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Harder and Stiffer Bone Osseointegrated to Roughened Titanium

The Jane and Jerry Weintraub Center for Reconstructive Biotechnology, Division of Advanced Prosthodontics, Biomaterials and Hospital Dentistry, UCLA School of Dentistry, 10833 Le Conte Avenue (B3-087 CHS), Box 951668, Los Angeles, CA 90095-1668, USA

^* corresponding author, tack{at}dent.ucla.edu

	ABSTRACT

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Mechanisms underlying the beneficial anchorage of roughened titanium implants have not been identified. We hypothesized that the implant surface roughness alters intrinsic biomechanical properties of bone integrated to titanium. Nano-indentation performed on two- and four-week post-implantation bone specimens of rats revealed that bone integrated to acid-etched titanium was approximately 3 times harder than that integrated to the machined titanium, both at the osseointegration interface and at the inner area of the peri-implant bone. The hardness of the acid-etched surface-associated bone was equivalent to that of untreated cortical bone at week 4, while the bone hardness around the machined surface was equivalent to that of the untreated trabecular bone. The elastic modulus of the integrated bone was 1.5 to 2.5 times greater around the acid-etched surface than around the machined surface. Analysis of the data suggests that the implant surface roughness affects the biomechanical quality of osseo-integrated bone, and that the bone integrated to the acid-etched surface is harder and stiffer than the bone integrated to the machined surface.

KEY WORDS: osseointegration • nano-indentation • acid-etching • bone hardness • elastic modulus

	INTRODUCTION

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The substantial benefit of titanium implants has been established for the replacement of teeth and the repair of fractured bone in dental and orthopedic reconstructions. Osseointegration is essential for clinical success and exhibits interesting biomechanics. Several measures—including removable torque (Klokkevold et al., 1997), pull-out (Baker et al., 1999), and push-in tests (Ogawa et al., 2000)—have been used to assess anchorage of implants. For instance, implants with rough surfaces are typically associated with the higher force that is required to break implant anchorage, when compared with smooth-surface implants (Klokkevold et al., 1997; Ogawa et al., 2000). However, whether the increased mechanical stability of roughened implants is due to an increased mechanical locking of tissue within the surface roughness, increased bone-implant contact, increased mass of surrounding bone, biologically modified bone bonding, or a combination of these, is still highly controversial or unknown (Wong et al., 1995; Caulier et al., 1997; Vercaigne et al., 1998). More importantly, the intrinsic biomechanical properties of the integrated bone, as represented by hardness and elastic modulus, have rarely been addressed.

Mineral and collagen deposition patterns primarily determine the biomechanical properties of bone (Fujii et al., 2000; Hoffler et al., 2000). Recent studies revealed that the expression of selected bone-related genes, including collagens and calcium-binding molecules, are up-regulated in bone healing with titanium implants, especially rough-surfaced implants, compared with osteotomy healing in vivo (Ogawa et al., 2002; Ogawa and Nishimura, 2003). More interestingly, recent in vitro studies with nano-indentation technology revealed an increased hardness and elastic modulus of mineralized tissue cultured on titanium, particularly on acid-etched roughened surfaces, compared with the mineralized tissue cultured on polystyrene, in association with enhanced collagen and mineral deposition (Saruwatari et al., 2005; Takeuchi et al., 2005).

The objective of this study was to assess the intrinsic biomechanical properties (hardness and elastic modulus) of in vivo bone tissue integrated to titanium with different surface topographies, using nano-indentation technology. We hypothesized that bone integrated to titanium has biomechanical properties different from those of trabecular or cortical bone, and that the surface roughness of titanium affects the biomechanical properties of the integrated bone.

	MATERIALS & METHODS

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Titanium Implants
Experimental T-shaped implants were fabricated from commercially pure titanium and had a hollow inner chamber (3.0 x 3.0 x 0.8 mm) (Fig. 1A). The implant surface was either turned by machining or treated by acid-etching with HCl and H₂SO₄. The surface morphology and roughness quantification have been reported previously (Nakamura et al., 2005; Takeuchi et al., 2005).

