J Oral Maxillofac Surg 68:1877-1883, 2010

Histomorphologic and Bone-to-Implant Contact Evaluation of Dual Acid-Etched and Bioceramic Grit-Blasted Implant Surfaces: An Experimental Study in Dogs Marcelo Suzuki, DDS,* Marcia V.M. Guimaraes, DDS, MS, PhD,† Charles Marin, DDS, MS, PhD,‡ Rodrigo Granato, DDS, MS, PhD,§ Carlos A.O. Fernandes, DDS, MS, PhD,㛳 Jose N. Gil, DDS, MS, PhD,¶ and Paulo G. Coelho, DDS, PhD# Purpose: The objective of this study was to histologically evaluate a bioceramic grit-blasted and

acid-etched surface (presenting calcium and phosphorous incorporation within the surface and its oxide) versus a dual acid-etched (no calcium and phosphorous, control) moderately rough implant surface in a dog tibia model. Materials and Methods: Implants 3 ⫻ 10 mm were placed bilaterally along the proximal tibia of 6 Doberman dogs and remained for 2 and 4 weeks in vivo. After the dogs were euthanized, the implants were nondecalcified processed to ⬃30-␮m-thick plates. Transmitted light optical microscopy was used to evaluate healing patterns and bone-to-implant contact. Statistical analysis was performed by 1-way analysis of variance at the 95% level of significance and by Tukey post hoc tests. Results: At 2 weeks, histologic evaluation showed woven bone formation throughout the perimeter of both implant surfaces. However, replacement of woven bone by lamellar bone was only observed around the test surface at 4 weeks in vivo. No significant differences in bone-to-implant contact were observed for the different groups (P ⬎ .27). Conclusion: Despite nonsignificant differences between bone-to-implant contact for the different surfaces and times in vivo, higher degrees of bone organization were observed for the test implants. Biomechanical testing is warranted to verify potential differences in biomechanical fixation effectiveness between surfaces. © 2010 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved. J Oral Maxillofac Surg 68:1877-1883, 2010 Osseointegration has been described as the intimate contact between bone and biomaterials occurring at the optical microscopy level, and this phenomenon allows implants to replace load-bearing organs, restor*Assistant Professor, Department of Prosthodontics, Tufts University School of Dental Medicine, Boston, MA. †Instructor, Department of Implantology, Escola de Aperfeicoamento Profissional (EAP), São Paulo, Brazil. ‡Instructor, Department of Oral and Maxillofacial Surgery, Universidade Federal de Santa Catarina, Florianopolis, Brazil. §Instructor, Department of Oral and Maxillofacial Surgery, Universidade Federal de Santa Catarina, Florianopolis, Brazil. 㛳Assistant Professor, Department of Restorative Dentistry, Universidade Federal do Ceara, Fortaleza, Brazil. ¶Assistant Professor, Department of Oral and Maxillofacial Surgery, Universidade Federal de Santa Catarina, Florianopolis, Brazil.

ing their form and function.1 Specific to dental implantology, where classic implant therapy success ratios often exceed 90%,2 alterations in implant surface have been extensively investigated with a substantial #Assistant Professor, Department of Biomaterials and Biomimetics, New York University, New York, NY. This work was partially funded by the Department of Oral and Maxillofacial Surgery at the Universidade Federal de Santa Catarina, Florianopolis, Brazil, and by Intra-Lock International, Boca Raton, FL. Address correspondence and reprint requests to Dr Coelho: Department of Biomaterials and Biomimetics, New York University, 345 24th St, Room 804, New York, NY 10010; e-mail: pgcoelho@ nyu.edu © 2010 American Association of Oral and Maxillofacial Surgeons. Published

by Elsevier Inc. All rights reserved. 0278-2391/10/6808-0026$36.00/0 doi:10.1016/j.joms.2009.09.050

