1.Introduction
2.Material and methods
3.Result and discussion
4.Conclusions


  Abstract

    Chemical acid etching alone of the titanium implant surface have the potential to greatlyenhance osseointegration without adding particulate matter (e.g. TPS or hydroxyapatite) or embedding surface contaminants (e.g. grit particles). The aims of the present studywere to evaluate anydifferences between the machined and dual acid etching implants with the removal torque as well as topographic analysis.

    A total of 40 custom-made, screw-shaped, commerciallypure titanium implants with length of 5mm and an outer diameter of 3.75mm were divided into 4 groups, 10 screws in each, and chemical modification of the titanium implant surfaces were achieved using HF and HCl/H2SO4 dual acid etching.

    The first exposure was to hydrofluoric acid and the second was to a combination of hydrochloric acid and sulfuric acid. The tibia metaphysics was exposed by incisions through the skin, fascia, and periosteum.

    One implant of each group was inserted in everyrabbit, 2 in each proximal tibia metaphysics. Everyrabbit received 3 implants with acid etched surfaces and 1 implant with a machined surface.

    Twelve weeks post-surgically, 7 rabbits were sacrificed, Subsequently, the leg was stabilized and the implant was removed under reverse torque rotation with a digital torque gauge (Mark-10 Corporation, USA) (Fig. 1). Twelve weeks after implant placement, the removal torque mean values were the dual acid etched implants (24%HF+HCl/H2SO4, group C) required a higher average force (34.7 Ncm), than the machined surface implants (group A) (p = 0.045) (Mann-Whiteneytest).

    Scanning electron micrographs of acid etching of the titanium surface created an even distribution of verysmall (1-2 µm) peaks and valleys, while machining of the titanium surface created typical microscopically grooved surface characteristics.

    Nonetheless, there was no difference in surface topographybetween each acid etched implant groups.

    Therefore, chemicallyacid etching implant surfaces have higher strengths of osseointegration than machined implant surfaces. There is less correlation between removal torque and the difference in HF volume%.

1. Introduction

    Through the studyof clinical and experimental data, it is clear that the long-term clinical success of dental implants depends largelyon osseointegration of the implant. The importance of osseointegration was first described byBrane mark et al. In addition, the original microstructural and macrostructural aspects of the standard Branemark implant (Nobel Biocare, Goteborg, Sweden) were initiallycon sidered essential to ensure adequate bone formation around any implant [1,2]. The term 'integration strength' refers to the force required to break the bond between the implant and the bone. The surface morphologyand bone-implant interactions determine the predictabilityof endosseous dental implant-bone integration.

    To obtain osseointegration of an implant, several factors are of importance: biocompatibility, the surface quality, the surgical technique, the status of the host tissue, and the loading conditions [3]. The 1996 World Workshop in Periodontics concluded that the surface characteristics of an implant, particularlyrou ghness, maydirect tissue healing. However, these macrorough surfaces could potentiallyc ause failure of the implant because of increased bacterial aggregation on the rough surface or breakdown of the hydroxapatite [4]. The interaction between implants and surrounding tissues can be affected bythe macroscopic surface topography as well as bythe surface morphology or roughness at a microscopic level [5,6]. Although machined commercial pure titanium surface were recommended for many years to ensure best results, more recent studies suggest that other materials and surface characteristics mayalso be beneficial [1]. Various procedures have been tested to improve the anchorage strength and mechanical interlocking of root-form dental implants bymodify ing implant characteristics, especiallythe implant surface texture, byroughening, coating or chemical treatment [7-10]. The development and use of these surfaces modifications have been based on the theorythat improved osseointegration can be achieved byincrea sing the topographyor roughness of the implant surface [11,12]. Several investigators have found that roughened surfaces had increased implant surface areas that resulted in greater surface coverage bybone when compared to smooth-polished surfaces [13,8]. Also, several studies have shown that a rough implant surface helps to promote osseointegration more than a smooth machined surface. Buser et al. reported that an increased surface area positivelyco rrelated with an increased bone-implant contact. Several investigators have demonstrated that implant surface roughness enhances the biomechanical anchorage (osseointegration) of implants to bone. Theyde termined that this fact through torque removal tests [5,14-16].

    Surface blasting and acid etching can increase the rate and amount of bone formation on the implant surface [5,16,9,17]. Moreover, one of the more effective bone- implant interfaces is sandblasting, followed byacidattacking surfaces [5,18]. No negative effects on cell adhesion have been shown using this technique [5,18]. Grit blasting followed byacid etching has produced surface characteristics resulting in the greatest bone contact percentage of metal surfaces tested. This appeared superior to grit blasting, acid-pickled and titanium plasma sprayed surfaces alone [5]. However, several investigators have reported that grit particles can remain impregnated in the implant material, and are potentiallya causative agent in observed tissue breakdown [19].