View larger version (65K): [in this window] [in a new window]   Figure 1. Images for experimental protocol. (A) T-shaped titanium implants having an inner chamber prepared with machined surface (bottom) or acid-etched surface (top). (B) Schematic description of implant site. (C) Schematic diagram of the preparation of the osseointegration interface specimen. The gray area indicates bone, and thick line represents where the implant was in place. We harvested the femoral area specimens, including the implant, by cutting at both ends of the implant, and the implant and bone tissue were then carefully separated. Nano-indentation was performed in the lower half of the bone tissue separated from the implant (*). (D) A photo of the osseointegrated bone interface separated from the implant as described in (C). Bar = 1 mm. (E) Schematic diagram of the preparation of the peri-implant bone. Rat femoral area specimens, including the titanium implant, were cut and ground to expose the cross-section of the implant chamber. The gray area indicates bone, and the solid black area represents the implant. Nano-indentation was performed in the lower half of the bone tissue formed inside the inner chamber along the implant surface (*). (F) A photo of the ground surface of the peri-implant bone, prepared as described in panel (E). (G) An optical microscope image of a dent made on copper by a preliminary nano-indentation with a fixed depth of 2 µm, depicting a precise reproduction of the tip of the Berkovich indenter. The well-defined three-sided pyramidal imprint is observed. Bar is 10 µm. (H) An optical microscope image showing the remaining three-sided pyramidal imprint (triangle) of 500-nm-depth nano-indentation applied on the osseointegration interface. Bar is 1 µm.

Specimen Preparation
We prepared tissue samples for nano-indentation, to analyze both the osseointegration interface and the inner area of the peri-implant bone. At 2 and 4 wks after implantation, the 6 femurs, including either the machined or acid-etched implants (3 femurs per implant type), were harvested and cut perpendicular to the long axis of the femur at sites 3 mm from the mesial and distal ends of the implant. After a crack was made at the bottom side of the bone, the implant and bone were carefully separated (Fig. 1C), leaving the interface of the integrated bone exposed (Fig. 1D). The other 6 femurs with implants were cut along the medial-side chamber opening of the implants (Fig. 1E), exposing the transverse section of the implant chamber (Fig. 1F).

To obtain untreated trabecular bone samples, we exposed a cross-section of the metaphysis by cutting the femur at 6 mm from the distal edge of the knee end, while untreated cortical bone samples were obtained from a transverse section of the diaphysis region made at 9 mm from the distal femur end. Trabecular and cortical samples were also prepared from the week 4 femur, which received an osteotomy.

All specimens were embedded in non-exothermic epoxy resin (PL-1; Photoelastic Division, Measurements Group, Raleigh, NC, USA), which penetrated trabecular pores and inter-structural space but not the tissue itself. This embedding procedure was demonstrated not to affect nano-indentation measurements (Hoffler et al., 2000). After the epoxy resin was cured for 2 days in humidified conditions, the surfaces of the specimens were polished with increasing grades of grinding papers, from 500 to 2500 grit (Exact Apparatebau, Norderstedt, Germany) and an alumina solution (Figs. 1D,1F), and then cleansed in an ultrasonic water bath for 20 min. The specimens were mounted onto an autopolymerizing resin (Unifast II, GC, Tokyo, Japan) with their target surfaces for nano-indentation parallel to the bottom surface of the resin. The parallelism was adjusted and confirmed under an incident microscope with digitizing capability along the vertical axis (Acoustic microscope, Olympus, Tokyo, Japan). The retrieved implant surfaces that faced the prepared bone specimens were examined by scanning electron microscopy (SEM), energy-dispersive spectroscopy (EDX) (Stereoscan 250, Cambridge Instruments, Cambridge, MA, USA), and micro-computed tomography (µCT 40, Scanco Medical AG, Bassersdorf, Switzerland), for tissue-surface morphology, elemental composition and tissue structure, respectively.

