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This study evaluated the effect of microgrooves in the surface of titanium implanted in rabbit calvaria. In total, 24 titanium cylinders were used in the experiments.
Tissue Engineering and Regenerative Medicine, Vol. 10, No. 6, pp 347-352 (2013) DOI 10.1007/s13770-013-1097-6

|Original Article|

The Effect of Microgrooves on Osseointegration of Titanium Surfaces in Rabbit Calvaria Eun-young Song1, Jiyoung Yoon1, Jungho Yoon1, Myunghyun Lee2, Suk-Won Lee3, and Namsik Oh1* 1

Department of Dentistry, Inha University School of Medicine, Sinheung-dong 3-ga, Jung-gu, Incheon, 400-711, Korea 2 Energy & Environmental Division, Korea Institute of Ceramic Engineering and Technology(KICET) 3 Department of Biomaterials & Prosthodontics, Kyung Hee University Hospital at Gangdong, Institute of Oral Biology, School of Dentistry, Kyung Hee University, Seoul, Korea (Received: February 12th, 2013; Revision: July 6th, 2013; Accepted: August 7th, 2013)

Abstract : The purpose of this study was to evaluate bone formation ability and bone-implant adhesion over time when microgrooves are present on the surface of cylindrical titanium implants. Eight New Zealand White rabbits with a weight of approximately 3.5 kg were used. Cylindrical titanium implants with a smooth surface (NE0), acidetched rough surface (E0), and microgroove surface (NE60/10) were placed in each rabbit’s skull, with one implant of each type placed in each skull. Experimental animals were sacrificed after 2 and 4 weeks, and tissue specimens were processed for histomorphometric analysis of bone formation by measuring bone-implant contact (BIC) and the degree of differentiation. The bone-implant contact ratio at 2 and 4 weeks was 15.9% and 29.6% (NE0), 15.6% and 34.5% (E0), and 15.3% and 52.7% (NE60/10), respectively. Good bone regeneration was observed in all experimental groups; at 4 weeks, in particular, the NE60/10 group showed high bone contact relative to the control group (p < 0.05), and other groups showed stable and mature bone. On the basis of the observed increase in bone-implant adhesion at the titanium surface, microgrooves with a width of 60 µm and depth of 10 µm are considered to promote implant osseointegration. Key words: titanium implants, acid-etching, micro-groove, osseointegration, bone-implant contact ratio

in this study have a width of 1-10 µm, which is smaller than the diameter of most cultured cells and produces changes in cell morphology, adhesion, and proliferation in vitro.11 Effects on human gingival fibroblasts were observed but effects on osteoblastic cells and osseointegration were not reported. Implants treated with hydrofluoric acid have also been reported to promote osseointegration12,13 because of the decreased healing period14 and improved bone-implant bonding. In addition, hydrofluoric acid-treated implants have been reported to improve blood clot formation by facilitating the activity of fibrinogen.15,16 In this study, the effects of acid-etched and microgrooved surfaces on osteoblast behavior and bone-implant adhesion were evaluated.

1. Introduction Dental implants are a universal treatment method for intraoral defects and esthetic restoration. Recently, to shorten the overall duration of treatment and to induce rapid osseointegration, surface treatments of titanium have been actively researched. Typical methods include the addition of bioactive materials (additive method),1-3 generation of roughness by corrosive removal of the surface (subtractive method),4-6 and adjustment of the characteristics of the oxide layer.7-9 During a recent study on the formation of microgrooves on the surface of the titanium, changes to the implant surface topography were reported to affect the chemical reactivity of an endosseous implant surface and alter the ionic and biomolecular interactions with the surface. Also the formation of microgrooves on titanium was reported to increase bone formation by promoting the organization of mineralized tissue in vivo and in vitro.10 The microgrooves used

2. Materials and Methods 2.1 Titanium Cylinder Preparation and Surface Modification Commercially pure titanium with a diameter of 4.0 mm was

