and the implant microthreads in the cervical region, and healing chambers were created at regions where larger threads were present. The order in which the implants with different surfaces were placed was alternated along the tibia, with the starting implant being interchanged in every tibia. Balanced surgical procedures were used to allow the comparison of the torque and histology of same number of implant surfaces, surgical site (1 through 3), and animal at 2 weeks.

To avoid any damage to the implant-bone interface due to removal of a callus overgrowth after limb retrieval, a cover screw was installed in each implant. Standard layered suture techniques were used for wound closure (4-0 vicryl, internal layers; 4-0 nylon, the skin). Postsurgical medication included antibiotics (penicillin, 20 000 UI/kg) and analgesics (ketoprophen, 1 mL/5 kg) for a period of 48 hours postoperatively. The euthanasia was performed by anesthesia overdose 2 weeks after placement.

At necropsy, the limbs were retrieved by sharp dissection, the soft tissue was removed by surgical blades, and initial clinical evaluation was performed to determine implant stability. Half of the implants (specimens from the right limb) were then referred to biomechanical testing, and the other half (specimens from the left limb) were processed for histology.

For the torque testing, the tibia was adapted to an electronic torque machine equipped with a 200-Ncm torque load cell (Test Resources, Minneapolis, Minn). Custom machined tooling was adapted to each implant's internal connection, and the bone block was carefully positioned to avoid specimen misalignment during testing. The implants were torqued in the counterclockwise direction at a rate of ∼0.196 radians/min, and a torque vs displacement curve was recorded for each specimen.

For histology processing, the bone blocks were kept in 10% buffered formalin solution for 24 hours, washed in running water for 24 hours, and gradually dehydrated in a series of alcohol solutions ranging from 70% to 100% ethanol. Following dehydration, the samples were embedded in a methacrylate-based resin (Technovit 9100, Heraeus Kulzer GmbH, Wehrheim, Germany) according to the manufacturer's instructions. The blocks were then cut into slices (∼300 μm thickness), aiming the center of the implant along its long axis with a precision diamond saw (Isomet 2000, Buehler Ltd, Lake Bluff, Ill), and glued to acrylic plates with an acrylate-based cement, and a 24-hour setting time was allowed prior to grinding and polishing. The sections were then reduced to a final thickness of ∼30 μm by means of a series of SiC abrasive papers (400, 600, 800, 1200, and 2400; Buehler Ltd) in a grinding/polishing machine (Metaserv 3000, Buehler Ltd) under water irrigation.²⁴  The sections were then stained with toluidine blue and referred to optical microscopy evaluation.

The BIC was determined at ×50 to ×200 magnification (Leica DM2500M, Leica Microsystems GmbH, Wetzlar, Germany) by means of computer software (Leica Application Suite, Leica Microsystems GmbH). The regions of BIC along the implant perimeter were subtracted from the total implant perimeter, and calculations were performed to determine the BIC. The bone area fraction occupied (BAFO) between threads in trabecular bone regions was determined at ×100 magnification (Leica DM2500M, Leica Microsystems GmbH) by means of computer software (Leica Application Suite, Leica Microsystems GmbH). The areas occupied by bone were subtracted from the total area between threads, and calculations were performed to determine the BAFO (reported in percentage values of BAFO).²⁵ 

Friedman's test at the 95% level of significance and Dunn's post hoc test were used for multiple comparisons between groups for torque, BIC, and BAFO.

The implant surfaces' electron micrographs and 3D atomic force microscopies are presented in Figures 1 and 2, respectively. The AB/AE presented a minimally rough surface without the presence of embedded alumina particles (Figure 1a), while the RBMa surface electron micrographs showed that the acid-etching procedure was partially effective at removing embedded blasting media particles (Figure 1b). The hybrid surface presented a morphology similar to the RBMa surface (Figure 1c). The AFM assessment showed that the AB/AE surface presented significantly higher mean S_a (P < .03) and S_q (P < .04) values compared with the RBMa (Figure 3). The hybrid surface roughness presented intermediate values (Figure 3).

The AB/AE XPS survey analysis showed peaks of Ti, Al, V, C, and O (Table), while the RBMa and hybrid surfaces presented Ti, Al, Ca, C, P, and N (Table). High-resolution spectrum evaluation showed that for all surfaces, titanium was found primarily as TiO₂ with a very low level of metallic Ti, and carbon was observed primarily as hydrocarbon (C-C, C-H) with lower levels of oxidized carbon forms. For both the RBMa and hybrid groups, calcium and phosphate were detected in varied atomic concentrations. For the RBMa and hybrid groups, calcium and phosphate atomic concentrations ranged from ∼1 to 3.5 and ∼1 to ∼2.5 atomic percentage, respectively.