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1878 amount of research devoted to increasing the implant surface biocompatibility and osteoconductivity.3 As such, basic and clinical investigations have shown that the incorporation of bioactive ceramics on surfaces (typically as coatings) present higher degrees of osteoconductivity and biomechanical fixation at earlier implantation times compared with uncoated implants.4-8 Most commercially available bioceramic coatings are processed as 20- to 50-␮m-thick plasma-sprayed hydroxyapatite (PSHA) coatings.5-7,9-11 Although proven to improve overall bone fixation, PSHA coatings normally rely on mechanical interlocking between the metallic surface (which usually is grit-blasted or etched before the application of the coating) and the ceramic-like PSHA material for physical integrity during implant placement and function.6 This interface among the bulk metal, metal oxide, and bioceramic coating has been regarded as a weak link, where adhesive failures have been reported to occur.5,6,9 In an attempt to overcome the potential problems encountered in PSHA coatings and concurrently achieve a similar biological response, thin-film bioceramics,11,12 discrete crystalline depositions,13 and incorporation of calcium and phosphorous through different methods such as bioceramic grit-blasting14 have been used. Previous research concerning bioceramic surface treatments in the micrometer8,11 and nanometer15,16 scales have shown bone-to-implant contact (BIC),7,8,16 osteoactivity,15 and biomechanical results at short7,8,15 and long8,11 implantation times greater than uncoated implants and somewhat comparable to thick PSHA-coated implants.3,4,11,17 The objective of this study was to histologically evaluate a bioceramic grit-blasted and acid-etched surface (BGB/AA; presenting calcium and phosphorous incorporation within the surface and its oxide) versus a dual acid-etched (no calcium and phosphorus), moderately rough, implant surface in a dog tibia model.

Materials and Methods The implants used were provided by the manufacturer (Milo WP Implant; Intra-Lock International, Boca Raton, FL) and presented as a dual acid-etched surface (control) and the BGB/AA test (Ossean) surface treatment. Scanning electron microscopy and atomic force microscopy of the control and test surfaces are presented in Figure 1. Specific details about surface composition and chemistry have been previously addressed14 and are subsequently discussed. Six adult male Doberman dogs ⬃4.5 years of age were acquired after the approval of the ethics committee for animal research at Universidade Estadual Paulista, São José dos Campos, Brazil.

HISTOLOGIC EVALUATION OF 2 IMPLANT SURFACES

The surgery was performed using the proximal tibia, and the implants were placed along each limb. The first implant was inserted 2 cm below the joint line at the central medial-lateral position of the proximal tibia. The remaining device was placed along a distal direction, 1 cm from the first implant along the central region of the bone. The right and left limbs provided samples that remained for 4 and 2 weeks in vivo, respectively. The animals, limbs, and surgical site distributions (proximal and distal implant surfaces were interpolated between animals) for the 2and 4-week comparisons for the test and control surfaces resulted in an equal number of implants per group (6 implants per surface and time in vivo). All surgical procedures were performed under general anesthesia. The surgery involved skin exposure by a sharp blade followed by antiseptic cleaning with iodine solution at the surgical site and surrounding areas and a 5-cm incision at the skin. Then, a mucoperiosteal flap was reflected and the proximal tibia plateau exposed. The initial drilling was performed with a pilot drill under saline irrigation to produce the osteotomies. Sequential drilling using burs of 1.5, 2, and 2.5 mm in diameter under abundant irrigation (the Milo WP Implant thread outer diameter is 3 mm and the inner diameter is 2 mm, resulting in a healing chamber after osteotomy) was performed. The implants were then inserted into the osteotomy sites with a torque wrench according to the manufacturer’s recommendations. 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 IU/kg) and analgesics (ketoprofen 1 mL/5 kg) for a period of 48 hours postoperatively. The euthanasia was performed by anesthesia overdose. At necropsy, the upper thirds of the limbs were retrieved, the soft tissue was removed, and initial clinical evaluation performed to determine implant stability. If an implant was clinically unstable, it would be considered a failure and excluded from the study. The limbs were reduced to blocks containing 1 implant in its center and were kept in 10% buffered formalin solution for 24 hours. The blocks were then washed in running water and gradually dehydrated in a series of increasing alcohol solutions from 70% to 100% ethanol. After dehydration, the samples were embedded in a methacrylate-based resin (Technovit 9100; Heraeus Kulzer GmbH, Hanau, Germany). The blocks were then cut into slices (⬃300-␮m thickness) aiming the center of the implant long axis with a precision diamond saw (Isomet 2000; Buehler Ltd, Lake Bluff, IL), glued to acrylic plates with an acrylatebased adhesive (Aron Alpha Industrial Krazy Glue; Elmer’s Products, Inc, Columbus, OH), and a 24-hour