    A new surface that produces a microroughness similar to the blasted/etched surface but uses onlyspecia l hightemperature dual acid etching without grit blasting has been developed. Chemical modification (acid etching alone) of the titanium implant surface is of particular interest because it appears to have the potential to greatlyenhance osseointegration without adding particulate matter (e.g. TPS or hydroxyapatite) or embedding surface contaminants (e.g. grit particles). The purpose of dual etching is to produce a microrough surface that provides rapid osseointegration, while maintaining the long-term success associated with a machined implant surface.

    The aims of the present studywere to evaluate any differences between the machined and dual acid etching implants with the removal torque as well as topographic analysis.

2. Material and methods

2.1. Animals and anesthesia

    Ten adult white rabbits weighing 3-3.5 kg were used in the present study. The animals were anesthetized with a combination of ketamine (Ketalars 45 mg/kg of body weight, YuHan, Korea) and xylazine (Rompuns, 7 mg/ kg of body weight, Bayer, Korea) intramuscularly. Prior to surgery, 1.8 ml of 2% lidocaine were injected locally into the tibia metaphases. Post-operatively, the animals received antibiotics at a dose of animal for 3 days. Twelve weeks after surgery, the animals were sacrificed using an overdose of carbon dioxide.

2.2. Implant surface treatment

    A total of 40 custom-made, screw-shaped, commerciallypure titanium implants with length of 5mm and an outer diameter of 3.75mm were used in this study. Theywere divided into 4 groups, 10 screws in each, and chemical modification of the titanium implant surfaces were achieved using HF and HCl/H2SO4 dual acid etching. The groups were as follows:
    Group A: 10 implants, left as machined.
    Groups B-D: 30 implants, (10 implants per group) acid-etching surface treatment.
    Group B: 12% hydrofluoric acid (HF)+70% hydrochloric acid/sulfuric acid (HCl/H2SO4).
    Group C: 24% hydrofluoric acid+70% hydrochloric acid/sulfuric acid.
    Group D: 48% hydrofluoric acid+70% hydrochloric acid/sulfuric acid.

    Chemicallytreated implant surfaces were accomplished bysoaking the implants for 120 s in HF at each different volume as well as five more minutes in 70% HCl/H2SO4 at a high temperature (80_C). [20] The first exposure was to hydrofluoric acid and the second was to a combination of hydrochloric acid and sulfuric acid. All implants were cleaned with a steam cleaner as well as an ultrasonic cleaner and then sterilized in an autoclave.

2.3. Implant placement

    Prior to surgery, the legs were shaved, washed and decontaminated with a mixture of Betadine and 70% ethanol. The tibia metaphysics was exposed by incisions through the skin, fascia, and periosteum. The flat surface on the anteromedial aspect of the tibia was selected for implant placement. One implant of each group was inserted in everyrabbit , 2 in each proximal tibia metaphysics. The screws were alternately inserted in the left or right leg and in a distal or proximal position. In addition, each group was subdivided into Sections 1 and 2. Section 1 received implants inserted into the proximal part of the tibia, while Section 2 received implants in the more distal part of the tibia. By intermittent drilling using a low rotarysp eed and profuse saline irrigation, 2 holes were drilled 7mm apart in the central portion of each tibia and sequentiallyenlarged to 3.2 mm. The implants were gently screwed into place, without tapping the sites, until the implant shoulder was level with the bone surface. All implants were allowed to penetrate the first cortical layer only. Every rabbit received 3 implants with acid etched surfaces and 1 implant with a machined surface. After the implants were seated and stable, the fascia and skin were closed in separate layers using resorbable sutures.

2.4. Removal torque measurements

    Twelve weeks post-surgically, 7 rabbits were sacri- ficed, while the other 3 rabbits die of infection. The implant sites were surgicallyexposed via sharp dissection and the bone and soft tissues that had formed on top of the implants were carefullyremove d. Subsequently, the leg was stabilized and the implant was removed under reverse torque rotation with a digital torque gauge (Mark-10 Corporation, USA) (Fig. 1). This machine was used to measure the peak value of resistance to reverse torque rotation, in Ncm.

2.5. Statistical analysis

    Mean values of removal torque were calculated and subjected to the Mann-Whitneytest and Kruskal-Wallis test. A comparison between the machined (group A) and 24%HF+70%HCl/H2SO4 (group C) groups was accomplished with the Mann-Whitneytest . On the other hand, a comparison between the dual acid etched implants was accomplished using the Kruskal-Wallis test. These tests evaluated the differences in removal torque between each group. All statistical testing were carried out at the 5% significance level.

