effects of ipriflavone on augmented bone using a guided bone regeneration procedure

Effects of ipriflavone on augmentedbone using a guided bone regenerationprocedure

Koichi ItoTadashi MinegishiTadahiro TakayamaTakanori TamuraYutaka YamadaShuichi Sato

Authors’ affiliations:Koichi Ito, Tadashi Minegishi, Tadahiro Takayama,Takanori Tamura, Yutaka Yamada, Shuichi Sato,Department of Periodontology, Nihon UniversitySchool of Dentistry, Tokyo, JapanKoichi Ito, Yutaka Yamada, Shuichi Sato, Divisionof Advanced Dental Treatment, Dental ResearchCenter, Nihon University School of Dentistry,Tokyo, Japan

Correspondence to:Koichi ItoDepartment of PeriodontologyDivision of Advanced Dental TreatmentDental Research CenterNihon University School of Dentistry1-8-13 Kanda-SurugadaiChiyoda-ku, Tokyo 101-8310JapanTel.: þ81 3 3219 8097Fax:þ 81 3 3219 8394e-mail: [email protected]

Key words: bone augmentation, calvarial bone, GBR, ipriflavone, rabbit

Abstract: This study investigated the effects of ipriflavone (IP) on augmented bone using a

guided bone regeneration (GBR) procedure. In 15 rabbits, two titanium caps were placed

into calvarial bone for GBR. The animals were divided into three groups: the No-IP (no

intake of IP), Post-IP (IP orally, 10 mg/kg/day after GBR), and Pre-IP (IP intake beginning

before GBR) groups. One cap was removed from each rabbit after 3 months, and the

remaining site was a control. One month after one cap removal, all the animals were

euthanized, and histologic and histomorphometric analyses were performed. In all of the

groups, the newly generated tissue was of varying size, and it consisted of thin pieces of

mineralized bone and large marrow spaces with fat cells and some hematopoietic cells. In

all of the control sites, the newly generated tissue was noted and almost filled the space

under the cap. There was a significant difference between groups No-IP and Pre-IP

(93.8 � 4.6% vs. 98.5 � 0.8%, Po0.05). The tissue generated at the test sites in all of the

groups was resorbed, and its original shape and volume were not maintained 1 month after

one cap removal. In particular, the greatest percentage, approximately 20% of the newly

generated tissue, was resorbed in the No-IP group (93.8 � 4.6% vs. 73.9 � 3.7%, Po0.05),

and approximately 11% and 15% in groups Post-IP and Pre-IP, respectively. The relative

amount of mineralized bone generated at the control and test sites was significantly larger

in groups Post-IP and Pre-IP when compared with group No-IP, except for the test site

between groups No-IP and Post-IP (Po0.05). Therefore, the amount of mineralized tissue

generated appeared to increase with an increase in the total IP dose. Within the limitations

of this rabbit experimental model, we conclude that the daily intake of IP before or after

GBR inhibits the resorption of augmented tissue and would be useful for improving the

quality of newly generated bone beyond the skeletal envelope.

The principle of guided tissue regeneration

(GTR) was originally developed and suc-

cessfully applied for the treatment of

periodontal defects (Karring et al. 1993).

Subsequently, the same principle, often

called guided bone regeneration (GBR) or

guided bone augmentation (GBA), has been

applied to the treatment of various bone

defects and to increasing the volume of

atrophic alveolar ridges with great success,

allowing the placement of dental implants

(Hammerle & Karring 1998). It is possible

to produce considerable amounts of bone in

areas where bone did not previously exist

(Kostopoulos et al. 1994; Kostopoulos &

Karring 1994; Schmid et al. 1994), and this

de novo bone formation is stable on a long-

term basis (Lioubavina et al. 1999; Donos

et al. 2005). However, clinical observations

have suggested that the extent of the initial

augmentation created using GBR does not

quantitatively equal the actual amount ofCopyright r Blackwell Munksgaard 2006

Date:Accepted 30 January 2006

To cite this article:Ito K, Minegishi T, Takayama T, Tamura T, Yamada Y,Sato S. Effects of ipriflavone on augmented bone using aguided bone regeneration procedure.Clin. Oral Impl. Res. 18, 2007; 60–68doi: 10.1111/j.1600-0501.2006.01284.x