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FIGURE 1. Scanning electron micrographs show the (A) control (dual acid-etch) and (B) BGB/AA test surface microtopographies. The 20- ⫻ 20-␮m area, 3-dimensional atomic force microscopic micrometer/nanometer scale roughness shows the observed (C) control and (D) BGB/AA test surfaces. Suzuki et al. Histologic Evaluation of 2 Implant Surfaces. J Oral Maxillofac Surg 2010.

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setting time was allowed before grinding and polishing. The sections were then reduced to a final thickness of ⬃30 ␮m by a series of SiC abrasive papers (400, 600, 800, 1,200, and 2,400; Buehler Ltd) in a grinding/polishing machine (Ecomet 300, Buehler Ltd) under water irrigation.18 The sections were then stained with toluidine blue and referred to optical microscopic evaluation. The different groups’ histomorphology was evaluated under a transmitted light mode in an optical microscope (Leica DM2500M; Leica Microsystems GmbH, Wetzlar, Germany) at various magnifications. Percentage of BIC was determined at ⫻50 to ⫻200 magnifications with computer software (Leica Application Suite, Leica Microsystems GmbH). One-way analysis of variance at a 95% level of significance and Tukey post hoc for multiple comparisons were used for BIC statistical evaluation.

Results Animal surgical procedures were uneventful and follow-up demonstrated no complications related to the procedure or postoperative infection. No implants were excluded from the study and all histologic sections confirmed all implants’ osseointegration. Qualitative evaluation of the histologic sections showed intimate contact between cortical and trabecular bone and both implant surfaces (Fig 2). General histomorphologic observations included appositional bone healing, where direct contact existed between the implant and bone immediately after placement, and intramembranous, such as bone healing comprising substantial amounts of woven bone in the chamber that resulted from the difference in the 2.5-mmdiameter osteotomy and the 2.0-mm implant inner diameter.1,19 However, temporal morphologic differences could be distinguished for the different implant surfaces. At 2 weeks, the control (Fig 3A) and test (Fig 3B) surface groups showed implants primarily surrounded by woven bone throughout the perimeter. At 4 weeks, the diffuse woven bone pattern surrounding control implants did not show signs of remodeling or woven bone replacement by lamellar bone (Fig 3C), whereas test implants showed replacement of the diffuse woven bone pattern observed at 2 weeks by a lamellar bone morphology (Fig 3D). One-way analysis of variance showed no significant differences in percentage of BIC between groups (P ⬎ .27; Fig 4). However, the test group presented a slightly increased percentage of BIC means at 2 and 4 weeks compared with control implants (Fig 4).

FIGURE 2. General observations of implant in bone show close contact between all implant surfaces and cortical and trabecular bone regions. Suzuki et al. Histologic Evaluation of 2 Implant Surfaces. J Oral Maxillofac Surg 2010.

Discussion Osseointegration has been defined as the close contact between bone and biomaterials at the optical microscopic level,20,21 and its success for oral rehabilitation is higher than 90% for as-turned titanium implants.22-24 Over the previous 2 decades, significant attention has been devoted to improving host-to-implant biocompatibility and biomechanical responses through implant design. Among design alterations, surface modification has been the most evaluated parameter because it is the first part of the implant to interact with the host biofluids, thus having the potential of significantly altering the short-term interaction between bone and implant.12,13,25-27 Previous research has shown that increasing the as-turned surface microtopography through varied processes results in enhanced biological response and biomechanical fixation.1,20,21 Although it has been speculated that the higher biomechanical fixation of

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FIGURE 3. Optical micrographs show the bone–implant interface surrounding the control implants at (A) 2 weeks and (B) 4 weeks and the BGB/AA implants at (C) 2 weeks and (D) 4 weeks. Higher degrees of bone organization (lamellar bone) were observed around the BGB/AA surface at 4 weeks in vivo compared with all other groups, which presented a diffuse woven bone pattern around the implant. Suzuki et al. Histologic Evaluation of 2 Implant Surfaces. J Oral Maxillofac Surg 2010.

