ORIGINAL ARTICLE

Bone response to immediate loading through titanium implantswith different surface roughness in rats

Naoko Sato • Toshie Kuwana • Miou Yamamoto •

Hanako Suenaga • Takahisa Anada •

Shigeto Koyama • Osamu Suzuki • Keiichi Sasaki

Received: 30 August 2012 / Accepted: 28 January 2013 / Published online: 7 April 2013

� The Society of The Nippon Dental University 2013

Abstract Because of its high predictability of success,

implant therapy is a reliable treatment for replacement of

missing teeth. The concept of immediate implant loading

has been widely accepted in terms of early esthetic and

functional recovery. However, there is little biological

evidence to support this concept. The objective of this

study was to examine the interactive effects of mechanical

loading and surface roughness of immediately loaded

titanium implants on bone formation in rats. Screw-shaped

anodized titanium implants were either untreated (smooth)

or acid-etched. Two implants were inserted parallel to each

other in the tibiae of rats, and a closed coil spring (2.0 N)

was immediately applied. Trabecular and cortical bone

around both implants was analyzed using microtomo-

graphic images, and a removal torque test was performed at

weeks 1, 2, and 4. Immediate loading of acid-etched

implants resulted in significant decreases in bone mineral

density, contact surface area, and cortical bone thickness.

These effects were not observed after immediate loading of

smooth implants. Conversely, loading did not influence

acid-etched implant fixation; however, smooth implant

fixation at week 1 was significantly reduced. These results

imply that surface roughness regulates bone response to

mechanical stress and that immediate loading might not

inhibit osseointegration for smooth and rough implants in

the late healing stages.

Keywords Immediate loading � Osseointegration �Bone reaction � Surface roughness � Rats

Introduction

Owing to improved surgical techniques and development

of macro- and micro-implant designs, implant therapy is a

reliable treatment for replacement of missing teeth. The

notion that a 3- to 6-month healing period is requisite for

successful osseointegration has been used as a general

standard based on the initial clinical experience of Brane-

mark et al. [1, 2] in 1977. The concept of immediate

implant loading has become an option for common treat-

ments because it provides many benefits for patients,

including decreased patient anxiety and discomfort and

early recovery of esthetics and masticatory function.

Despite this, in vivo and clinical studies in this field are

sometimes confusing and contradictory, and the underlying

biological evidence is still unknown.

Surface roughness of implant fixtures influences bone

formation around implants. Rough titanium (Ti) surfaces

enhance osteoblast differentiation, with increased alkaline

phosphatase activity and upregulation of bone-related

genes [3–5]. The rate and degree of bone–implant contact,

bone volume, mechanical properties, and primary stability

are increased by rough implants compared with machined-

surface implants in vivo [6–10].

Osteoblasts are known to respond to mechanical

stress by increasing the production of matrix proteins,

prostaglandin E2, nitric oxide, intracellular calcium, and

N. Sato (&) � S. Koyama

Tohoku University Hospital, Maxillofacial Prosthetics Clinic,

4-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan

e-mail: kotani-n@dent.tohoku.ac.jp

T. Kuwana � M. Yamamoto � H. Suenaga � K. Sasaki

Division of Advanced Prosthodontics, Tohoku University

Graduate School of Dentistry, Sendai, Japan

T. Anada � O. Suzuki

Division of Craniofacial Function Engineering,

Tohoku University Graduate School of Dentistry,

Sendai, Japan

123

Odontology (2014) 102:249–258

DOI 10.1007/s10266-013-0107-4

cyclooxygenase-2 (COX-2) [11]. A large number of studies

have reported that mechanical stress influences bone

modeling and remodeling. The responsiveness of bone

tissue has been shown to vary based on (1) the nature of the

mechanical loading, including stress amplitude, frequency,

and total load repetitions [12–14], and (2) the mode of

mechanical stress, including fluid shear, stretching, and

compression stress [15]. Appropriate mechanical stimuli

therefore increases bone density, volume, and mechanical

properties, while excessive stress can have negative effects

on bone formation [16, 17]. Vandamme et al. [18] reported

that micro-motion of up to 90 lm in implant displacement

increased bone formation around immediately loaded

implants with a rough surface in rabbits. De Smet et al.

examined the effect of a sinusoidally varying bending

moment on peri-implant bone in guinea pigs using a force-

controlled electromechanical shaker. They showed that a

strain rate amplitude of 1.620 microstrain s-1 stimulated

optimal osteogenesis in cortical bone around acid-etched

implants [19]. Isidor [20, 21] reported that implant mobility

was caused by progressive peri-implant bone loss after

occlusal overload for 18 months in monkeys.