60

bone remaining after healing. A review of

the literature disclosed several studies of

GBR that documented a loss of alveolar

height and width during healing (Simion

et al. 1994; Lekovic et al. 1997, 1998; Zitz-

mann et al. 2001). Thus, controversy exists in

the results of animal and clinical studies.

Ipriflavone (7-isopropoxyisoflavone) (IP),

which is derived from the soy isoflavone

daidzein, has important effects on bone

metabolism. IP is effective in reducing the

bone turnover rate, mainly by inhibiting

bone resorption (Bonucci et al. 1992;

Notoya et al. 1993; Albanese et al. 1994),

but also by stimulating bone formation

(Benvenuti et al. 1991; Cheng et al.

1994). Therefore, IP appears to have several

mechanisms of action, all of which en-

hance bone density; it possesses bone-

forming properties and is considered anti-

resorptive. Studies in humans also suggest

that IP prevents bone loss (Agnusdei et al.

1989; Adami et al. 1997; Gennari et al.

1998; Ohta et al. 1999) and increases bone

mass in postmenopausal women (Passeri

et al. 1992; Moscarini et al. 1994; Agnus-

dei et al. 1997).

The effect of IP on bone formation in

vivo was studied in rat peri-alveolar bone

after surgically producing a hole in the

mandible; the results were consistent

with a role of IP in stimulating osteogen-

esis and suggest that IP is a potential

therapeutic tool for promoting the repair

of injured peri-alveolar bone (Martini et al.

1998). Furthermore, a study of the direct

effects of IP on bone augmentation in rabbit

calvarial bone (Minegishi et al. 2002) sug-

gested that IP affects the quality of bone

augmentation at an early stage.

However, there are insufficient histo-

morphometric data on the in vivo effect of

IP administration on the augmented bone

formation beyond the skeletal envelope. In

addition, it is not clear whether IP prevents

the resorption of augmented tissue and

promotes mineralized bone in tissue newly

generated using GBR. Therefore, this study

investigated the potential pharmacological

effect of IP on augmented bone using GBR.

Material and methods

Animals and surgical procedure

Forty adult male Japanese white rabbits,

weighing from 2.4 to 3.1 kg, were tested

in order to choose suitable animals for

the oral administration of IP. After a

2-week acclimation test, 15 rabbits were

selected for surgery. This study was ap-

proved by the Animal Experimentation

Committee at Nihon University School

of Dentistry.

IP (Takeda Chemical Industries Ltd.

Osaka, Japan) was prepared at 10 mg/ml

using 1% hydroxypropyl cellulose solu-

tion. The animals were randomly assigned

to one of three groups: the No-IP (no intake

of IP), Post-IP (10 mg/kg IP daily orally

after GBR), and Pre-IP (IP daily orally

beginning 1 month before GBR) groups.

General anesthesia was induced by inhala-

tion of halothane gas (1.5–2%, vol%)

(Fluothanes

, Takeda Chemical Industries)

and by injection of pentobarbital sodium

(0.4 ml/kg) (Nembutals

, Abbot Labora-

tories, North Chicago, IL, USA) via an

ear vein. In addition, approximately 1 ml

of local anesthesia with lidocaine HCl

containing epinephrine 1 : 80,000 (2% Xy-

locains

, Astra Japan, Fujisawa Pharmaceu-

tical Co., Osaka, Japan) was used to reduce

hemorrhaging. After the forehead of the

rabbit was shaved, a cutaneous flap was

created and lifted to expose the calvarial

bone on both sides of the midline.