*Corresponding author Tel: +82-32-890-3594; Fax: +82-32-890-2475 e-mail: [email protected] (Namsik Oh)

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Table 1. Titanium cylinders with various surface topographies. NE0

E0

NE60/10

Groove width (µm)

0

0

60

Groove depth (µm)

0

0

10

Hydrofluoric acid treatment

Non-etched Acid-etched Non-etched

NE0: smooth titanium. E0: NE0 with subsequent acid-etching. NEα/β: titanium substrate with surface microgrooves of α µm width and β µm depth.

of titanium implanted in rabbit calvaria. In total, 24 titanium cylinders were used in the experiments. The titanium cylinders were divided into three groups: one control group and two experimental groups. The groups were as follows: titanium cylinder with a smooth surface (NE0), titanium cylinder with a rough surface formed from acid etching (E0), and titanium cylinder with microgrooves (NE60/10) (Table 1). Each animal was implanted with three titanium cylinders, one from each group.

Figure 1. Confocal laser scanning microscope images of the three different cylinder groups. (A) Surface of NE0, (B) Surface of E0, (C) Surface of E60/10

produced by machining (cp-Ti) titanium cylinders (Megagen Implant Co Ltd., Gyeongsan, Korea). The surface of the cylinder had a surface roughness of Ra ≤ 0.1 µm, as determined by mechanical polishing using an atomic force microscope (AFM). Micro-grooves were photolithographically formed in the polished titanium on the surface of the cylinder. The photoresist pattern ensured that the sub-micro-mechanical polishing direction was consistent with the direction of the texture. Next, microgrooves with a width of 60 µm and depth of 10 µm were formed in the titanium surface by etching in 1% hydrofluoric acid (NE60/10). Acid etching of the titanium surface was performed by additional acid etching in 1% hydrofluoric acid (HF) on a surface with a sub-micron texture (E0). A titanium surface with a sub-micron texture that was formed by mechanical polishing was used as a control group (NE0) (Fig 1). After surface treatment, the cylinders were cut to a height of 5.0 mm, and a bevel with a height of 1.0 mm was formed at the bottom of the cylinder. As a result, cylinders with a diameter of 4.0 mm and height of 5.0 mm were fabricated (Fig 2).

2.3 Animal Experiments The Inha University Animal Experiment Ethics Committee approved the experimental protocol. Eight mature male New Zealand White rabbits weighing 3.5 to 4.0 kg were used in this study. The rabbits were anesthetized by intramuscular injection of 1 mg/kg of Zoletil® (Virbac SA, France 5-10 mg/kg) mixed with Rompun® (Bayer Korea Ltd., Korea 0.15 mL/kg). Local anesthesia with 2% lidocaine and 1:100,000 epinephrine (Yuhan, Seoul, Korea) was performed to control bleeding and to provide additional local anesthesia. Surgical sites were exposed using a sagittal incision through the skin and the periosteum at the midline of the calvaria. Cylinders were placed on both sides of the calvaria using a dental low-speed round burr under sterile saline irrigation. The titanium cylinders were placed, being careful to avoid meningeal perforation.The surgical site was closed in layers and sutured for full coverage with Vicryl 4-0 (Ethicon Inc., Somerville, NJ, USA). Antibiotics and steroids were i.m. injected to prevent infection and pain. Adrenocortical hormones (0.1 mL/ kg), lincomycin antibiotic (0.2 mg/kg), thioctic acid (0.2 mg/ kg) were given intramuscularly after surgery.

2.2 Experimental Groups This study evaluated the effect of microgrooves in the surface

2.4 Specimen Preparation and Histomorphometric Analysis Rabbits were sacrificed and examined at 2 and 4 weeks after implantation. The skin was dissected and calvaria were obtained as a 2.0×3.0 cm rectangular shape with a safe margin of approximately 5.0 mm surrounding the titanium cylinder. The samples were then immediately immersed in a 10%

Figure 2. Cylinder design. (A) The bevel helps to stabilize the cylinder.