Mechanical stress transmitted through Ti surface mic-

rotopography potentially alters the local mechanical envi-

ronment [22, 23]. In addition, Ti surface roughness

influences focal adhesive components, which are thought to

play critical roles in stress transmission to bone cells [24,

25]. Although the individual effects of mechanical stress

and Ti surface roughness on bone tissue response have

been widely studied, there is little information about their

interaction [26].

We hypothesized that the degree of Ti surface roughness

influences osseointegration and marginal bone formation

around immediately loaded implants. The objective of this

study was to examine the interactive effect of mechanical

loading and Ti surface roughness on bone formation around

immediately loaded implants.

Materials and methods

The guidelines for animal use (NIH Animal Research

Advisory Committee, 1995) and specific national laws

were followed. The preparation of animals was performed

according to animal protocols approved by Tohoku

University.

Implants

Screw-shaped Ti implants (Orthoanchor; Dentsply-Sankin,

Tokyo, Japan), 1.2 mm in diameter and 9.25 mm in length,

were used in this study. Implant surfaces were anodized in

accordance with the manufacturer’s instructions to prevent

oral discoloration. Implants were left untreated (Smooth,

Sm) or were acid-etched (AE). The AE surface was created

by immersion in 67 % (w/w) sulfuric acid (H2SO4) at

120 �C for 75 s, washing with deionized water, and storage

in a dark room for 3 weeks. All implants were autoclave-

sterilized prior to surgery. There are few studies on how

implant surface roughness and loading affect peri-implant

bone formation [26, 27]. In this study, the roughness of AE

implants was determined with reference to a previous study

[26], in which an Ra value of 2.75 lm was chosen for the

roughened surface.

Low- and high-magnification images using scanning

electron microscopy (XL30; Philips, Eindhoven, The

Netherlands) showed that the Sm surfaces were relatively

smooth with machining grooves and that the AE surfaces

had uniform 0.5 to 1.0 lm grooves surrounded by sharp

peaks and troughs (Fig. 1). Using a noncontact, three-

dimensional (3D) measuring device (NH-3; Mitaka Kohki,

Tokyo, Japan), the average roughness (Ra), root mean

square roughness (Rq), and average maximum height of the

profile (Rz) were measured. Ra = 0.341 ± 0.08 lm,

Rq = 0.462 ± 0.1357 lm, and Rz = 1.884 ± 0.699 lm

for the Sm implants, and Ra = 2.223 ± 0.264 lm,

Rq = 2.776 ± 0.326 lm, and Rz = 9.742 ± 1.497 for the

AE implants.

Animals and surgical procedure

Sixty 12-week-old male Wistar rats received Ti implants.

The surgery was performed under intraperitoneal anesthe-

sia (sodium pentobarbital, 50 mg/kg) and aseptic condi-

tions. A full-thickness flap was made on the medial side of

the right tibia, and implant holes were created using a

1 mm surgical drill at a speed of [1000 rpm with irriga-

tion. Implants were inserted with low rotational speed

(300 rpm) until the implant heads were exposed to about

5 mm. Each rat received two implants: a test implant (Sm

or AE implant) placed 5 mm distal to the growth line of the

knee joint, and an anchor implant (Sm implant) placed

13 mm distal to the test implant (Fig. 2). The flaps were

closed with resorbable sutures, leaving the implant heads

protruding.

Immediate loading with coil springs

Nickel-Ti alloy closed coil springs (Sentalloy; Tomy

International, Okuma, Japan) were attached to the implant

heads immediately after implant insertion. Coil springs

10 mm in length (3 mm tension coil and 7 mm hook

attachment) provided a continuously compressed load of

2.0 N within the effective length (10–22 mm). In this

study, the unloaded group (without spring) and loaded

group (with spring) with the Sm and AE implants were

250 Odontology (2014) 102:249–258

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examined. There were no observed inflammatory reactions

at the implant sites and no movement of implants during

the experimental period.