The experimental device was a custom-

made, standardized stiff titanium hemi-

spherical cap (Ti 499.5%; JIS H6400,

Sankin, Tokyo, Japan) with a smooth sur-

face. The cap was 4 mm high, 8 mm in

diameter, and 0.2 mm thick. A circular

groove with an inner diameter of 8 mm

was prepared in the bone on each side of

the midline with a trephine drill (Bone

trephine 131001 Technica, Tokyo, Japan).

Then, nine small holes were drilled with a

No. 2 round bur to induce bleeding from

the marrow space. A standardized titanium

cap was placed in the circular groove using

press-fit.

The skin tissue of each reflected flap was

repositioned to cover the titanium cap and

sutured. Postoperatively, the animals re-

ceived antibiotic therapy (2,500,000 IU/ml

penicillin G, Yamanouchi, Tokyo, Japan)

at a dose of 0.1 ml/kg, given as a single

intramuscular injection. One cap in each

rabbit was removed after 3 months, under

general anesthesia as described above. This

site was defined as the test site, and the site

with the remaining cap was the control site

(Fig. 1a–c).

Specimen preparation

One month after one cap removal, all the

animals were euthanized with an overdose

of pentobarbital. The calvarial bone was

dissected, fixed in 10% neutral-buffered

formalin, dehydrated, and embedded in

polyester resin (Rigolac-2004, Rigolac-

70F, Nisshin EM, Tokyo, Japan). One

sagittal undecalcified ground section (ap-

proximately 200mm thick) from the mid-

portion was prepared using a low-speed

diamond saw (Micro cutter, MC-201,

Maruto, Tokyo, Japan).

Histological and histomorphometricanalyses

Histological examination, photography,

and histomorphometric assessment of the

sections were performed as reported else-

where (Yamada et al. 2003). The histo-

morphometric data obtained from each

specimen were recorded with a computer-

ized image analysis system (Adobe Photo-

shops

, 4.01 J, Adobe Systems, Tokyo,

Japan). Slides taken at � 3 magnification

were digitized with a solid state, 35 mm

slide scanner and a CCD linear photo diode

array interfaced with a computer.

The percent area of newly generated

tissue that consisted of mineralized bone

and marrow space in each histological sec-

tion of the control site was calculated

relative to the area bounded by the hemi-

spherical shape of the titanium cap and the

host bone; this latter volume was taken as

100%. In addition, we determined the

cross-sectional area of the generated miner-

alized bone expressed as a percentage of the

total tissue area generated within each

space. At the test site, where one cap was

removed, a hypothetical cap (the semicir-

cular line) with the same shape and size as

the original cap was created in histological

sections using computer generation.

Statistical analysis

All the data are presented as means and

standard deviation. The data were sub-

jected to statistical analysis using the

Mann–Whitney test following the Krus-

kal–Wallis test (intergroup comparison),

and with the Wilcoxon test (intragroup

comparison). Differences at Po0.05 were

considered significant. The statistical pro-

gram SPSSs

Base 10.0J (SPSS Japan,

Tokyo, Japan) was used for all analyses.

Ito et al . Effects of ipriflavone on augmented bone

61 | Clin. Oral Impl. Res. 18, 2007 / 60–68

Results

The reproducibility of the measurements

by one examiner (T. M.) was tested for

sections from each control and test site in

each group. The measurements were re-

corded twice for the same sections on two

different days, 2 weeks apart. Paired differ-

ences were calculated and tested for nor-

mality using the Kolmogorov–Smirnoff

goodness-of-fit test. The differences be-

tween the first and second recording were

found not to have a normal distribution.

The results of the two recordings were then

analyzed statistically using the Wilcoxon

test for paired observations. No statistically

significant differences were found between

the two recordings.

Histological observations

Healing was uneventful at all of the surgi-

cal sites in all of the rabbits, except that one

animal in the Post-IP group died before the

end of the study for unknown reasons.