Nano-indentation
Hardness and elastic modulus of the bone specimens were measured by means of a nano-indenter (Nano Hardness Tester, Micro Photonics, Allentown, PA, USA). The details of the procedure were reported previously (Saruwatari et al., 2005; Takeuchi et al., 2005). The instrument, equipped with a Berkovich diamond three-sided pyramid probe, is capable of measuring load and depth with 10-µN and 1.0-nm resolutions, respectively. A typical indentation that reflects the shape of the indenter tip is shown in Fig. 1G.

The samples were placed on the stage of the nano-indenter. Bone specimens were pressed at a loading rate of 10 mN/min until the probe reached a 500-nm depth. The sample was then unloaded at the same rate after a 15-second pause. The indentation sites were selected under a light microscope, and the imprinted spot was confirmed after the testing (Fig. 1H). The bone tissue (darker area) and epoxy resin (white area) were clearly discriminated under the microscope (Fig. 1H). We performed the indentation in the lower half (bone marrow side) of the interfacial tissue specimens (osseointegration interface) (Figs. 1C, 1D) and transverse bone specimen (peri-implant bone in the implant chamber) (Figs. 1E, 1F), to ensure that the measurement of newly formed bone avoided pre-existing bone or ’migrated’ cortical bone. As for the nano-indentation for the peri-implant bone inside the chamber, we performed the measurement within the range of 10 µm to 60 µm from the titanium surface, to avoid the interface and surrounding bone (Fig. 1E). Based on established theory and equations (Oliver and Pharr, 1992), the elastic modulus and hardness were calculated with an assumed Poisson ratio of 0.3 (Hoffler et al., 2000). Two areas on each of the 3 independent specimens—that is, 6 areas per experimental group—were measured.

Statistical Analysis
We used one-way analysis of variance (ANOVA) to evaluate the effects of specimen types on the hardness and elastic modulus of bone. Bonferroni multiple comparison was used for post hoc testing; p < 0.05 was considered statistically significant. Comparisons of the µCT parameters between the machined and acid-etched surfaces were performed with the Student’s t test.

	RESULTS

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Hardness of Implant-associated and Non-implant-associated Bone
The maximum load required to reach the 500-nm-depth indentation varied greatly among the specimen types at both weeks 2 and 4, as displayed in the representative load-displacement curves (Figs. 2A–2D). Accordingly, hardness values calculated from the load-displacement curves differed significantly with the specimen type (one-way ANOVA, p < 0.0001) (Figs. 2E, 2F). The untreated cortical bone was approximately 4 times harder than the untreated trabecular bone at both weeks 2 and 4. Hardness of the osseointegration interface was nearly 3 times greater for the acid-etched surface than for the machined surface; this tendency was consistent at weeks 2 and 4. The acid-etched surface-associated hardness was equivalent to the cortical bone hardness at week 4, while the machined surface-associated bone and the trabecular bone were comparable in their hardness. The hardness of the peri-implant bone, measured at the inner area of bone tissue adjacent to titanium, was also 2.5 times greater around the acid-etched surface than the machined surface. Regarding the effect of surgery on trabecular and cortical bone hardness, there was no difference between the untreated and osteotomized tissues (Fig. 2G).

