J Periodontol May 2007

Inuence of Different Implant Surfaces on Peri-Implant Osteogenesis: Histomorphometric Analysis in Sheep

sire Marco Franchi,* Beatrice Bacchelli,* Gianluca Giavaresi, Viviana De Pasquale,* De e Martini,* Milena Fini, Roberto Giardino, and Alessandro Ruggeri*
Background: The present study investigated peri-implant osteogenesis and implant biologic xation in different zirconia sandblasted endosseous titanium surfaces (SLA-60 and SLA120) and a turned titanium surface (T) 2 and 4 weeks after surgery. Methods: Seventy-two implant screws were implanted in tibia of six sheep. Histologic sections of implants (2 and 4 weeks after surgery) were analyzed with light microscopy for histomorphometric analysis of bone-to-implant contact (BIC), bone ingrowth (BI), and bone surface (BS/BV). Histologic blocks were used to perform bone microhardness studies next to the implants. Some implants were also observed with scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Results: In general, the highest values of BIC, BI, BS/BV, and Vickers hardness number (HV) were measured in SLA-60 samples, followed by SLA-120 and T implants. Two weeks after surgery, all the implants appeared biologically xed by a newly formed woven bone arranged in thin bone trabeculae and lling the gap between implant and host bone. Four weeks after implantation, the thickness of the woven bone trabeculae had increased, especially around the SLA-60 and SLA-120 implants by a gradual deposition of parallel-ber bone. Conclusions: Our results suggest that, in the early period of peri-implant healing, the implant surface morphology that seemed to inuence the increase of peri-implant osteogenesis, bone turnover, and peri-implant bone maturation was SLA60. We suggest that this surface, characterized by moderately deep titanium cavities very similar to the osteocyte lacunae, could act as a microscopic scaffold for mesenchymal and/or osteoblast-like cells adhesion. J Periodontol 2007;78:879-888. KEY WORDS Dental implantation, endosseous; histology; microscopy, electron; osseointegration; titanium; zirconium oxide.

* Department of Human Anatomical Sciences and Physiopathology of Locomotory Apparatus, University of Bologna, Bologna, Italy. Department of Experimental Surgery, Codivilla-Putti Research Institute, Rizzoli Orthopedic Institute, Bologna, Italy.

hape, chemical composition, and macro/microtopography of the implant surface have been widely studied as major factors positively inuencing implant osseointegration. Titanium is the most commonly used material for orthopedic and endosseous dental implants because of its good mechanical properties and biocompatibility,1,2 good resistance to corrosion, no cell toxicity, and very poor inammatory response in peri-implant tissues.1,3-5 Implant surface topography plays an important role in favoring early periimplant bone deposition.6-11 Rough surfaces increase the implant area in contact with the host bone, favoring both implant primary stability12,13 and peri-implant bone formation more than smooth surfaces.14-20 Titanium implant screws with roughened surfaces, with or without a coating, can enhance the osteoblast activity, favoring the rate and the degree of osseointegration through the deposition of new bone directly on the implant surface.10,14,15,21,22 All experimental studies on peri-implant osseous healing around different implant surfaces aim to nd the implant surface that can best improve osseointegration of the medical device, allowing early implant loading.14,22-31 In recent years, various roughened uncoated surfaces obtained by removal techniques such as sandblasting treatment have been proposed to reduce the time of peri-implant healing. Structural and functional properties of components of osteoblast adhesion

