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RESEARCH REPORT |
Laboratory for Bone and Implant Sciences (LBIS), 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 (B3-081 CHS), Box 951668, Los Angeles, California 90095-1668, USA
* corresponding author, tack{at}dent.ucla.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: osseointegration • glycosaminoglycan (GAG) • interface • nanoscratch • osteoblast
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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In the phenomenon of osseointegration, bone faces the titanium surface without intervening tissue at the light microscopic level (Albrektsson and Linder, 1981), while there is an amorphous layer intervening between the bone and titanium at the electron microscopic level (Thomsen and Ericson, 1985). The amorphous layer ranges from 50 to 400 nm in thickness (Sennerby et al., 1991), appears granular under the transmission electron microscope, and may be abundant in osteocalcin (Ayukawa et al., 1998), where collagen deposition is rarely observed (Albrektsson and Hansson, 1986). The biological mechanism of establishing the layer and, more importantly, the significance of this layer in establishing the mechanical stability of implants are unknown.
Proteoglycans are complex molecules comprised of a core protein and glycosaminoglycans, such as chondroitin sulfate, keratin sulfate, heparin sulfate, and dermatan sulfate. Proteoglycans play a role in the attachment and adhesion between osteoblastic cells and the extracellular matrix (Mizuno et al., 1996; Imai et al., 1998; Wendel et al., 1998). Several studies have implied that proteoglycans permeate the interfacial layer of the tissue facing titanium (Linder et al., 1983). The localization of proteoglycans at the titanium interface was presumed by light microscopic observation of positive periodic acid-Schiff/alcian blue reactivity for glycosaminoglycan, and the ultrastructural detection of a ruthenium-red-positive layer (Albrektsson and Hansson, 1986; Linder, 1992). Elemental analyses at the bone-titanium interface exhibited a high percentage of sulfur, a component of various glycosaminoglycans (Squire et al., 1996; Ogawa et al., 2000). However, the existence of proteoglycans around implant surfaces has not been proven, due to the lack of specificity of histochemical techniques and the possible existence of artifacts during sectioning and fixation (Klinger et al., 1998). More importantly, the localization and potential role of proteoglycans associated with roughened titanium surfaces, which are in use in the majority of current dental implant systems, have rarely been investigated.
The hypothesis to be tested was that proteoglycans play an important role in establishing tissue-titanium adhesion. The objective of this study was to investigate transcriptional and localizational outcomes for proteoglycans during the establishment of the osteoblastic mineralized tissue-titanium interface, and to examine the knock-down effect of the proteoglycan/GAG complexes on the interfacial strength between mineralized tissue and titanium.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Osteoblastic Culture
Bone marrow cells isolated from the femurs of eight-week-old male Sprague-Dawley rats were placed into alpha-modified Eagle’s medium supplemented with 15% fetal bovine serum, 50 µg/mL ascorbic acid, 10 mM Na-ß-glycerophosphate, 10–8 M dexamethasone, and antibiotic-antimycotic solution. Cells were incubated in a humidified atmosphere of 95% air, 5% CO2 at 37°C. At 80% confluence, the cells were detached and seeded onto either 12-well culture plates, or the machined titanium or acid-etched titanium disks at a density of 3 x 104 cells/cm2. This study protocol was approved by the University of California at Los Angeles Chancellor’s Animal Research Committee.
Reverse-transcription/Polymerase Chain-reaction (RT-PCR)
Expression of proteoglycan genes in the osteoblastic cultures was analyzed by the reverse-transcription/polymerase chain-reaction at culture days 14, 21, and 28, as described previously (Nakamura et al., 2005). We performed preliminary PCR trials to determine the annealing temperature and the optimal number of cycles that yielded the linear range of PCR amplification for each primer set. We performed the PCR amplification at least 3 times with the determined condition to verify the consistency. Further, identification of PCR products was confirmed through cloning and sequencing of the bands. The primer sequences and PCR conditions are described in the APPENDIX.
Immunochemistry
The osteoblastic cells were cultured on the polystyrene culture dishes, with and without titanium coating. The titanium coating was carried out with the use of electron-beam physical vapor deposition technology (SLONE Technology Co., Santa Barbara, CA, USA) (Saruwatari et al., 2005). At day 28, osteoblastic cultures were fixed with paraformaldehyde, dehydrated, and then embedded in Q.C.T. compound for frozen tissue sectioning (5 µm thickness).
Different types of sulfated glycosaminoglycans were detected immunochemically. Following incubation in diluted normal goat serum (50 µL/10 mL), the sections were incubated with a primary antibody, each of monoclonal anti-chondroitin-6-sulfate, anti-heparin sulfate, anti-keratan sulfate, and anti-dermatan sulfate (Seikagaku Corporation, Tokyo, Japan) (1:100 dilution in PBS), for 30 min. Negative control was prepared according to the same protocol, but without the addition of these primary antibodies. The sections were then incubated with biotinylated secondary antibody, followed by additional incubation with an avidin-biotin-peroxidase complex (Vectastain ABC reagent, Vector Laboratories, Burlingame, CA, USA). The signal was detected with the use of a substrate kit for peroxidase. At least 5 histology sections were prepared from each of the 3 independent culture dishes per experimental group.