View larger version (26K): [in this window] [in a new window]   Figure 2. Load-depth curves obtained by nano-indentation of (A) the untreated trabecular bone, (B) untreated cortical bone, (C) the week 4 osseointegration interface to the machined titanium, and (D) the week 4 osseointegration interface to the acid-etched titanium. The indentation continued to the fixed depth of 500 nm with a loading rate of 10 mN/min. Note that the maximum load required to reach the 500-nm depth varied greatly among the specimens tested. (E) Biomechanical properties (hardness and elastic modulus) of week 2 post-surgery tissues at the osseointegration interface and peri-implant bone, along with the untreated trabecular and cortical bone tissues. Data are shown as the mean ± SD (n = 6). Results from a Bonferroni multiple-comparison test are indicated. Levels of statistical significance: *p < 0.05, **p < 0.01, and ***p < 0.001. (F) Biomechanical properties (hardness and elastic modulus) of week 4 post-surgery tissues. Data are shown as the mean ± SD (n = 6). *p < 0.05, **p < 0.01, and ***p < 0.001. (G) Biomechanical property comparisons of the trabecular and cortical bone between the untreated and osteotomized areas. The tissue samples were harvested 4 wks after osteotomy.

Morphology and Elements on Retrieved Bone-Implant Interface
The machined implant surfaces revealed little or no tissue remnant after the tissue separation (Figs. 3A, 3C), and the bare machined surface was recognizable in high magnification (Figs. 3E, 3G). Energy-dispersive spectroscopy (EDX) spectra showed almost no calcium or phosphorus peaks on the machined surfaces (Figs. 3K, 3N).

View larger version (119K): [in this window] [in a new window]   Figure 3. Morphology and elemental composition of retrieved bone-implant interface. Scanning electron microscopic (SEM) images of the week-2- and -4-retrieved machined implant surfaces (A,C) and acid-etched implant surfaces (B,D). Magnified images (E-J) were obtained from the circled areas of e-j in panels A-D, respectively. Bar = 1 mm for panels A-D, 100 µm for panels E-J. (K-P) Energy-dispersive spectroscopic (EDX) elemental analysis of the retrieved implant surfaces for Ti, Ca, P, and S elements. The spectra K, L, and M were obtained from the images E, F, and I, respectively, and the spectra N, O, and P were from the images G, H, and J.

3-D Structure of Osseointegrated Bone
MicroCT images of the tissue separated from the implants clearly depicted the 3-D architecture of peri-implant bone (Fig. 4A). The tissue separated from the machined surface showed an extensive, preserved bony structure (Fig. 4A), while parts of the tissue separated from the acid-etched surface were missing (data not shown). The polished, flattened surfaces of the retrieved interfacial tissues prepared for nano-indentation were also seen (Fig. 4B). When bone tissues were imaged under the VOI (volume of interest) of 300 µm x 300 µm x 100 µm, the quantity of bone appeared greater for the tissue obtained from the acid-etched surface (Fig. 4D) than from the machined surface (Fig. 4C). The quantitative assessment showed that the bone volume/tissue volume was significantly greater for the tissue from the acid-etched surface than from the machined surface (Fig. 4E), while there were no differences in the trabecular number and trabecular thickness values. When analyzed in the bone tissues at a size of 100 µm x 100 µm x 100 µm, the bone tissues from the machined and acid-etched surfaces were equivalent in bone volume/tissue volume (Fig. 4F). The trabecular number and trabecular thickness could not be calculated.

View larger version (33K): [in this window] [in a new window]   Figure 4. Three-dimensional (3-D) bone morphology and morphometry of osseointegrated bone. (A) A microCT image of the detached tissue from the machined implant at week 4 post-implantation, depicting the trabecular architecture of newly formed bone tissue. (B) A microCT image of the prepared tissue specimen for nano-indentation. The tissue was separated from the week 4 machined implant, embedded in epoxy, and polished as described in the text. The flattened and smoothened bone surface is seen at the lower half of the tissue, where the nano-indentation was perfomed. Magnified microCT images of the tissue separated from the machined surface (C) and acid-etched surface (D). The volume of interest (VOI) for these 2 images was set at 300 µm x 300 µm x 100 µm. (E) Quantitative assessment of 3-D parameters performed in the VOI of 300 µm x 300 µm x 100 µm. Data are shown as the mean ± SD (n = 6). Statistical significance, *p < 0.01. (F) Bone morphometry performed in the VOI of 100 µm x 100 µm x 100 µm. Data are shown as the mean ± SD (n = 6).