Specimen preparation and micro-CT evaluation

At 1, 2, and 4 weeks after implant insertion, rats in the

loaded and unloaded groups (n = 5/group) were killed and

the tibias were harvested. Implant-tibia blocks were created

by sharp dissection of the implant sites. All specimens were

scanned with an X-ray micro-CT system (ScanXmate-

E090; Comscantecno, Yokohama, Japan) with a tube

voltage of 90 kV and tube current of 0.1 mA. After scan-

ning, the TRI/3D-BON system (Ratoc System Engineering,

Tokyo, Japan) was used to build 3D models from serial

tomographic datasets for observation and morphometric

analysis. The 3D images were segmented into voxels

(isotropic voxel size of 15.3 lm) identified as bone and

implant (Fig. 3). The gray-scale images were segmented

using a strict filter to remove noise and a fixed threshold to

extract the mineralized bone phase. The following param-

eters were examined:

1. Bone mineral density of trabecular bone around the

implant (t-BMD).

2. Trabecular bone surrounding the implant was seg-

mented into three zones (circumferential zone, within

150 lm of the implant surface; middle zone, from 150

to 300 lm; and far zone, from 300 to 450 lm). The

t-BMD in the three zones was analyzed.

3. Bone mineral density of cortical bone around the

implant (co-BMD).

4. Cortical bone 15.3 lm from the implant surface was

analyzed. We focused on cortical bone on the side of

Fig. 1 Scanning electron

micrograms of smooth and acid-

etched titanium implant

surfaces. Bars = 100 lm

(upper), 2 lm (middle), and

0.5 lm (lower). Average

roughness (Ra) is shown for

both implants

Fig. 2 Right loaded rat tibia. Photographs show two implants inserted perpendicular to the long axis of the tibia at an interval of 13 mm with a

coil spring

Odontology (2014) 102:249–258 251

123

the implant head in contact with the loaded implants

because the marginal bone profiles around the implant

heads are critical for esthetics.

5. Contact surface area between cortical bone and the

implant (co-area)

6. The reconstructed 3D images of the implant and

superficial cortical bone of the implant were extracted.

The implant and cortical bone were identified based on

each CT number. The surface area of the cortical bone,

which overlaps the surface area of the implant, was

calculated as the co-area.

7. Thickness of cortical bone in contact with the implant

(co-thickness).

8. Two-dimensional reconstructed images along the long

axis of the bone (sagittal image) were used for

analysis. The central axis of the implants divided the

cortical regions into the region in the direction of the

load (compression side) and the region in the opposite

direction (tensile side). In these regions, the co-

thickness was measured (Fig. 4).

Biomechanical testing of the implant–bone interface

(stability measurement)

Biomechanical testing of the implant–bone interface was

carried out using the removal torque test. Implant-tibia

blocks were perpendicularly fixed with a jig. The implant

driver with a torque gauge (AHV-11A; Tohnichi, Tokyo,

Japan) was attached to the implant head. The peak loosening

torque (Ncm) was then recorded. Implants were carefully

removed to preserve integrity of the bone structure.

Statistical analysis

After tests for normality and equality of variance, statistical

analysis of the data was conducted using two-way analysis

of variance (ANOVA). When necessary, the post hoc

Bonferroni test was used as a multiple comparison test. A

p value of \0.05 was considered statistically significant.

Results

Bone mineral density of trabecular bone (t-BMD)

around implants

After immediate loading, no significant differences were

observed in t-BMD around both the Sm and AE implants in

all three zones (Fig. 5). The t-BMD in the middle and far

zones of unloaded AE implants was significantly increased

at week 2 and maintained at week 4. However, there were

no significant differences over time in t-BMD for Sm

implants, regardless of load application.

Fig. 3 Left, three-dimensional

reconstructed image of the tibia

with implant. Right, two-

dimensional reconstructed

image of the tibia with implant

(transverse image)

Fig. 4 Schematic views of cortical bone with loaded implant. The

thicknesses of cortical bone in contact with the implant on the

compression and tension sides (co-thickness) were measured using

two-dimensional reconstructed images along the long axis of the bone

(sagittal image)

252 Odontology (2014) 102:249–258

123

Bone mineral density of cortical bone (co-BMD)

around implants

Immediate loading significantly decreased co-BMD around

AE implants at week 4 (p \ 0.01, Bonferroni); no effect on

Sm implants was observed (Fig. 6). The co-BMD of

unloaded AE implants at week 4 was significantly higher

than that at weeks 1 and 2. Conversely, there were no

significant changes in co-BMD around Sm implants with

and without loading at all time points.