Consequently, there were five specimens

for each control and test site in groups No-

IP and Pre-IP, and four specimens for each

site in group Post-IP. The percentage of

newly generated tissue and mineralized

bone in each section was determined in

the 28 specimens. Upon specimen retrieval

and while dissecting and removing the

cutaneous layer overlaying the caps, no

signs of inflammation, pathological pro-

cesses, or adverse reactions were noted at

the surgical sites. In the control groups, the

titanium caps were placed in the host bone

tightly.

The percentage of newly generated tissue

and mineralized bone in each section was

determined in the 28 specimens. In all of

the groups, the newly generated tissue was

of varying size, and it consisted of thin

pieces of mineralized bone and large mar-

row spaces with fat cells and some hema-

topoietic cells underneath the titanium cap

or hypothetical cap. No connective tissues

were present under the cap. The generated

mineralized bone layer tended to climb

along the inner wall of the cap in close

contact with the titanium surface and cov-

ered the generated tissues (Fig. 2a–f). Cu-

boidal osteoblast-like cells were generally

found to be actively laying down bone to

varying degrees along the mineralized bone

in all of the groups. More obvious findings

of osteoblast-like cells were often found in

the Post-IP and Pre-IP groups (Fig. 3a–c).

Histomorphometric evaluation

Using the Kruskal–Wallis test, there was a

significant (Po0.05) difference in the


bone at the control and test sites among

all groups, except for the test site in the

newly generated tissue. At all of the control

sites, newly generated tissue was noted,

which almost filled the space under the cap.

There was a significant difference in the

amount of generated tissue between groups

No-IP and Pre-IP (93.8� 4.6% vs.

Fig. 1. (a) A cutaneous flap was lifted and the calvarial bone was exposed. A slit was made and nine holes were

put in the host bone. (b) The titanium cap was placed in the bone. (c) One cap was removed after 3 months.

This site was defined as the test site, and the site with the remaining cap as the control site.



98.5� 0.8%, Po0.05). Furthermore, the

tissue generated at the test sites in all of

the groups was resorbed, and its original

shape and volume were not maintained 1

month after one cap removal. A reduction of

approximately 20% was found in the No-IP

group, and approximately 11% and 15% in

groups Post-IP and Pre-IP, respectively. In

groups No-IP and Pre-IP, there was a sig-

nificantly greater amount of newly gener-

ated tissue at the control site compared with

the test site (Po0.05) (Table 1 and Fig. 4).

The relative amount of mineralized bone

generated at the control and test sites was

significantly larger in groups Post-IP and

Pre-IP when compared with group No-IP,

except for the test site between groups No-

IP and Post-IP (Po0.05). Furthermore, there

was a significant difference in the relative

amount of generated mineralized bone be-

tween the control and test sites in the No-IP

group (Po0.05) (Table 2 and Fig. 5).

Discussion

In our study, we demonstrated that it is

possible to augment the generation of bone

beyond the skeletal envelope and into areas

where there was previously no bone pre-

sent, by using a titanium cap. This finding

concurs with studies in which a titanium

device (cap, dome, or cylinder) had been

used to evaluate the potential for new bone

formation (Schmid et al. 1994; Majzoub

et al. 1999; Nanba 1999; Lundgren et al.

2000; Takaoka 2001; Minegishi et al. 2002;

Slotte & Lundgren 2002; Yamada et al.

2003; Tamura et al. 2005). In the present

study, newly generated tissue occupied 92–

99% of the maximal possible space created

by the caps after 4 months of healing with

Fig. 2. Typical sagittal histological section of the control (a) and test (b) sites in the No-IP group. Newly generated tissue with no signs of the presence of connective tissue

grew under the cap to various degrees. A slender layer of mineralized bone climbed along the inner wall of the cap and covered the generated tissue. The titanium cap was

removed one month before euthanization. Some resorption of the newly generated tissue was observed when compared with the control site. A typical sagittal

histological section of the control (c) and test (d) sites in the Post-IP group. Newly generated tissue has grown under the cap, and almost fills the space in the control site.