	DISCUSSION

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A previous report of tests on human femoral bone by a 500-nm-depth indentation showed that the hardness was 0.25–0.4 GPa, 0.45–0.6 GPa, and 0.55–0.65 GPa for the trabecular, osteon, and interstitial bone tissues, respectively (Hoffler et al., 2000). The elastic modulus was reported as 5–15 GPa, 10–20 GPa, and 15–20 GPa for the trabecular, osteon, and interstitial bone tissues, respectively (Zysset et al., 1999; Hoffler et al., 2000). The hardness and elastic modulus of the trabecular and cortical bone obtained in this study fall within the reported range, validating the appropriateness of the measurement conditions and reliability of the data.

To study the biomechanical properties of the interfacial tissue, we detached the tissue from the implant surface. The flat design of the implant exterior facilitated the detachment of tissue compared with the cylinder shape, as shown in the completely exposed machined surface with little tissue remnant. Although the prepared acid-etched surfaces were partially associated with the remnant tissue, some areas exhibited little or no calcium, phosphorus, or sulfur elements, indicating the complete detachment of the tissue on which the nano-indentation was performed. Therefore, it can be assumed that the effect of sample preparation on the nano-indentation results between the 2 different implant surfaces was minimal. The bone occupation percentage, defined as bone volume/tissue volume, did not differ between the tissues from the machined and acid-etched surfaces in the analysis of the small-size tissue samples (100 µm x 100 µm x 100 µm). Considering the fact that nano-indentation with 500-nm depth was performed in the area of 0.5 to 1 µm², as shown in the image of the indent, the differences in tissue structure around the machined and acid-etched surfaces were unlikely to have affected the nano-indentation results.

Only the machined surface-associated bone showed a layer-dependent elastic modulus at both weeks 2 and 4. The elastic modulus was smaller at the machined surface interface than that measured at the inner area of the adjacent bone. There seems to be a 50- to 400-nm-thick amorphous layer intervening between the bone and titanium at the electron-microscopic level (Thomsen and Ericson, 1985). Nano-indentation requires highly polished surfaces on the test materials; a majority of the amorphous layer may have been removed during the sample preparation for nano-indentation. Therefore, the presence or absence of the amorphous layer probably had no effect on the biomechanical differences in the osseointegration interface.

To estimate the maturity of bone-healing, we evaluated the mechanical quality of the post-osteotomy bone tissues. The week 4 post-osteotomy trabecular bone showed hardness and elastic modulus levels similar to those of the untreated trabecular bone, indicating that bone healing was complete at week 4. Therefore, the revealed differences in biomechanical properties of bone between the machined and acid-etched may be interpreted as a consequence of the distinct biological processes of osseointegration, not to the maturity level of bone-healing. Recent in vitro and in vivo studies that demonstrated the existence of gene regulation at the local level of implant surfaces may support this interpretation (Ogawa et al., 2002; Ogawa and Nishimura, 2003; Takeuchi et al., 2005).

This study has revealed that the intrinsic biomechanical properties of peri-implant bone can be enhanced by an acid-etched titanium implant surface, providing novel evidence that supports the dominance of anchorage of roughened implants over those with a relatively smooth, machined surface. Although bone volume and bone-implant contact percentage have been used as parameters to assess the biological potential of implants, evaluating the intrinsic biomechanical properties of bone around implants may provide a new approach for assessing and developing implant surfaces for improved osseointegration.

	ACKNOWLEDGMENTS

 
This study was supported by 3i Implant Innovations, Inc., 3i Implant Innovations, Inc., Japan, JIADS, and API Japan.

Received November 12, 2004; Last revision February 10, 2006; Accepted March 3, 2006

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