The animal surgical procedures and follow-up demonstrated no complications regarding procedural conditions, postoperative infection, or other clinical concerns. The biomechanical testing results showed that implant surface did not have a significant effect on torque to interface fracture (P = .51; Figure 4).

Qualitative evaluation of the toluidine blue–stained thin sections showed intimate contact between cortical and trabecular bone (Figure 5) for all implant surfaces, showing that all 3 surfaces are biocompatible and osseoconductive. At 2 weeks, newly formed woven bone was observed in close proximity to all implant surfaces (Figure 6). The histomorphometric results demonstrated that the implant surface also did not have a significant effect on BIC and BAFO (P = .46 and P = .64; Figures 7a and 7b, respectively).

As a result of years of implant dentistry research, the implant surface modification has evolved from as-machined, smooth surfaces to microscopically moderately roughened surfaces that have shown to enhance bone healing after the placement of implants.^3,4,7,26,27  Subsequent surface chemistry modifications such as the incorporation of bioactive ceramics have long been the focus of investigations as a positive factor for improved early bone healing. However, considering that surface chemistry modifications typically involve changes in surface topography,²⁸  it is still unclear whether resulting topography changes alone and/or the combination with chemical modifications leads to improved osseointegration.⁵ 

The surface treatments investigated in this study composed an alumina-blasted/acid-etched control, an RBMa, and a hybrid surface that combined AB/AE followed by RBMa treatment. A previous study has shown that subsequent etching RBM (RBMa) substantially reduced Ca and P amounts on the surface yet with no differences in removal torque, BIC, and BAFO when compared with a rougher control AB/AE.²¹  Another investigation has observed improved torque at 2 times in vivo for an RBMa compared with an overall smoother dual acid-etched surface used as a control.²⁰  Because the dual-acid etched surface in the latter study resulted in lower torque values compared with the RBMa,²⁰  it was speculated that surface roughness, rather than chemistry, was likely the factor influencing improved bone response. The rationale for evaluating the different surfaces at 2 weeks in vivo in the beagle dog model was based on our previous investigations, which have shown that high degrees of biomechanical fixation occur as early as 3 weeks in the beagle dog long bones.^21,29–40  The bone-healing pattern and long-term remodeling dynamics have been shown to be remarkably similar between species.⁴¹  However, the literature is sparse concerning the quantification of healing kinetics around implants between species. Although osseointegration rates are known to be higher in animal models compared with humans, its quantification is yet to be experimentally determined.

The rationale of testing the hybrid surface was to bring the surface roughness of this group to an approximate scale to the AB/AE group combined with the presence of CaP achieved with subsequent RBMa treatment. Despite the not significantly different roughness between AB/AE and the hybrid surfaces, the expected synergistic effect of combined roughness and chemical bonding involving implants with CaP remnants from blasting,²⁰  early fixation, BIC, and BAFO of all three surfaces could not be confirmed. Future studies incorporating higher amounts CaP on implant surfaces are warranted.

There have been studies showing promising results concerning reduced-length scale bioactive ceramic integration on implant surfaces.^20,35,36,40  However, specific to RBM surfaces, various factors such as particle size, blasting media composition, blasting pressure, distance, and the subsequent acid etching have been shown to affect the early host response to the implant,^3,20,21  and thus, the current design rationale for the surface incorporation of Ca-P still remains to be determined.^5,42 

The general results from this study show that all 3 surfaces investigated are biocompatible and osseoconductive, resulting in a similar bone-healing pattern with the cortical and trabecular bone in close interaction with the implant surface.^25,43  Woven bone formation was observed in all 3 surfaces after the 2 weeks allowed after the initial placement.

In agreement with the biomechanical testing results, no significant differences were found among the 3 surfaces with regard to BIC and BAFO measurements. Thus, within the limitations of the present study, we conclude that the availability of Ca and P on the surface did not hasten early integration of the investigated surfaces.

Abbreviations

AB/AE

alumina-blasting + acid-etching

AFM

atomic force microscopy

BIC

bone-to-implant contact

BAFO

bone area fraction occupied

PSHA

plasma-sprayed hydroxyapatite

RBM

resorbable-blasting media

RBMa

resorbable-blasting media + acid-etched

SEM

scanning electron microscope

XPS

x-ray photoelectron spectroscopy