doi: 10.1902/jop.2007.060280

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are determined by different modications of the surface topography.32 Osteoblasts attach to the sandblasted surfaces from day 1 after implantation, depositing bone-related proteins.31 In addition, calcium-phosphate needle-like crystallites in an abrillar cement line matrix or lamina limitans have been described directly at the implant surface in the early period of implant healing.8,24,31,33-35 These ndings suggest that osteoblast adhesion to the implant surface is particularly important in early biologic implant xation. Chronologic peri-implant tissue responses and different animal model studies investigated early bone formation adjacent to different roughened implant surfaces, particularly in the very early period after implantation.9,21,22,36,37 However, the biologic xation of an implant requires the deposition of a well-structured calcied tissue (woven bone arranged in arches of bone trabeculae), which begins only 10 to 14 days after surgery. A network of newly formed bone trabeculae supporting a suitable mechanical and biologic anchorage to the implant was described only 1 month after implantation.11,38-40 Supporting these histologic data, other studies reported that the mean removal torque values in acid etched and sandblasted implants 4 weeks after implantation decreased41 or did not increase much over time.42 Our previous in vitro research showed that zirconia sandblasted titanium surfaces better inuenced osteoblast metabolic activity by modifying phenotype, surface adhesion levels, and proliferation rate compared to smooth, plasmasprayed, or alumina sandblasted titanium surfaces.43 The present study investigated the rate and degree of peri-implant osteogenesis occurring in vivo around two different zirconia sandblasted titanium surfaces compared to a turned titanium surface. In particular, we aimed to evaluate which implant surface best favors biologic implant xation within an early period of 4 weeks of peri-implant healing. MATERIALS AND METHODS Medical Device Preparation Seventy-two conic screw-shaped implants of commercial grade 2 Ti (ISO 5832-2), with a length of 8 mm and an outer diameter of 3.8 mm resulting from the machining process and prepared on a turning lathe (T; arithmetic average of the absolute values of all points of the prole [Ra] = 0.56 mm), followed these surface treatments: 1) 24 implants were sandblasted with ZrO2 particles obtaining a microroughened surface with Ra = 1.52 mm, maximum peak-to-valley height of the entire measurement trace (Rt) = 12.06 mm, and arithmetic average of the maximum peak-to-valley height of the ve greatest values (Rz) = 11.54 mm (SLA-60); 2) 24 implants were sand880

blasted with similar ZrO2 particles obtaining a microroughened surface with Ra = 1.32 mm, Rt = 8.76 mm, and Rz = 8.86 (SLA-120); 3) the remaining 24 implants with turned titanium surfaces were used as controls. The SLA-120 surface was treated with a double number of zirconia particles that struck the implant surface during sandblasting versus the SLA-60 surface. All implants were cleansed by ultrasound in a detergent solution for 30 minutes and in distilled water for another 20 minutes to degrease and clean the surface from contaminants. Finally, all implants were sterilized by means of g rays. Surface roughness was analyzed on some samples to conrm declared mean values, whereas micro-Raman spectroscopy was used to detect any impurities on the surface of the plates caused by manufacturing. Surgical Procedures This study was performed according to European and Italian Legislation on animal experimentation, the principles stated in the Guide for the Care and Use of Laboratory Animals, and the Animal Welfare Assurance A5424-01 by the National Institutes of Health, Rockville, Maryland. The animals used for this project were obtained from a larger study currently in progress. Six cross-bred (BergamascaMassese) sheep, 3.0 0.5 years old and 70 5 kg body weight, were submitted to bilateral implantation of 72 implant screws in the tibial diaphysis. Animals were premedicated with an intramuscular injection of 10 mg/kg ketamine and 0.3 mg/kg xylazinei and a subcutaneous injection of 0.0125 mg/kg atropine sulfate. General anesthesia was induced with 10 mg/kg intravenous sodium thiopentone (2.5% solution) and maintained with 60%/ 40% O2/N2O and 1.5% to 2% isourane. The medial surface of tibial mid-diaphyses was exposed, and six 3.9-mm-diameter holes, transversally oriented, were drilled at low speed under sterile 0.9% NaCl. The 72 different screws were implanted with an insertion torsial moment of 1.6 0.2 Nm. Antibiotics (cephalosporin 1 g/day for 5 days) and analgesics (ketoprofen 500 mg/day for 3 days) were prescribed in the immediate postoperative period. Two and 4 weeks after surgery, respectively, three animals were pharmacologically euthanized. Tibiae were excised and cleaned of soft tissue. Cylindrical bone segments containing each implant were obtained from the tibial middiaphyses. Bone segments were xed in Karnovsky solution (para-aldehyde, 4%; glutaraldehyde, 2.5%; 0.1 M cacodylate buffer) for 30 minutes for histomorphometric evaluation and ultrastructural studies that were carried out by masked operators.
i Or-Vit, Castelmaggiore-Bologna, Italy. Ketavet, Farmaceutici Gellini, Aprilia, Italy. Rompun, Bayer Italia, Milan, Italy. Torque wrench, CITIEFFE, Bologna, Italy.