GAG Degradation Assay
We used chondroitinase AC, which degrades chondroitin sulfate A and C, and chondroitinase B, which degrades dermatan sulfate, as well as keratanase and heparinase to degrade keratan sulfate and heparan sulfate, respectively. These enzymes were purchased from Seikagaku America (East Falmouth, MA, USA). At day 21 of culture, the enzymes were added at a concentration of 50-m units for chondroitinase AC, 5-m units for chondroitinase B, 5-m units for heparinase, and 500-m units for keratanase, prepared in 100 mM NaCl/1 mM CaCl2/50 mM Hepes in 100 µg/mL BSA (pH adjusted to 7.0). After a three-day incubation period (at day 24 of culture, post-seeding), the samples were washed with distilled water 3 times and subjected to nano-indentation and nanoscratch testing. The entire culture study, including nano-indentation and nanoscratch tests, was repeated at least twice (3–4 experiments in total) for confirmation, with independent cell batches from different animal pools.
Nano-indentation for the Intrinsic Biomechanical Properties of Tissue
To determine whether GAG degradation affected the intrinsic biomechanical properties of the mineralized tissue, we measured the hardness and elastic modulus of the mineralized culture, using a nano-indenter (Nano Hardness Tester, Micro Photonics, Allentown, PA, USA) (Saruwatari et al., 2005; Takeuchi et al., 2005). The mineralized tissues from the day 24 cultures were pressed at a loading rate of 400 mN/min, until the maximum load of 200 mN was reached. The sample was unloaded at the same rate following a 15-second pause. A computer algorithm gave the hardness and elastic modulus sums, based on established equations (Oliver and Pharr, 1992). Three areas in each of the 3 independent cultures were measured.
Nanoscratch Test
The adhesion property of the mineralized tissue on the substrate was evaluated by a nanoscratch test. The details of the procedure have been reported previously (Butz et al., 2005; Saruwatari et al., 2005). Tissues from day 24 cultures were placed on the motorized sliding stage of the nanoscratch tester (Microphotonics, Irvine, CA, USA). Scratches with a spherical stylus were generated with a progressive loading rate of 140 mN/min, at a scratching speed of 50 µm/sec. Mineralized areas representing a white nodule larger than 1.5 mm in diameter were selected for the scratch. The scratch path was subsequently observed by a light microscope equipped with the nanoscratch tester, and the point of delamination was determined. The critical load (delaminating force of mineralized tissue), defined as the minimal frictional force for recognizable failure in the tissue (i.e., exposure of the substrate surface under microscopic observation), was measured. Three areas were measured per culture specimen, and the measurement was performed in 3 independent culture specimens. To validate the determined delamination spots, we examiend surfaces of the selected scratches by scanning electron microscopy (SEM) and an energy-dispersive x-ray detector (EDX) (Stereoscan 250, Cambridge Co., Cambridge, MA, USA).
Statistical Analyses
We used analysis of variance (ANOVA) to evaluate the effects of culture conditions on the critical load, hardness, and elastic modulus of the mineralized tissue; < 0.05 was considered statistically significant. When appropriate, Bonferroni multiple-comparison testing was used.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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): (1) up-regulated expression for both titanium surface types compared with polystyrene culture; (2) early-stage up-regulation for both titanium surface types (machined and acid-etched titanium). followed by later-stage up-regulation for acid-etched titanium; (3) up-regulation for acid-etched titanium; and (4) unaffected expression by titanium or its surface types. Expression type #1 was seen for osteoadherin, #2 for fibromodulin, syndecan, and biglycan, and #3 for aggrecan. The rest of the tested genes, including glypican, lumican, decorin, and versican, were classified as type #4.
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), while some of the tissue outer surface was also chondroitin-sulfate-positive. The tissue image accidentally dissociated from the titanium surface clearly depicted the intense localization of chondroitin sulfate at the tissue immediately facing the titanium surface (Fig. 2B). The chondroitin sulfate signal was weaker and limited in the area on the tissue on the polystyrene (Fig. 2C). Although the trend was not as remarkable as with chondroitin sulfate, positive staining results specific to keratan sulfate, heparan sulfate, and dermatan sulfate were found at the tissue area closer to the titanium interface (Figs. 2D, 2F, 2H), while the tissue-polystyrene interface did not present such immunoreactivity (Figs. 2E, 2G, 2I).