Fig. 5 Bone mineral density of

trabecular bone around smooth

(Sm) and acid-etched (AE)

implants following loading at

weeks 1, 2, and 4. Three zones

(within 150 lm of the implant

surface, from 150 to 300 lm,

and from 300 to 450 lm) were

analyzed. Data are shown as the

mean ± SD (n = 5). *p \ 0.05,

**p \ 0.01

Odontology (2014) 102:249–258 253

123

Contact surface area between cortical bone

and the implant (co-area)

The co-area for AE implants was significantly decreased by

load application at week 2 (p \ 0.05, Bonferroni) (Fig. 7).

For Sm implants, there were no significant effects of

loading on co-area at all time points.

Thickness of cortical bone in contact with loaded

implants (co-thickness)

The co-thickness was significantly increased on the com-

pression and tension sides of unloaded Sm and AE

implants and on the tension side of loaded Sm implants at

week 2 compared with week 1. The co-thickness was sig-

nificantly lower on the tension side in loaded AE implants

compared with unloaded AE implants at week 2 (p \ 0.05,

Bonferroni). There were no significant differences in the

co-thickness between the compression side and tension side

at all time points (Table 1).

Biomechanical strength of the implant–bone interface

Immediate loading significantly decreased the biome-

chanical strength of the implant–bone interface for Sm

implants at week 1 (p \ 0.01, Bonferroni) (Fig. 8). There

were no significant differences in reverse torque values

between loaded and unloaded AE implants at all time

points. For each group, the biomechanical strength at

weeks 2 and 4 was significantly higher than that at week 1.

Discussion

The present study demonstrated that immediate loading did

not influence AE implant biomechanical strength. How-

ever, immediate loading did result in negative effects on

cortical bone around the AE implant, including decreased

co-BMD at week 4, co-area at week 2, and co-thickness on

the compression side at week 2. Conversely, cortical bone

around the Sm implant was unaffected by loading, whereas

Fig. 6 Bone mineral density of

cortical bone around smooth

and acid-etched implants under

mechanical stress at weeks 1, 2,

and 4. Data are shown as the

mean ± SD (n = 5). *p \ 0.05,

**p \ 0.01

Fig. 7 Contact surface area

between cortical bone and

smooth (Sm) and acid-etched

(AE) implants following loading

at weeks 1, 2, and 4. Data are

shown as the mean ± SD

(n = 5). *p \ 0.05, **p \ 0.01

254 Odontology (2014) 102:249–258

123

the biomechanical strength of Sm implants at week 1 was

significantly reduced by load application. These results

provide insight that surface roughness regulates bone

response to mechanical stress.

According to the proposal of Frost’s minimum effective

strains, when bone tissue is subjected to loads that cause

intraosseous peak strains in the 100–1500 le,

1500–3500 le, and[3500 le ranges, bone mass will show

no change, an increase, and a decrease, respectively [16,

28, 29]. In the present study, immediate loading failed to

improve the profile of trabecular and cortical bone around

both implants compared with unloaded implants. This

suggests that the applied mechanical stress might not have

been of the optimal magnitude to accelerate bone forma-

tion. Furthermore, negative effects of immediate loading

on cortical bone around AE implants were observed,

including decreased co-BMD at week 4, co-area at week 2,

and co-thickness on the compression side at week 2. Other

studies have reported the existence of an appropriate strain

range for triggering bone cells to form bone and that the

strain generated through roughened surfaces more fre-

quently reaches the trigger level than that generated

through smooth surfaces [23]. The increased surface area

of the AE implant compared with the Sm implant could

have amplified the stress transmitted to adjacent bone tis-

sue, which might account for our results.

Table 1 Thickness of cortical bone in contact with smooth (Sm) and acid-etched (AE) implants under loading on the compression and tension

sides at weeks 1, 2, and 4

The Compression side The tension side

Sm implant

1W Unload 235 ± 87 342 ± 149

Load 351 ± 51 301 ± 140

2W Unload 686 ± 55 669 ± 102

Load 430 ± 131 668 ± 132

4W Unload 420 ± 162 523 ± 194

Load 420 ± 118 565 ± 86

AE implant

1W Unload 425 ± 218 455 ± 91

Load 379 ± 49 378 ± 75

2W Unload 744 ± 51 806 ± 142

Load 539 ± 91 671 ± 268

4W Unload 668 ± 96 476 ± 225

Load 573 ± 66 483 ± 103

Data are shown as the mean ± SD (n = 5). * p \ 0.05, ** p \ 0.01

Fig. 8 Biomechanical strength

of the implant–bone interface

for smooth (Sm) and acid-etched

(AE) implants following

mechanical stress at weeks 1, 2,

and 4 measured by the removal

torque test. Data are shown as

the mean ± SD (n = 5).