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Histology and Histomorphometry Bone segments englobing 36 implants were dehydrated in ethanol series and embedded in methylmethacrylate. Three bone implant sections of 100 to 200 mm in thickness were obtained parallel to the long axis of the xture by means of the diamond saw microtome and further ground to 60 10 mm with a grinding system.# Two sections for each surface treatment were stained with toluidine blue and acid fuchsin. Histomorphometric analyses were performed using an optic microscope** connected to an image analyzer system. Each measurement was taken semiautomatically at an original magnication of 10. The following histomorphometric parameters were measured only in the portion of each implant engaged in the cortical bone of tibia diaphysis, eluding the implant apex protruding in the medullary canal: 1) boneto-implant contact (BIC, %; i.e., the percentage of intraosseous implant surface directly covered by bone, independently from the thickness or the quantity of the new bone in the gap between the implant and host bone),12,39,44 differentiating among newly formed bone (BICNEW), host bone (BICOLD), and both (BICTOT); 2) bone ingrowth (BI, %), representing the amount of bone growth inside the gap between the implant and the host bone, measured in an area located between the bottom and the top of the threads independently from bone-to-implant direct contact; and 3) bone surface (BS/BV, %), which is the ratio between the newly formed bone perimeter and the new bone area by multiplying by 4/p, which is correct for isotropic structures, indicating the geometry of the newly formed bone.45 Microhardness Test Resin-embedded specimens, ground and polished with diamond paste of 3-KD-C3 grade, were used to measure bone hardness by means of an indentation test. The microhardness measurements were taken tangentially to the interface with an indenter applied to the bone at a load of 0.05 kgf and dwell time of 5 seconds.46,47 The average value for each sample was calculated with a mean of 10 for each examined area in the regrown bone within 200 mm from the interface (HV200). Scanning Electron Microscopy (SEM) Another 18 implants with the surrounding periimplant tissues were removed after 2 and 4 weeks and processed for SEM observation. Immediately after removal, the peri-implant host bone was mechanically detached from each screw. Both the implants and detached peri-implant tissues were dehydrated in an ascending scale of ethanol and nally treated with hexamethyldisilazane for 2 10 minutes. The screws were mounted on stubs with carbon bioadhesive lm, gold/palladium coated, and observed with

an SEMii tted with secondary electron (SE) probes, at voltages of 15 kV. Transmission Electron Microscopy (TEM) Another 18 implants with the surrounding peri-implant tissues were removed after 2 and 4 weeks and processed for TEM observation. The xed implants with the surrounding host bone were postxed in 1% osmium tetroxide, dehydrated in an ascending scale of ethanol, and embedded in resin araldite. The ne cross-sections from these samples were contrasted with uranyl acetate and lead citrate and nally examined with an electron microscope. Statistical Analysis Statistical analysis was performed using statistical software.## Data are reported as means SD at a signicance level of P <0.05. After having veried the normal distribution and the homogeneity of the variance, the analysis of variance (ANOVA) test, followed by the Scheffe post hoc multiple comparison test, were used to analyze histomorphometric and microhardness data between tested surfaces. Student t test was used to compare histomorphometric and microhardness data between experimental times within each tested surfaces. RESULTS All the animals survived until the nal experimental time, and no operative or postoperative complications were encountered. Before the removal of the implants with the surrounding bone, clinical and macroscopic evaluation revealed that all the medical devices appeared well xed in their endosseous position. Two Weeks After Implantation Light microscopic analysis showed a good implant healing in all sample groups. Most of the implant surface was engaged in the bone tibiae, whereas the apex of the implants protruded in the bone marrow of the medullary canal (Fig. 1). Around all implants, a few chips of host bone that probably arose from the bur friction were present in the peri-implant environment. Most of the screw threads were well xed in the host bone, presumably allowing good primary stability of the implants. No newly formed bone was observed where the threads were in contact with the host bone. On the contrary, newly formed bone consisting of woven bone trabeculae was discernible in the peri-implant environment between two adjacent threads, where a gap of ;250 to
# ** ii ## Saw and Grinding, Remet, Bologna, Italy. BX41, Olympus Optical Europa, Hamburg, Germany. Qwin, Leica Imaging Systems, Cambridge, U.K. Microhardness VMHT 30, Leica, Vienna, Austria. Sigma-Aldrich Srl, Milan, Italy. Philips 515, Philips, Eindhoven, The Netherlands. Philips CM-10, Philips. SPSS v.12.1, SPSS, Chicago, IL.