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). In terms of the tissue hardness or elastic modulus, there was no significant effect of adding the GAG degradation enzymes, except for the significantly reduced hardness of the acid-etched titanium culture with the treatment of heparinase and keratanase (p < 0.05).
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). An SEM image of a delamination point on the untreated control machined surface vividly displayed the tissue peeling away from the substrate, exposing the substrate with little tissue remnant (Figs. 4B, 4C). EDS elemental analysis of selected specimens detected titanium, but practically no calcium or phosphate, immediately after the point of delamination (Fig. 4E), while the spot located immediately before the delamination point exhibited calcium and phosphate peaks (Fig. 4D). Although less apparent, the tissue on the acid-etched surface also displayed the penetrating pathway of the scratch into the tissue in the series of images, eventually resulting in the exposure of the titanium surface (Figs. F, G, H). EDS spectra validated the point of delamination, showing a lack of calcium and phosphate elements (Fig. J), which was detected before the point of delamination (Fig. 4I).
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). Administration of any of the 4 enzymes reduced the critical load between the tissue and titanium by up to 45%, compared with the respective untreated control cultures (Bonferroni, p < 0.05). There were no differences in the effect among the 4 different enzymes for both of the titanium surface types. The critical load of the polystyrene-tissue interface was reduced by heparinase and keratanase, but not by chondroitinase AC or B. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Proteoglycans containing different GAGs show different functions (Moyano et al., 1999; Denholm et al., 2000). Chondroitin sulfate is the most common member of the GAG family and has 3 major forms: chondroitin sulfate A, chondroitin sulfate B (dermatan sulfate), and chondroitin sulfate C. Two different chondroitinases have been purified and characterized as chondroitinase AC, which digests chondroitin sulfate A and C, and chondroitinase B, a dermatan sulfate-degrading enzyme. This study used the 2 enzymes and keratanase and heparinase, as well, to degrade keratan sulfate and heparin sulfate, respectively. Proteoglycan core molecules bind to one or more sulfated GAG chains; for instance, those of biglycan and decorin bind to both chondroitin sulfate and dermatan sulfate, while aggrecan contains chondroitin sulfate and keratan sulfate. Fibromodulin and osteoadherin contain keratan sulfate. All of these proteoglycans are known to be expressed in bone tissue. This study did not yield any differences in the tissue-detaching effect among the GAG degradation enzymes tested; the enzymes tested uniformly reduced the critical load of the mineralized tissue cultured on titanium. In fact, the proteoglycans whose gene expressions were modulated by titanium, including osteoadherin, fibromodulin, syndecan, biglycan, and aggrecan, encompassed all of the GAGs tested in their molecular structure (APPENDIX Table 2). Given the present loss-of-function results of the role of GAG in tissue-titanium adhesion, future gain-in-function studies, by both in vitro and in vivo approaches, will be of great interest toward the establishment of a way to improve bone-titanium interfacial properties.
The nanoscratch outcome obtained from the substrates having different surface topographies needs careful attention. Although direct comparison is difficult, the higher critical load for the acid-etched surface than for the machined surface was consistent with the tissue-titanium interfacial strength measured by the laser spallation technique (Nakamura et al., 2006). This study also showed that the interfacial strength of the titanium cultures was greater than that of the polystyrene culture. With only these presented results, one is not able to determine whether the increased interfacial strength is due to titanium as a material or to the rougher surface topography of the titanium surface used, compared with polystyrene. It is known that the machined titanium shows greater surface roughness than the cell-culture-grade polystyrene surface. A recent study addressed this issue by comparing the polystyrene and titanium cultures having equivalent surface roughness, and demonstrated that titanium as a material enhanced the tissue interfacial strength (Saruwatari et al., 2005). In this model, the validation of the nanoscratch test for use in cultured mineralized tissue was examined by EDX elemental analysis on the surface, as well as by cross-sectional SEM (Saruwatari et al., 2005). Although the area of elemental detection by EDX is relatively large, this study also used EDX, as well as SEM images, to confirm the absence of remnant tissue at the site of delamination. With this experimental set-up, consistent datasets with a coefficient of variation [(standard deviation/mean) x 100] of < 15% have been obtained (Butz et al., 2005; Saruwatari et al., 2005).
Transitionally, following the definition of osseointegration, "direct bone contact to titanium", the biological potential of titanium implants has been evaluated primarily by the percentage area of bone-implant contact. Although this variable arguably provides a clinically useful measure for implants, the bone-implant contact area percentage is not necessarily correlated with the biomechanical anchorage of the implants (Vercaigne et al., 1998). The suggested GAG/proteoglycan-mediated mechanism for the tissue-titanium interfacial strength may be a novel tool to assess the osteoconductive potential of the implant surface, and also may be a valuable target to improve the bonding capability of implant surfaces to bone.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received November 10, 2005; Last revision August 31, 2006; Accepted October 17, 2006
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