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tempered solution of formaldehyde. For dehydration, the formaldehyde solution was replaced with 100% alcohol and acetone. Samples were then embedded using Spurr resin (Polyscience Inc. Warrington, USA) according to the manufacturer's instructions. The embedded blocks were cut and polished parallel to the long axis of the cylinder. Decalcified specimens were fabricated. The sections were stained with hematoxylin &eosin, and histological analysis was conducted using a light microscope (OLYMPUS BX51, taxane MS) under ×12.5, ×40, and ×100 magnification.

2.5 Bone-to-Implant Contact Length Ratio (%) Histomorphometric analysis was conducted using a computer program (DP manager) with an image analysis system (Image Pro Plus; Media Cybernetics, MD, USA). The surface area of newly formed bone tissue in contact with the cylinder was computed for the sections of rabbit calvaria. The area of new bone was measured as the area of newly formed bone within the specified cylinder contact area (from the upper part of the cylinder to the starting point of the cylinder bevel). Additionally, the amount of newly formed bone was expressed as a percentage of the specified cylinder area. The degree of bone-implant contact (BIC) as a percentage was also determined (Fig 3). Figure 4. NE0 group (H&E stain) (A), (D) Histological findings in the NE0 group after 2 and 4 weeks (×12.5). (B), (C) The cylinder was in contact with new bone, which was in contact with the surrounding cancellous bone (×100). (E), (F) The cylinder was lined with newly formed bone (×100). Oc, osteocyte; ob, osteoblast.

2.6 Statistical Analysis The data for BIC% were evaluated statistically using the Kruskal-Wallis test and Mann-Whitney test. Data are presented as the mean±SD. p < 0.05 was considered statistically significant.

3. Results healing. No signs of post-operative infection or exposure of the surgical area were reported during clinical observation.

3.1 Histologic Evaluation The eight rabbits did not experience complications for

3.1.1 Control group (NE0 group) At 2 weeks, no new bone formation was observed (Fig 4A, 4B and 4C). The healing process tended to be somewhat delayed, and less formation of trabecular bone was observed relative to other surface treatments (Fig 4D). At 4 weeks, more immature bone was observed at the interface of the cylinder than at 2 weeks. Increased new bone growing toward the cylinder surface was observed (Fig 4E and 4F). 3.1.2 E0 group In the E0 group, new bone formation was progressing toward the surface of the cylinder at 2 weeks. On part of the cylinder surface, because of space at the interface between the cylinder and bone, new bone was attached loosely (Fig 5A and 5C). At 4 weeks, the formation of immature bone was increased relative

Figure 3. Photomicrograph of the ground section (H&E stain; magnification ×12.5) The length of the area that was attached to bone (A, B to C, D to E, F) was measured first and then divided by the total length of the cylinder (H1-H2, H4-H3).

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Figure 5. E0 group (H&E stain) (A), (D) Histologic findings of the E0 group after 2 and 4 weeks (×12.5). (B), (C) New bone occurred around the cylinder. (E), (F) A large portion of the cylinder surface was in contact with newly formed bone (×100).

Figure 6. NE60/10 group (H&E stain) (A), (D) Histologic findings in the NE60/10 group after 2 and 4 weeks (×12.5). (B), (C) The calvaria had a regular trabecular pattern of cancellous bone. (E), (F) The threads of the cylinder were lined with newly formed bone and many of the threads were filled with newly formed bone (×100). nb, new lamellar bone.

to 2 weeks, and bone maturity was also higher than that at two weeks. As a result, new bone was in close contact with a large portion of the cylinder and surrounded the cylinder. The new bone was observed to grow toward the acid-corroded surface of the cylinder (Fig 5D, 5E and 5F).

Table 2. Bone-implant contact (BIC)(%) and standard deviation of each group at 2 and 4 weeks.