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surface, with no soft tissue interposition. The arrangement of these bone trabeculae consisted of a network of calcied tissue surrounding large blood vessels in marrow spaces (Figs. 2B and 2C). The implant surface of the apical portion of the implants and protruding in the medullary canal of the tibiae diaphysis was covered by small newly formed bone trabeculae (Fig. 1). Very few multinucleated cells/osteoclasts were observed. Two weeks after surgery, the histomorphometric analysis showed that the best and highly statistically signicant value of BICNEW (%) (P <0.05) was observed in SLA-60 implants (19.8 1.2), followed by T (18.0 1.8) and SLA-120 samples (17.5 1.0). Both BICOLD and BICTOT were higher in SLA-120. ReFigure 1. Two weeks after surgery. T implant. Light microscope analysis shows garding peri-implant BI (%), the best results were seen good implant healing with part of the implant surface engaged in the in SLA-60 (28.9 2.7) versus SLA-120 (22.5 2.4) bone tibiae. The implant apex protruding into the bone marrow of the and T (21.7 2.4; P <0.05). The highest value for medullary canal is covered by small newly formed bone trabeculae. BS/BV (%) was detected in SLA-60 implants (23.2 2.4), followed by SLA-120 (23.1 2.0) and T samples (13.9 1.5; Table 1). Microhardness tests showed that 2 weeks after surgery, the most mineralized newly formed periimplant bone, measured as HV200, was detectable in SLA60 versus T and SLA-120 (Table 2). SEM and TEM evaluation of the implants surfaces detached from the surrounding bone conrmed the data reported by microscopic analysis. In particular, on some areas of the Figure 2. SLA-60 and SLA-120 implant Light microscopy. Two weeks after surgery in cortical bone. In T implants, newly formed woven perisurfaces, very at and thin implant bone trabeculae appear thin, very rich in osteoblasts and osteocytes, and delimit marrow newly formed trabeculae of wospaces (A). These bone trabeculae prevalently start from the host bone and run toward the implant surface. In SLA-60 (B) and SLA-120 implants (C), the woven bone trabeculae arise both from the ven bone were bounded to the host bone and directly onto the implant surface, with no interposed soft tissue. Bar = 100 mm. metal surface that appeared covered by an amorphous biologic material (Figs. 3 and 4). 350 mm was present between the implant and the host Rounded osteoblast-like cells in high activity of depobone. The bone trabeculae appeared thin and very sition showing a well-developed ergoplasmatic reticrich in rounded osteocytes, which were located in wide ulum and many cytoplasmic elongations were lacunae. At the surface of the newly formed bone, trapresent both on the surface of the bone trabeculae beculae rounded osteoblasts were aligned, producing and on the implant rough surfaces (Figs. 5 and 6). osteoid tissue. Around the T implants, the thin bone Only very few bone trabeculae were observed in contrabeculae started from the host bone and ran to the tact with the T implant surface. implant surface, sometimes anchoring to the implant surface. The bone trabeculae delimited blood and preFour Weeks After Implantation sumably lymphatic vessels of different sizes dispersed Histologic observations showed that both in T and SLA in marrow lacunae rich in mesenchymal cells (Fig. (60 and 120) implants, a peri-implant newly formed 2A). In contrast, around the SLA-60 and SLA-120 bone regularly arranged in a three-dimensional netimplants, the woven bone trabeculae appeared to work was present, delimiting the well-dened marrow arise from the host bone and directly onto the implant lacunae described in 2-week samples. The newly
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Table 1.