*p \ 0.05, **p \ 0.01

Odontology (2014) 102:249–258 255

123

There are some negative aspects of micro-CT evalua-

tion. The effect of artifacts produced by metals similar to

Ti should not be ignored. Metal artifacts greatly affect

micro-CT images in terms of halation. In this study, to

reduce this influence on the CT number of peri-implant

bone as much as possible, the inclination of the implants

was standardized in CT imaging; thus, the data compari-

sons were performed under the same conditions. Although

quantitative evaluation of new bone alone is difficult, it is

important to observe biological events (i.e., bone resorption

and new bone formation) in the healing phase. Because the

CT number is calculated by the X-ray absorption coeffi-

cient, new bone and preexisting bone were identified as

cortical bone based on the level of mineralization. Thus,

data based on micro-CT alone is insufficient for compre-

hensive understanding of peri-implant bone reactions in

healing. Further histological investigations are required to

observe biological events (i.e., bone resorption and new

bone formation) in peri-implant bone under loading

conditions.

The mechanism by which bone cells on Ti detect

mechanical stress and process the information is largely

unknown [30]. It has been proposed that application of

stress drives conformational changes in cell membranes,

focal adhesion complexes [e.g., extracellular matrix (ECM)

and integrins], cell–cell adhesions (e.g., cadherin), and gap

junctions, which induce cytoskeletal rearrangement and

signal transduction for proliferation and differentiation [31,

32]. ECM connections to the cytoskeleton may act as

amplifiers of mechanical signals [33]. In other studies,

bone cell growth on rough surfaces resulted in upregulation

of integrins, enhanced production of ECM proteins such as

fibronectin, and altered focal contacts compared with bone

cell growth on machined surfaces [34–36]. Moreover,

Simmons et al. [22] demonstrated that implant surface

topography modulated a local mechanical environment that

affected bone formation as determined using finite element

analysis. Taken together, these findings show that it is

reasonable to assume that modified focal adhesion com-

plexes on rough Ti surfaces and substrate features such as

increased surface area and 3D configurations account for

differences in responsiveness of bone tissue around Sm and

AE implant surfaces to stress conditions.

Generally, bone adaptation to mechanical stress is

observed as bone remodeling. Improvement of bone

mechanical properties due to receiving optimal mechanical

stress is attributed to tiny structural changes in bone [37].

Our results demonstrate that loading deteriorates implant

fixation of Sm implants and cortical bone profiles of AE

implants at early time points. This suggests that the

mechanical stress applied in this study may not have

exceeded the physiological threshold of bone adaptation,

allowing bone to adapt to the stress. Previous studies

reported that completion of osseointegration in the rat tibia

occurs 4–6 weeks after implantation based on nuclear

medicine, histological, morphologic, and radioautographic

examinations [38–42]. In the present study, it is unknown

how a decrease in co-BMD of loaded AE implants at week

4 could subsequently change. Further studies are needed to

observe the bone reaction around loaded implants beyond

4 weeks.

It is well known that implant surface roughness influ-

ences the biomechanical quality of osseointegrated bone

and that Ti implants with rough surfaces induce more

pronounced osseointegration than do Ti implants with

smooth surfaces. Ogawa et al. [10] investigated the bio-

mechanical strength of the cylindrical implant–bone

interface for acid-etched surfaces and turned surfaces in

rats using the implant push-in test. The acid-etched

implants showed significantly higher push-in test values

than did the turned implants throughout the experimental

period. Butz et al. examined the hardness and elastic

modulus of the integrated bone around acid-etched surfaces

and turned surfaces in rats using nano-indentation tech-

niques [7]. The data showed that the bone integrated to the

acid-etched surface was harder and stiffer than the bone

integrated to the machined surface. They concluded that

implant surface roughness affects the biomechanical qual-

ity of osseointegrated bone. In the present study, to clarify

the bone response to immediate loading through each Sm

and AE Ti implant surface, statistical comparison was

performed between loaded and unloaded groups of each

Sm and AE implant. Despite no statistical comparison, the

biomechanical strength of the implant–bone interface for

the AE implants seemed to be higher than that for the Sm

implants in unloaded and loaded conditions. These data are

consistent with the results of the study by Ogawa et al.