3.1.3 NE60/10 group The cylinder was well maintained within the bone. Abnormalities such as osteonecrosis were not observed. At 2 weeks, distance osteogenesis was observed where newly formed bone entered the cylinder surface (Fig 6A). Relative to the other surface treatment group, the amount of new bone had increased because the trabeculae had combined with each other (Fig 6B and 6C). Extensive contact with adjacent grooves was observed. At 4 weeks, the new bone consisted of mature bone and trabeculae on the cylinder surface. Generally favorable osseointegration was observed according to the slope of the cylinder (Fig 6E and 6F).

NE0

E0

NE60/10

2 weeks

15.91±10.45

15.06±9.81

15.31±4.45

4 weeks

29.56±12.54

34.98±19.62

52.65±8.96

(N = 4)

3.2 Histomorphometric Evaluation At 2 weeks, the NE60 group had a similar bone-implant contact ratio to the other groups. At 4 weeks, the bone-implant contact ratio was increased in all groups. The NE60 group showed the highest increase (Table 2). Histomorphometric analysis revealed a significant difference in bone-implant contact ratio between the NE60 group and the control NE0 group at 4 weeks (p < 0.05) (Fig 7).

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On the basis of these studies, we hypothesized that an etched microgroove surface would provide increased bone-implant contact relative to a smooth, polished titanium surface. The microgroove surface treatment was thus shown to provide better osseointegration than the smooth, polished titanium surface. Histological sections after 2 weeks of healing showed more active bone formation at the implant surface than that observed in the other two groups. Furthermore, at 4 weeks, increased new bone formation was observed relative to the 2week sections. The BIC ratio for the microgroove titanium cylinder (NE60) value was 15.31% at 2 weeks, which was unexpectedly similar the value in the control group (NE0). However, the BIC ratio increased to 52.65% at 4 weeks, which is consistent with previous research on the efficacy of a microgroove surface. The E0 group showed an apparent increase relative to the control group at 2 and 4 weeks but the increase was not significant. In an in vitro study, Lamolle et al.20 submerged Ti implants in a weak HF solution and concluded that time-dependent specific surface changes were related to the improved biocompatibility of these surfaces. In another study, Rupp et al.21 evaluated dynamic wettability and fibronectin interactions on acid-etched Ti implant cylinders and found that the initial surface roughness and wettability of implants may influence initial macromolecular biological responses. On the basis of these prior findings, we expected increased osseointegration of acid-etched surfaces. However, the differences relative to the control group were not significant. Wollman et al.22 noted that the bone growth rate of rabbit is approximately 3 times higher than that of humans. Gameleya et al.23 also noted that the growth of rabbit bone was at least 3 times higher than that of humans. In this study, bone growth at 2 weeks in rabbits was identical to that at 6 weeks or more in humans. Therefore, at this time, rabbit tissues should begin to undergo bone matrix absorption and bone matrix remodeling. However, bone remodeling was not observed in some acidetched specimens after 2 weeks. This variability is thought to be caused by the experimental populations. It is also likely that the insertion angle was not constant and that there was a lack of adjustment of the insertion when the implant cylinder was placed. With standardization of the insertion angle and implantation site in the skull, further research will reduce the deviation among specimens in each group. In conclusion, titanium surfaces that were altered through acid etching and microgroove formation were placed in the calvaria of rabbits. The animals were sacrificed for histological and histomorphometric analyses at different time points, and osteoblast behavior and bone-implant adhesion were evaluated.

Figure 7. Comparison of the bone-implant contact (BIC) ratio among groups.

Figure 8. Masson Trichrom Staining. Blue : Old bone, Red : New bone (A) NE0 group, (B) E0 group (C) NE60/10 group.