Histomorphometric Parameters for All Tested Surfaces at Different Experimental Times

2 Weeks T BICNEW (%) BICOLD (%) BICTOT (%) BI (%) BS/BV (%) 18.0 1.8 16.4 4.5 34.4 8.2 21.7 2.4 13.9 1.5** SLA-60 19.8 1.2 14.7 3.9 34.5 5.5 28.9 2.7 23.2 2.4

4 Weeks SLA-120 17.5 1.0 22.4 7.0 39.9 7.7 22.5 2.4 23.1 2.0 T 23.4 1.8* 14.6 5.1 38.0 7.9 31.8 2.8 11.3 1.3 SLA-60 32.2 1.3 10.8 1.1i 43.0 6.9 43.0 2.4
#

SLA-120 19.2 1.8 20.0 6.5 39.2 8.3 37.9 2.7 13.9 1.1ii

12.5 2.2

Values are mean SD; n = 4. * Scheffe post hoc multiple comparison test between surface treatments within each experimental time: T versus SLA-60 and SLA-120 (P <0.005). Student t test between experimental times within each surface treatment (P <0.05). Student t test between experimental times within each surface treatment (P <0.0005). Scheffe post hoc multiple comparison test between surface treatments within each experimental time: SLA-120 versus SLA-60 (P <0.0005). i Scheffe post hoc multiple comparison test between surface treatments within each experimental time: SLA-60 versus SLA-120 (P <0.05). Scheffe post hoc multiple comparison test between surface treatments within each experimental time: SLA-60 versus T and SLA-120 (P <0.005). # Scheffe post hoc multiple comparison test between surface treatments within each experimental time: SLA-60 versus T (P <0.05) and SLA-120 (P <0.001). ** Scheffe post hoc multiple comparison test between surface treatments within each experimental time: T versus SLA-60 and SLA-120 (P <0.05). Scheffe post hoc multiple comparison test between surface treatments within each experimental time: T versus SLA-60 (P <0.05) and SLA-120 (P <0.01). Student t test between experimental times within each surface treatment (P <0.05). Student t test between experimental times within each surface treatment (P <0.0005). ii Student t test between experimental times within each surface treatment (P <0.0005).

Table 2.

Bone Microhardness Results for Type of Implants at 2 and 4 Weeks

2 Weeks Parameter HV200 (HV) BMI (%) T 34.9 0.8 47.8 1.2 SLA-60 39.2 0.6 53.5 0.9 SLA-120 35.2 0.3 48.0 1.1 T 46.4 1.8* 60.4 1.5i 4 Weeks SLA-60 46.3 1.8 63.3 1.3 SLA-120 44.3 1.3 63.1 1.2#

Values are mean SD; n = 4. HV200 = bone microhardness within 200 mm from the interface; BMI = bone maturation index. * Student t test between experimental times within each surface treatment (P <0.001). Student t test between experimental times within each surface treatment (P <0.05). Scheffe post hoc multiple comparison test between surface treatments within each experimental time: SLA-60 versus T and SLA-120 (P <0.005). Scheffe post hoc multiple comparison test between surface treatments within each experimental time: T versus SLA-60 and SLA-120 (P <0.05). i Student t test between experimental times within each surface treatment (P <0.0005). Student t test between experimental times within each surface treatment (P <0.0005). # Student t test between experimental times within each surface treatment (P <0.0005).