3. Result and discussion

3.1. Removal torque

    Twelve weeks after implant placement, the average removal torque was 12.2 Ncm (A1) and 36.1Ncm (A2) for the machined implants, 18.5Ncm (B1) and 31.6 Ncm (B2) for the 48%HF+HCl/H2SO4 acid etching implants, 25.8Ncm

Fig. 1

Digital torque gauge(Mark-10).

    (C1) and 64.7Ncm (C2) for the 24%HF+HCl/H2SO4 acid etching implants and 27.6Ncm (D1) and 44.1Ncm (D2) for the 12%HF+HCl/H2SO4 acid etching implants. For the Mann-Whiteneytest and Kruskal-Wallis test, the removal torque mean values were of 15.2, 34.3, 34.7 and 38.7Ncm for groups A, B, C and D, respectively. The highest removal torque corresponded to the 24%HF+HCl/H2SO4 acid etching implants, while the lowest was demonstrated bythe machined implants. In the Mann-Whiteneytest, the dual acid etched implants (24%HF+HCl/H2SO4, group C) required a higher average force (34.7 Ncm), than the machined surface implants (group A). This was statisticallysigni ficantly (p ¼ 0:045). However, the data between dual acid etched implants were not statisticallysigni ficant (po0:05). The mean values obtained for maximum removal torque forces for each implant surface are shown in Tables 1 and 2 as well as Figs. 2 and 3.

3.2. Topographic evaluation

    Scanning electron micrographs of the chemically etched and machined surfaces demonstrated microscopic differences in surface topography. Acid etching of the titanium surface created an even distribution of verysmall (1-2 mm) peaks and valleys, while machining of the titanium surface created typical microscopically grooved and relativelysmoo th surface characteristics. In addition, acid etched implant groups appeared rougher than the machined implant groups. Nonetheless, there was no difference in surface topographybetween each acid etched implant groups (Figs. 4-7).

Table 1
Removal torque values in Newton's
Rabbit No.	Machined	48%HF	24%HF	12%HF
	(RP,AI)	(RD,BI)	(LP,CI)	(LD,DII)
1		37.8	12.4	52.9
2	8.8	12.0
3	14.9	34.3	42.4	35.9
4	13.0	42.3	22.5	43.6
Mean values	12.2	31.6	25.8	44.1
Rabbit No.	48%HF	Machined	12%HF	24%HF
	(RP,BI)	(RD,AI)	(LP,DI)	(LD,CII)
5	34.4	62.4	41.4	124.7
6	12.0	15.4	14.3	37.3
7	9.2	30.6	27.0	32.0
Mean values	18.5	36.1	27.6	64.7
RP: proximal part of the right tibia. RD: distal part of the right tibia.
LP: proximal part of the left tibia. LD: distal part of the left tibia.

Table 2
Mean values of the removal torque in each group (UNIT:Ncm)
	Group A	Group B	Group C	Group D
Mean values	15.2	34.3	34.7	38.7
SD	20.1	14.3	20.4	13.6
Group A: 10 implants left as machined
Group B: 12% hydrofluoric acid+70% hydrochloric acid/sulfuric acid.
Group C: 24% hydrofluoric acid+70% hydrochloric acid/sulfuric acid.
Group D: 48% hydrofluoric acid+70% hydrochloric acid/sulfuric acid.

    In the present study, it was found that chemically acid-etched titanium implants achieved greater resistance to reverse torque rotation than machined implant surfaces 3 months post-surgeryin the rabbit tibia. Resistance to reverse torque rotation has been used as a measure of implant anchorage or osseointegration. Greater torque rotation forces required to remove implants maybe interpreted as an increase in the bone-implant contact leading to higher osseointegration strength.

    In vivo experiments investigating bone growth into hollow test chambers lined with acid-etched and machined surface demonstrated that acid-etched surfaces support more and closer bone growth. Several in vivo studies have also reported that osseointegration is better facilitated byrou gh implant surfaces than by machined implants composed of identical material [5,21,22].