[10]. Interestingly, the mechanical strength of the AE

implant–bone interface was not affected by loading, while

cortical bone profiles were observed to deteriorate. This

conflict may have some possible explanations: (1) other

undetected factors in the cortical bone area (e.g., immature

bone) might be related to implant–bone fixation according

to micro-CT evaluation. (2) The increased surface area of

AE implants is assumed to increase the removal torque

resistance of the implants and improve the biomechanical

quality of osseointegrated bone. This might counteract the

loading influence of the deteriorated cortical bone profile

on osseointegration of AE implants. Because of the limited

data from this study, further experiments are necessary.

Positive and negative bone responses to mechanical

loading have been reported. Bone chamber models in

rabbits have been used to examine de novo bone formation

and tissue differentiation under loading conditions [18, 43,

44]. In a series of studies, Vandamme et al. histologically

analyzed the effects of implant surface roughness on peri-

256 Odontology (2014) 102:249–258

123

implant bone formation in unloaded and loaded conditions:

30 lm displacement loading for 400 cycles at 1 Hz, 3

times a week for 9 weeks in rabbits [26]. The data showed

no significant differences in the incidence of osseointe-

gration between turned implants and roughened implants

under loaded conditions. Melsen et al. [45] demonstrated

that orthodontic forces increased cortical bone turnover and

alveolar bone density. The tissue turnover characteristics

were not affected by the magnitude of the loading force

between 100 and 300 cN. Gotfredsen et al. [46–48] showed

that implants subjected to a static lateral expansion load

increased bone density and mineralized bone–implant

contact compared with control implants. Hoshaw examined

the hypothesis that mechanical loading of implants and the

consequent stress and strain fields influence bone modeling

and remodeling at the bone–implant interface in dogs [17].

They demonstrated a decreased percentage of mineralized

bone tissue around implants following axial loading with a

triangular waveform (300 N maximum, 10 N minimum,

330 N/s for 500 cycles per day for 5 consecutive days) for

12 weeks. The results support the premise that bone loss

observed around the neck of loaded implants at 12 weeks

postloading was a consequence of bone modeling and

remodeling secondary to bone microdamage caused by the

loading protocol. Thus, bone reactions to mechanical

loading depend on the loading condition: (1) the nature of

the mechanical loading, including stress amplitude, fre-

quency, and total load repetitions [12–14], and (2) the

mode of mechanical stress, including fluid shear, stretch-

ing, and compression stress [15]. Therefore, a direct

comparison of data is difficult because of different exper-

imental designs.

The present study focused on the cortical bone profiles for

the following reasons: (1) Most studies using finite element

analysis demonstrated high stress concentration in cortical

bone when implants were loaded [49, 50]. (2) Other studies

have reported limited contact with trabecular bone and main

contact with cortical bone 4 weeks after loading in guinea

pig tibiae [19]. Because the load was applied in one direc-

tion, the thickness of cortical bone (co-thickness) was

examined on the compression and tension sides. Although

the co-thickness for loaded AE implants was significantly

lower than that for unloaded AE implants at week 2, there

were no significant differences in the co-thickness between

on the compression and tension sides in the presence of

loading. These results might be attributed to the fact that

loading also tended to decrease the co-thickness on the

tension side at week 2 despite no statistical differences.

A large number of previous studies have investigated

mechanical loading in vivo. However, surface microto-

pography likely alters the local mechanical environment

[22, 23] and focal adhesive components, which are thought

to play critical roles in stress transmission to cells [32].

Therefore, we should discriminate bone behaviors acti-

vated by stress through Ti from bone behaviors affected by

mechanical stress alone. Nevertheless, few studies have

considered the interactive effects of surface roughness and

mechanical factors on bone reactions [26, 27, 44]; there is

no evidence of an association between surface roughness

and bone response under loading conditions. The results of

the present study imply that surface roughness regulates

cortical bone reactions to mechanical stress and that

immediate loading might fail to deteriorate osseointegra-

tion for both implants at late healing time points. Further

studies of different loading protocols will help to eluci-

date the regulation effects of stress transmitted through

Ti implants with different surface roughness on bone

formation.

Acknowledgments This work was supported by a Grant-in-Aid for

Scientific Research (grant no. 21791876) from the Ministry of Edu-

cation, Culture, Sports, Science and Technology of Japan.

Conflict of Interest None of the authors have any conflicts of

interest associated with this study.

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