formed bone trabeculae seemed to be more developed than in 2-week samples, and more numerous woven bone trabeculae appeared in direct contact with the implant surface. The thickness of the woven bone trabeculae was increased especially around the SLA-60 and SLA-120 implants by a progressive deposition of a lamellar parallel-bered bone. Some host bone chips, resulting from the friction of the surgical bur with the host bone during cavity preparation, were partially or completely enveloped by the newly formed bone trabeculae. In some areas of SLA implants, the woven bone was partially replaced by lamellar bone, not only in contact with the host bone but also adjacent to the surface of the device. At this

stage, signs of remodeling were observed together with development of secondary osteons, in particular in regions close to the parent bone (Fig. 7). Histomorphometric analysis showed that 4 weeks after surgery, the implant surface with the highest percentage of newly formed bone in direct contact with the implant surface (BICNEW [%]) was SLA-60 (32.2 1.3), followed by T (23.4 1.8) and SLA-120 (19.2 1.8). The lower value of BICOLD and the higher BICTOT (%) were recorded for SLA-60. The periimplant BI average was 43.0 2.4 in SLA-60, 37.9 2.7 in SLA-120, and 31.8 2.8 in T implants. BS/ BV (%) average was 13.9 1.1 in SLA-120, 12.5 2.2 in SLA-60, and 11.3 1.3 in T samples (Table 1).
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Figure 3.
SEM picture of a SLA-60 implant detached from the surrounding bone 2 weeks after surgery. On a thread of the screw, a peri-implant tissue in close contact with the implant surface shows some at and thin newly formed bone trabeculae bound to the metal surface. The metal surface is covered by an amorphous biologic material. Bar = 100 mm.

Figure 5.
SEM picture of a SLA-60 implant detached from the surrounding bone 4 weeks after surgery. A newly formed bone trabecula is in contact with the implant surface. Bridges of osteoblast cytoplasmatic elongations run from the bone trabecula to the implant surface. Bottom right: a rounded osteoblast-like cell (arrow) with cytoplasmic elongations is detectable on the metal surface covered with biologic material. Bar = 10 mm.

Figure 4.
TEM picture of peri-implant tissue detached from SLA-60 2 weeks after surgery. A thin newly formed trabecula of woven bone with interweaved collagen brils is detectable in proximity of the implant surface. On the right, collagen brils of osteoid tissue are visible. Bar = 2 mm.

Figure 6.
TEM picture of peri-implant tissue detached from a SLA-120 implant 2 weeks after implantation. In proximity of the implant surface, a rounded osteoblast depositing new bone shows a well-developed ergoplasmatic reticulum and cytoplasmic elongations. Bar = 2 mm.

Microhardness tests showed that 4 weeks after surgery, the most mineralized newly formed peri-implant bone, measured as HV200, was equally detectable in SLA-60 (46.3 1.8) and T (46.4 1.8) versus SLA120 (44.3 1.3; Table 2). SEM and TEM observations conrmed the histologic data: thick bone trabeculae of lamellar mature bone regularly arranged in a tri-dimensional network lled the peri-implant environment. In SLA implants, some bone trabeculae were intimately bound to the implant surface. Round osteoblast-like cells in depositional activity were readily detectable, even in the proximity of rough implant surfaces (Figs. 8 and 9).
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DISCUSSION All experimental studies9,14,22-29,31 on peri-implant osseous healing around different implant surfaces aimed to establish the implant surface best favoring osseointegration of the medical device, allowing early loading of the implant. In recent years, different roughened uncoated titanium surfaces obtained by removal techniques such as sandblasting treatment have been proposed to shorten the time of periimplant healing.