There are no obvious differences in the appearance of the newly generated tissue, other than the obvious reduction in the amount of tissue at the test site. Typical sagittal

histological sections of the control (e) and test (f) sites in the Pre-IP group. The amounts and appearance of the newly generated tissue at both the control and test sites

were similar when compared with the Post-IP group. (black line: the outline of the hypothetical titanium cap with the same dimensions as the original cap in (b), (d), and

(f). Staining with basic fuchsin and methylene blue. Original magnification, � 3).



or without IP. This proportion is similar to

the 3-month results (80–97%) in studies

using the same titanium cap model (Nanba


Yamada et al. 2003).

Using a Teflon capsule at the lateral

border of the rat mandible also resulted in

the formation of new extra-skeletal man-

dibular bone. At 120 days, the mean

amount of bone obtained was 56% of the

total space created by the oval Teflon

capsule (Kostopoulos et al. 1994). In addi-

tion, studies of the hemispherical Teflon

capsule model demonstrated that newly

formed bone occupied 24–40% of the max-

imal possible space created by the capsules

at days 60 and 120 of healing (Stavropoulos

et al. 2001; Donos et al. 2005). The fact

that the amount of newly formed bone

after 120 days did not significantly exceed

the amount after 60 days of healing sug-

gests that the total amount of bone gener-

ated using GBR is limited by factors other

than the healing time.

The use of titanium devices on the

calvaria of rabbits has resulted in a more

dramatic amount of bone formation (72–

100% after 2–3 months of healing) than

the use of the Teflon capsule (Majzoub

et al. 1999; Nanba 1999; Lundgren et al.


Yamada et al. 2003). Therefore, it appears

that a titanium surface has advantages over

Teflon in the formation of new extra-ske-

letal bone. Generally, studies report that a

thin layer of mineralized bone climbs up

the inner wall of the titanium dome/cap.

Recently, three-dimensional images and

histological specimens have revealed three

basic shapes in generating tissues from one

to 3 months: flat, cup-shaped, and domed.

Ultimately, trabecular bone forms along

the wall of the cap, and the bone fills the

inside of the cap within 3 months (Tamura

et al. 2005).

Our results demonstrated that the tissue

newly generated on the surface of the rabbit

calvaria using GBR is not stable over time.

Some resorption, equivalent to 11–20% of

the newly generated tissue, was observed

within the first month after removing the

titanium cap. This resorption can probably

be ascribed to the surgical trauma that

occurs during cap removal, which exposes

the newly generated tissue in all groups.

There was a significant difference in

the resorption of newly generated tissue

Fig. 3. Typical histological sections of the control sites in the (a) No-IP, (b) Pre-IP, and (c) Post-IP groups.

Generally, cuboidal osteoblast-like cells (arrow) were actively laying down bone on the mineralized bone at the

control sites in all groups. A similar amount of osteoblastic activity was observed at the test site in all groups.

Large marrow spaces (n) with fat cells and some hematopoietic cells were found in all of the groups (staining

with basic fuchsin and methylene blue. Original magnification, � 100).



between the control and test sites in the

No-IP and Pre-IP groups. In particular, the

greatest percentage, approximately 20% of

the newly generated tissue, was resorbed in

the No-IP group (93.8� 4.6% vs.

73.9� 3.7%, Po0.05). Therefore, IP

may inhibit further resorption of the tissue

generated using GBR.

IP appears to have anti-resorptive and

bone-forming mechanisms of action. The

anti-resorptive mechanism involves the

inhibition of both the activation of mature

osteoclasts and the formation of new os-

teoclasts (Bonucci et al. 1992; Notoya et al.