rougher compared with smoother surfaces is due to mechanical interlocking between the surface and bone, a study using nano-indentation demonstrated that surface micro-roughness results in higher bone mechanical properties compared with as-machined surfaces at early implantation times.28 Recently, it has been reported that increases in surface roughness and inclusion of calcium and phos-

phorus onto the implant surface in substantially lower quantities compared with PSHA significantly change the bone-to-implant response at early implantation times.8,11,14,29,30 According to the literature, the dual acid-etched surface (control) and the BGB/AA surface (test; incorporation of calcium and phosphorus through variations of resorbable blasting media, acid-etching, and cleaning

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FIGURE 4. One-way analysis of variance (mean ⫾ 95% confidence interval) summary for percentage of BIC for the test (BGB/AA) and control (dual acid-etched) surfaces. Despite the higher means observed for the test surface at both times in vivo, no significant differences existed between groups (P ⬎ .27). Suzuki et al. Histologic Evaluation of 2 Implant Surfaces. J Oral Maxillofac Surg 2010.

procedures) usually yield a micrometer roughness that ranges from a roughness average (Ra) ⫽ 0.5 to 2 ␮m, and is known to present better in vivo performance than as-turned surfaces.20,21 Preliminary characterization14 of the implants used in the present study showed that, from a qualitative and quantitative perspective, the BGB/AA presented higher submicrometer texturing with the presence of calcium and phosphorus in its surface chemistry and within its oxide layer, whereas higher degrees of texturing at a shorter scale was observed for the control (dual acid-etched) implant surfaces that presented primarily titanium oxide in its surface chemistry. Thus, both surfaces used in the present study have provided the wound healing site the surface roughness and chemical properties known to lead to osteoconduction and osseointegration (Figs 2, 3).8,11,14,29,30 The animal model29,31-34 (dog tibia) used in the present study provided histologic evaluation of the interaction between trabecular and cortical bone for the control and test surfaces (Fig 2). The model used may be advantageous over other models typically restricted to mostly cortical bone,19,27 because the analysis of trabecular bone response is desirable owing to regions of lower bone density often requiring more time for osseointegration establishment.35 The histologic assessment of the implant surfaces used in the present experiment showed that all specimens from all groups presented intimate cortical and trabecular bone contact to the implant, supporting both surfaces’ biocompatible and osteoconductive properties.1,2,22,27 Although percentage of BIC between groups was not significantly different (Fig 4), replacement of the diffuse woven bone present at 2 weeks around both implant surfaces by lamellar bone could only be noted for the test implants at 4 weeks in vivo (Fig 3). This suggests that the test implant surface positively influ-

enced early bone healing, resulting in the presence of lamellar bone morphology in proximity to test implants. According to these histomorphologic observations, we speculate that higher degrees of bone organization (lamellar bone replacing woven bone) should translate into better biomechanical fixation for the test implants. However, to better assess if test implants could be loaded at earlier implantation times in different clinical scenarios, in vivo laboratory studies concerning biomechanical testing should be conducted.