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In our research, all implants appeared biologically xed by a newly formed woven bone arranged in thin bone trabeculae lling the gap between the implant and host bone 2 weeks after surgery. However, both the rough surfaces obtained by zirconia sandblasting techniques (SLA-60 and SLA-120) showed a more complete peri-implant osteogenesis than the machined one (T), showing contact osteogenesis (i.e., bone Figure 7. trabeculae directly formed on Light microscope analysis. Four weeks after surgery, the peri-implant tissue around T implant (A) shows the implant surface) over disthick bone trabeculae arranged in a tridimensional network in direct contact with the implant surface. tance osteogenesis.48 The woDespite the wider periimplant space in SLA-60 (B) and SLA-120 (C) implants, the gap between the ven bone detectable at 2 weeks metal and the host bone is lled by a new woven bone with bone trabeculae that are clearly thicker because of the apposition of lamellar bone in an osteon arrangement. Bar = 100 mm. was very rich in rounded osteoblasts/osteocytes located in wide bone lacunae, and this supported the idea that static osteogenesis had occurred.49 Four weeks after implantation, the thickness of the woven bone trabeculae was increased, especially around the SLA-60 and SLA-120 implants, by a progressive deposition of a parallel-bered bone, indicating dynamic osteogenesis.49 The trabecular woven bone represents the calcied tissue that may ll the gap between the implant and the host bone faster and more uniformly and is very important to support the implant for an early prosthetic loading.11,21,39 It is probable that at 2 weeks after insertion, the implants reached a good biologic integration that could allow a real functional implant loading. Buser et al.42 also showed that removal torque values of SLA versus machined titanium implants from maxilla of miniature Figure 8. pigs were higher in the early period of implant healing SEM picture of peri-implant tissue and an SLA-60 implant 4 weeks but were similar in time (4, 8, and 12 weeks). The ulafter surgery. On the right, a thread of the implant (I) is recognizable. The gap between the host bone (HB), in the top part of the picture, trastructural observations conrmed the more evident and the implant is lled by a three-dimensional network of newly direct bone deposition and cell adhesion in the zircoformed thick trabeculae of lamellar mature. Some bone trabeculae nia SLA surfaces versus machined ones. Osteoblasts are intimately bound to the implant surface. Bar = 100 mm. were reported to attach to the sandblasted surfaces from day 1 after implantation, depositing bone-related proteins.31 Calcium-phosphate needle-like This histologic and ultrastructural study described crystallites in an abrillar cement line matrix or lamina the rate of peri-implant healing around different zircolimitans similar to the incremental lines in bone or rania sandblasted (SLA-60 and SLA-120) and turned dicular cement have also been described directly at titanium implant surfaces (T) to establish which the implant surface.8,24,31,33-35 These ndings sugimplant surface improves peri-implant osteogenesis gest that ideal macro/microtopography of an implant in the best and fastest possible way. The period of obshould be very similar to the morphologic surface of servations of this study was appropriately limited to 2 calcied bone to better favor a scaffold effect of the and 4 weeks after surgery because a previous study22 implant surface. The zirconia SLA-60 surface tested showed that there were no differences in the bone in this research showed regular concavities of 10 to pattern and tissue components between turned and 15 mm in diameter, with a peak to valley of 12.06 sandblasted titanium surfaces 6 to 12 weeks after mm. It was shown that moderately rough surfaces betimplantation. ter favor peri-implant bone growth than smoother or
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Figure 9.
TEM picture of the peri-implant bone in an SLA-120 4 weeks after surgery. The newly formed peri-implant bone shows a lamellar mature bone with an osteoblast. Bar = 5mm.