Fig. 2 (1)		Fig. 2 (2)
Machined versus 24%HF+70%HCl/H2SO4		Machined versus 24%HF+70%HCl/H2SO4
A1: machined surface, proximal part of the tibia		A: machined surface, proximal part of the tibia
A2: machined surface, distal part of the tibia		C: 24%HF + 70%HCl/H2SO4 treated surface, distal part of the tibia
C1: 24%HF + 70%HCl/H2SO4 treated surface, proximal part of the tibia
C2: 24%HF + 70%HCl/H2SO4 treated surface, distal part of the tibia

Fig. 3

Comparison of removal torque as difference of volume% HF

B1: 12% HF + 70% HCl/ H2SO4, C1: 24 % HF + 70% HCl/ H2SO4 D1: 48% HF + 70% HCl/ H2SO4

B2: 12% HF + 70% HCl/ H2SO4, C2: 24% HF + 70% HCl/ H2SO4, D2: 48% HF + 70% HCl/ H2SO4

B: 12% HF + 70% HCl/ H2SO4, C: 24% HF + 70% HCl/ H2SO4, D: 48% HF + 70% HCl/ H2SO4

Fig. 4		Fig. 5
Machined implant (Group A).		48%HF + HCl/H2SO4 (Group B).
Fig. 6		Fig. 7
24%HF + HCl/H2SO4 Group C).		12%HF + HCl/H2SO4 (Group D).

    In vitro, Bowers et al. also found that surface roughness significantlyincrea ses the degree of cellular attachment of osteoblast-like cells. At the cellular level, the differences between acid-etched and machined surfaces maybe explained bythe microrough topographyof acid-etched surfaces, which could favorably affect angiogenesis as well as cellular migratorybeh avior, cell alignment, orientation, attachment, and finally, cell activityand function [23,9,24]. Wennerburg et al. also reported improvements in bone anchorage of cp titanium and titanium alloyimpl ants when the surfaces were roughened with chemical etching [14].

    Thus, it has been suggested that even minimal changes in material or design of an implant mayha ve a significant impact on the clinical outcome. The mean torque values obtained in this studya re comparable with mean torque values observed for similar healing times in other studies using screw shaped implants in the rabbit [25-27]. Improved bone-implant interface and greater resistance of failure has been reported in studies that tested an acid etched implant surface in combination with other surface treatments. Specifically, screw shaped implants with surfaces that were sandblasted and acid etched (HCl/H2SO4) achieved higher resistance to reverse torque rotation than screw shaped implants with surfaces that were electro-polished, sandblasted and acid pickled (HF/HNO3) or titanium plasma spraycoated [16]. This is in agreement with the present findings.

    In a histomorphometric study, implants with surfaces that were sandblasted with large grit (25-50 mm) and acid etched were found to have a 50-60% mean value of bone-implant contact at 6 weeks while implants that were titanium plasma sprayed had a 30-40% mean value of bone-implant contact for the same time period [16].

    A direct comparison of sandblasted (large grit 25- 50 mm) implant surfaces with those that were sandblasted (25-50 mm) and acid etched (HCl/H2SO4) revealed a significant increase in bone contact for surfaces that were sandblasted and acid etched [16].

    The authors also stated that the increase can most likelybe attributed to the acid treatment with HCl and H2SO4. This demonstration is in agreement with this experiment. In this study, the gradient difference of HF volume% was no statisticallysigni ficant (po0:05), but the difference in the removal torque between the machined implants and acid etched implants was significant (po0:05). Thus these results mayindica te that HF is less effective in acid etching treatment than HCl/H2SO4.

    Increased surface roughness mayenha nce the mechanical interlocking between the macromolecules of the implant surface and the bone, resulting in a greater resistance to compression, tension and shear stress, as some authors have demonstrated [13].

    A rougher surface has been shown to result in a faster as well as stronger bone integration [13]. However, there has been some confusion about what is rough and what is smooth due to a lack of an appropriate measuring technique and evaluation system especially for screwshaped oral implants [21]. In this present study, the acid etched implants yielded higher removal torque values than the machined implants [17,18]. The results are in agreement with those of Klokkevold et al., who reported that chemicallyetched implant surfaces conferred a resistance to reverse torque removal in the rabbit femur that was 4 times greater than that of machined surfaces 2 months post-placement [16]. The greater removal torque values achieved with the acid-etched group may be related primarilyto the higher bone-to-implant contact. Several investigators demonstrated this positive correlation between the degree of bone in contact with the implant and removal torque [25,17].

4. Conclusions

    This studyindica tes that rough acid etched implants achieve greater resistance to reverse torque removal than machined surface implants. In addition, there is less correlation between removal torque and the difference in HF volume%. The greater torque rotation forces required to remove implants maybe interpreted as increases bone-implant contact, leading to higher strengths of osseointegration.

    Therefore, chemicallyacid etching implant surfaces have higher strengths of osseointegration than machined implant surfaces. Further research is needed to study chemical subtraction methods to define the optimal implant surface topologythat enhances osseointegration.

Acknowledgements

    This studywas supported bya grant of the Korea Health 21 R&D Project, Ministryof Health & Welfare, Republic of Korea (00-PJ-PG3-31400-0053).

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