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SUZUKI ET AL 11. Yang Y, Kim KH, Ong JL: A review on calcium phosphate coatings produced using a sputtering process—An alternative to plasma spraying. Biomaterials 26:327, 2005 12. Coelho P, Freire J, Coelho A, et al: Nanothickness bioceramic coatings: Improving the host response to surgical implants, in Liepsch D (ed). World Congress of Biomechanics Conference Proceedings. Munich, Germany, Medimont, 2006, p 253-258 13. Mendes VC, Moineddin R, Davies JE: The effect of discrete calcium phosphate nanocrystals on bone-bonding to titanium surfaces. Biomaterials 28:4748, 2007 14. Marin C, Granato R, Suzuki M, et al: Removal torque and histomorphometric evaluation of bioceramic grit-blasted/acidetched and dual acid-etched implant surfaces: An experimental study in dogs. J Periodontol 79:1942, 2008 15. Coelho PG, Suzuki M: Evaluation of an IBAD thin-film process as an alternative method for surface incorporation of bioceramics on dental implants. A study in dogs. J Appl Oral Sci 13:87, 2005 16. Orsini G, Piatelli M, Scarano A, et al: Randomized, controlled histologic and histomorphometric evaluation of implants with nanometer-scale calcium phosphate added to the dual acidetched surface in the human posterior maxilla. J Periodontol 77:1984, 2006 17. Burgess AV, Story BJ, Wagner WR, et al: Highly crystalline MP-1 hydroxylapatite coating. Part II: In vivo performance on endosseous root implants in dogs. Clin Oral Implants Res 10:257, 1999 18. Donath K, Breuner G: A method for the study of undecalcified bones and teeth with attached soft tissues. The Sage-Schliff (sawing and grinding) technique. J Oral Pathol 11:318, 1982 19. Berglundh T, Abrahamsson I, Lang NP, et al: De novo alveolar bone formation adjacent to endosseous implants. Clin Oral Implants Res 14:251, 2003 20. Albrektsson T, Wennerberg A: Oral implant surfaces: Part 1—Review focusing on topographic and chemical properties of different surfaces and in vivo responses to them. Int J Prosthodont 17:536, 2004 21. Albrektsson T, Wennerberg A: Oral implant surfaces: Part 2—Review focusing on clinical knowledge of different surfaces. Int J Prosthodont 17:544, 2004 22. Branemark PI, Hansson BO, Adell R, et al: Osseointegrated implants in the treatment of the edentulous jaw. Experience from a 10-year period. Scand J Plast Reconstr Surg Suppl 16:1, 1977 23. Henry PJ, Laney WR, Jemt T, et al: Osseointegrated implants for single-tooth replacement: A prospective 5-year multicenter study. Int J Oral Maxillofac Implants 11:450, 1996

1883 24. Jemt T, Chai J, Harnett J, et al: A 5-year prospective multicenter follow-up report on overdentures supported by osseointegrated implants. Int J Oral Maxillofac Implants 11:291, 1996 25. Abrahamsson I, Cardaropoli G: Peri-implant hard and soft tissue integration to dental implants made of titanium and gold. Clin Oral Implants Res 18:269, 2007 26. Berglundh T, Abrahamsson I, Albouy JP, et al: Bone healing at implants with a fluoride-modified surface: An experimental study in dogs. Clin Oral Implants Res 18:147, 2007 27. Buser D, Broggini N, Wieland M, et al: Enhanced bone apposition to a chemically modified SLA titanium surface. J Dent Res 83:529, 2004 28. Butz F, Aita H, Wang CJ, Ogawa T: Harder and stiffer bone osseointegrated to roughened titanium. J Dent Res 85:560, 2006 29. Coelho PG, Cardaropoli G, Suzuki M, et al: Early healing of nanothickness bioceramic coatings on dental implants. An experimental study in dogs. J Biomed Mater Res B Appl Biomater 88:387, 2009 30. Mendes VC, Moineddin R, Davies JE: The effect of discrete calcium phosphate nanocrystals on bone-bonding to titanium surfaces. Biomaterials 28:4748, 2007 31. Coelho PG, Cardaropoli G, Suzuki M, et al: Histomorphometric evaluation of a nanothickness bioceramic deposition on endosseous implants: A study in dogs. Clin Implant Dent Relat Res 2008 Sept. 9 (Epub ahead of print) 32. Coelho PG, Lemons JE: Physico/chemical characterization and in vivo evaluation of nanothickness bioceramic depositions on alumina-blasted/acid-etched Ti-6Al-4V implant surfaces. J Biomed Mater Res A 90:351, 2009 33. Granato R, Marin C, Suzuki M, et al: Biomechanical and histomorphometric evaluation of a thin ion beam bioceramic deposition on plateau root form implants: An experimental study in dogs. J Biomed Mater Res B Appl Biomater 90:396, 2009 34. Marin C, Granato R, Suzuki M, et al: Removal torque and histomorphometric evaluation of bioceramic grit-blasted/acidetched and dual acid-etched implant surfaces: An experimental study in dogs. J Periodontol 79:1942, 2008 35. Albrektsson T, Zarb G, Worthington P, et al: The long-term efficacy of currently used dental implants: A review and proposed criteria of success. Int J Oral Maxillofac Implants 1:11, 1986