1993). In contrast, the osteoblastic effect of

IP and its metabolites stimulated the pro-

liferation of an osteoblast-like cell line

(UMR-106a), increased alkaline phospha-

tase activity, and enhanced collagen forma-

tion (Benvenuti et al. 1991). Bone marrow

osteoprogenitor cells and trabecular bone

osteoblasts were isolated from human do-

nors and incubated with IP and its meta-

bolites. These substances were found to

regulate osteoblastic differentiation by en-

hancing the expression of important bone–

matrix proteins and by facilitating miner-

alization (Cheng et al. 1994). Although

different numbers and degrees of cuboidal

osteoblast-like cells were observed on the

mineralized bone in all of the groups, the

changes were more obvious in the Post-IP

and Pre-IP groups.

Martini et al. (1998) provided further

evidence of the direct action of IP on

osteoblastic activity. A small, circular cav-

ity (3 mm in diameter) was created in a rat

mandible surgically, and was filled with

powdered IP. The local application of IP

was recommended for enhancing and ac-

celerating bone formation to repair a surgi-

cally created hole. They concluded that IP

stimulated osteogenesis and that it was a

potential therapeutic tool for promoting

the repair of injured peri-alveolar bone.

Although the in vivo effects of IP on bone

formation, particularly bone augmentation

using GBR, have not been investigated

thoroughly, they observed a positive effect

of IP on bone augmentation using GBR.

Minegishi et al. (2002) used a single 400-

mg dose of IP with GBR in a rabbit calvarial

model. They suggested that IP affected the

quality of bone augmentation at an early

stage. Light microscopic examination of

the test sites packed with IP in collagen

gel showed that newly generated minera-

lized bone was formed surrounding the IP.

Furthermore, IP was effective in reducing

the bone turnover rate, primarily by stimu-

lating bone formation and possibly by

inhibiting bone resorption. Although IP

appeared to be resorbed gradually, it was

not completely resorbed 3 months after

surgery. Therefore, incompletely resorbed

IP residue in the titanium cap may inhibit

further bone formation within a limited

space. These findings were consistent

with those with bone graft materials,

such as BIO-OSSs

(Osteohealth, Shirley,

NY, USA) and b-tricalcium phosphate re-

sidue, in the titanium cap (Nanba 1999;

Takaoka 2001), and were also in agreement

Table 1. Percentage areas of newly generated tissue under the titanium cap or hypothe-tical cap in all groups

Group N Control Test

No-IP 5 93.8 � 4.6 73.9 � 3.7Post-IP 4 92 � 7 81.3 � 8.4Pre-IP 5 98.5 � 0.8 83.4 � 5.2

Mean � SD, unit¼%.nPo0.05, analysed by the Mann–Whitney test after the Kruskal–Wallis test.

wPo0.05, analysed by the Wilcoxon test.

IP, ipriflavone; No-IP, no intake of IP; Post-IP, 10 mg/kg IP daily orally after GBR; Pre-IP, IP daily orally

beginning 1 month before GBR.

Control0

50

100

(%)

No-IP Post-IP Pre-IPTest Control Test Control Test

Fig. 4. Percentage areas of newly generated tissue at the control and test sites in all groups. Horizontal lines

indicate the mean values of each site.

Table 2. Percentage areas of mineralized bone in the newly generated tissue under thetitanium cap or hypothetical cap in all groups

Group N Control Test

No-IP 5 22.1 � 1 28.9 � 2.9

Post-IP 4 25.7 � 1.8 28.8 � 2.7

Pre-IP 5 36.2 � 0.9 34.3 � 2.3

Mean � SD, unit¼%.nPo0.05, analysed by the Mann–Whitney test after the Kruskal–Wallis test.

wPo0.05, analysed by the Wilcoxon test.

IP, ipriflavone; No-IP, no intake of IP; Post-IP, 10 mg/kg IP daily orally after GBR; Pre-IP, IP daily orally

beginning 1 month before GBR.

w

n

w

w

n

n

n

n

n



with the observations of other studies

using biomaterials (Stavropoulos et al.

2003, 2004; Donos et al. 2005).