rougher surfaces.50 We suggest that the moderately deep titanium cavities of SLA-60 implants, very similar to the osteocyte lacunae, could operate as a microscopic scaffold for mesenchymal cells or osteoblast-like cells, which can be favored in adhering on these roughened titanium surfaces.43 According to other authors,12,22,29,51 we measured BICTOT that was similar in all surfaces 2 weeks after surgery. The relative higher value observed in SLA120 was related to its higher BICOLD. Four weeks after implantation, the BICTOT was higher in SLA-60 versus SLA-120 and T because of its higher value of BICNEW. To better quantify the rate of peri-implant osteogenesis observed in the different implant surfaces, we measured BICNEW to indicate the percentage of intraosseous implant surface covered by newly formed bone,12,39,44 independently from the quality or the quantity of the new bone between the implant and host bone. At 2 weeks after insertions, T, SLA-60, and SLA-120 implant surfaces, respectively, showed a BICNEW of 18.0%, 19.8%, and 17.5%. The values increased at 4 weeks to become 23.4%, 32.2%, and 19.2% (Table 1), respectively. We focused the measurement to the newly formed bone in direct contact with the implant surface because this parameter better shows the real rate of the biologic integration of the implant, (i.e., the newly formed bone linked to the implant surface). The higher and signicant decrease of BICOLD in SLA-60 versus SLA-120 and T from 2 to 4 weeks after surgery suggests that the higher bone resorption of peri-implant host bone (i.e., a more developed periimplant bone turnover) occurs around the SLA-60 surface. Nevertheless, some authors16,35,51,52 suggested that increased proportions of direct-toimplant-contact is the only factor to evaluate the
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early xation of the implants; we believe that BIC (both BICTOT and BICNEW) alone may not be representative of such a solid anchorage of the implant to allow functional loading. The ideal early biologic implant xation should consist of thick well-mineralized bone trabeculae in direct contact with most of the implant surface but also uniformly lling most parts of the peri-implant space. In this research, BICNEW was higher in SLA-60 implants both at 2 and 4 weeks after surgery. However, a functional implant xation depends not only on the amount of the new bone in direct contact with the implant surface but also on the quantity and geometry/ morphology of the newly formed bone in the periimplant gap. In this study, the amount of the periimplant newly formed bone was measured by BI, independently from the extension of bone-to-implant direct contact area. Both at 2 and 4 weeks, the highest BI was reported in SLA-60 implants. The morphology of the new peri-implant bone in the peri-implant environment was evaluated by BS/BV.45 The highest value of this parameter (i.e., maximum perimeter and minimal area) corresponded to a well-developed tri-dimensional network of thick bone trabeculae that can ll the gap between the implant and the host bone in the fastest, most uniform way.11,40,53 The reduction of BS/BV values from 2 to 4 weeks after implantation in all samples suggests a remodeling of the newly formed peri-implant bone trabeculae into a lamellar bone consisting of thicker bone trabeculae with fewer medullary spaces. The reduction of BS/BV, indicating a maturation of the peri-implant bone in time, was higher in SLA-60 (46.1%) versus SLA-120 (39.8%) and T (18.7%). Both at 2 and 4 weeks after surgery, the best rate of peri-implant osteogenesis evaluated by the addition of these three peri-implant healing parameters (BIC, BI, and BS/ BV), all expressed in percentage, was found in SLA60 implants. At 2 weeks after surgery, the microhardness results showed that the highest mineralization of the newly formed bone was around the SLA-60 implants. An increased mineral content in the newly formed periimplant bone in time (4 weeks) occurred in all implant surfaces, with no highly signicant differences. Our results suggest that the implant surface morphology inuenced the rate of mineralization of the newly formed peri-implant bone mainly in the early period of peri-implant healing (2 weeks). The implant surface that best favors peri-implant osteogenesis, bone turnover, and maturation is SLA-60. ACKNOWLEDGMENTS The authors thank Or-Vit, Castelmaggiore-Bologna, Italy, for providing the implants; Massimo Gamberini,

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technician, Department of Human Anatomical Sciences and Physiopathology of Locomotory Apparatus, University of Bologna, for histologic assistance; and Gianfranco Filippini, technician, Department of Agroenvironmental Sciences and Technologies (DISTA), DISTA, University of Bologna, for SEM. This research project was supported by Italian Ministry of University and Research (MIUR) (grant 2005058998-001). REFERENCES
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Correspondence: Dr. Marco Franchi, Human Anatomy Institute, Via Irnerio 48, 140126 Bologna, Italy. Fax: 39051-2091659; e-mail: marco.franchi3@unibo.it. Accepted for publication November 21, 2006.

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