3.3 Immunohistochemical Evaluation At 4 weeks, new bone formation was observed in all groups (Fig 8A, 8B and 8C).

4. Discussion The present animal study was designed to evaluate the impact of microgrooves on osseointegration of titanium surfaces. For this study, a rabbit calvarial model was used. Titanium cylinders with a diameter of 4.0 mm and height of 5.0 mm were implanted in the calvaria of New Zealand White rabbits. The rabbits were sacrificed and examined at 2 and 4 weeks after implantation, and histomorphometric evaluation was performed. This study compared two titanium surface treatments (i.e., an acidetched surface and a microgroove surface) with an untreated titanium surface. A recent study reported that etched microgrooves promoted surface hydrophilicity and osteoblast cell proliferation.17 In addition, previous studies on the effect of etched microgrooves on the hydrophilicity of titanium and osteoblast response reported that MC3T3 cell proliferation on the surface after 24 h of incubation was higher for E60/10 than for E0 or E15/3.5 (p < 0.05).18 In another study, etched microgrooves with a width of 60 µm and depth of 10 µm on Ti triggered the proliferation of human gingival fibroblasts as well as gene and protein expression.19

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Within the limits of the present study, it is concluded that microgrooves with a width of 60 µm and depth of 10 µm promote effective implant osseointegration.

11. SW Lee, SY Kim, IC Rhyu, et al., Influence of microgroove dimension on cell behavior of human gingival fibroblasts cultured on titanium substrata, Clin Oral Implants Res, 20, 56 (2009). 12. LF Cooper, Y Zhou, J Takebe, et al., Fluoride modification effects on osteoblast behavior and bone formation at TiO2 gritblasted c.p. titanium endosseous implants, Biomaterials, 27, 926 (2006). 13. T Berglundh, I Abrahamsson, JP Albouy, et al., Bone healing at implants with a fluoride-modified surface: an experimental study in dogs, Clin Oral Implants Res, 18, 147 (2007). 14. M Monjo, SF Lamolle, SP Lyngstadaas, et al., In vivo expression of osteogenic markers and bone mineral density at the surface of fluoride-modified titanium implants, Biomaterials, 29, 3771 (2008). 15. J Guo, RJ Padilla, W Ambrose, et al., The effect of hydrofluoric acid treatment of TiO2 grit blasted titanium implants on adherent osteoblast gene expression in vitro and in vivo, Biomaterials, 28, 5418 (2007). 16. A Thor, L Rasmusson, A Wennerberg, et al., The role of whole blood in thrombin generation in contact with various titanium surfaces, Biomaterials, 28, 966 (2007). 17. JP Cooney, A Sanz, A Oyarzu, et al., Experimental study of bone response to a new surface treatment of endosseous titanium implants. Implant Dent, 10, 126 (2001). 18. JA Park, R Leesungbok, SJ Ahn, et al., Effect of etched microgrooves on hydrophilicity of titanium and osteoblast responses: A pilot study, J Adv Prosthodont, 2, 18 (2010). 19. SY Kim, N Oh, MH Lee, et al., Surface microgrooves and acid etching on titanium substrata alter various cell behaviors of cultured human gingival fibroblasts, Clin Oral Impl Res, 20, 262 (2009). 20. SF Lamolle, M Monjo, M Rubert, et al., The effect of hydrofluoric acid treatment of titanium surface on nanostructural and chemical changes and the growth of MC3T3-E1 cells, Biomaterials, 30, 736 (2009). 21. F Rupp, L Scheideler, D Rehbein, et al., Roughness induced dynamic changes of wettability of acid etched titanium implant modifications, Biomaterials, 25, 1429 (2004). 22. Y Wollman, S Rochkind, In vitro cellular processes sprouting in cortex microexplants of adult rat brains induced by low power laser irradiation, Neurol Res, 20, 470 (1998). 23. NF Gameleya, Laser biostimulation: In. Wolbarsht ML. ed Laser Biomedical Research in the USSR, Plenum Press, New York, USA, 114 (1997).

Acknowledgments: This work was supported by an Inha University Hospital Research Grant.

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