To assess the potential impact of IP on


bone using GBR, a daily oral dose (10 mg/

kg) of IP was administered to rabbits for at

least 4–5 months. Generally, 200 mg IP is

administered orally three times daily in

osteoporosis therapy (Passeri et al. 1992;

Adami et al. 1997; Gennari et al. 1998;

Ohta et al. 1999), which is equivalent to an

IP dose of 10 mg/kg for an average 60 kg

human. Therefore, the daily intake of IP

used in this study (10 mg/kg) is a reasonable

dose when compared with human intake.

One must consider whether the bone

tissue generated beyond the skeletal envel-

ope will be maintained on a long-term basis

after removing the titanium cap. In our

study, some bone remodeling of the newly

generated tissue that formed under the tita-

nium caps occurred after cap removal; the

amounts of newly generated tissue remain-

ing at the test site 1 month after cap

removal differed from that at the control

site. This counters the results of a previous

study showing that the bone tuberosities

formed on the lateral aspect of the rat

mandible under originally empty Teflon

capsules are stable at least 12 months after

capsule removal (Lioubavina et al. 1999).

The stability of the bone produced by GTR

in that study was based on planimetric

measurements and subtraction radiography

data and showed that no further resorption

of the bone tuberosities occurred after 3

months following capsule removal. At 12

months, the tuberosities had maintained

91–98% of the capsule volume. In their

study, a small (4–8%) but statistically sig-

nificant, reduction in the amount of newly

formed bone was observed at 3 months after

capsule removal, while no further resorption

occurred for up to 12 months. A similar

study reported that some remodeling of the

bone that formed under the empty control

capsules had obviously occurred after cap-

sule removal, but the amounts of bone at 3

and 6 months after capsule removal did not

differ significantly from that observed at

baseline (Stavropoulos et al. 2004). The

discrepancy between the observations of

Lioubavina et al. (1999) and Stavropoulos

et al. (2004) regarding early resorption of the

newly formed bone may be attributable to

the fact that the capsules in the former

study were removed after 6 months of

healing and those in the latter study were

left in place for 12 months, which resulted

in more mature bone.

It has been reported that there is a strik-

ing difference between the morphology of

the host bone and the newly generated

bone tissue in the rabbit calvarial model.

Generally, the rabbit calvarial bone is char-

acterized by an area of elongated, central

spongious bone with large sinusoids sur-

rounded by comparatively thick inner and

outer compact laminae. In contrast, the

morphology of the newly generated tissue

within the dome or cap is quite different,

with a thin layer of mineralized bone and

large marrow cavities (Schmid et al. 1994;

Lundgren et al. 1995; Nanba 1999;

Takaoka 2001; Minegishi et al. 2002; Ya-

mada et al. 2003). This appearance is

characteristic of the primary spongiosa of

immature bone and may be related to the

comparatively short healing time period (2–

3 months). Schmid et al. (1994) reported

that after 8 months of healing, all domes

were completely filled with newly gener-

ated bone tissue, although the generated

bone was characterized by slender areas of

mineralized bone and large cavities filled

with bone marrow (approximately 31% vs.

69%, respectively). Conversely, Donos et

al. (2005) stated that at 60 days of healing,

the Teflon capsule was incompletely filled

with newly generated bone (approximately

36%) characterized by the presence of thin

areas of mineralized bone and large marrow

spaces, and the percentages of mineralized

bone and marrow space were approxi-

mately 22% and 14%, respectively. At

120 days of healing, the percentages of

newly generated bone, mineralized bone,

and marrow space were approximately

40%, 30%, and 10%, respectively. Thus,

the newly generated bone in the rabbit

calvarial model is characterized by a lower

percentage of mineralized bone with large

marrow spaces compared with the rat

ramus model, in which there is a large

percentage of mineralized bone with small

marrow spaces. The difference in the ratio

of mineralized bone to marrow space may

influence the long-term volume stability of

the newly generated bone.

In the Teflon capsule model, data indi-

cate that newly generated bone remains

stable or even becomes denser in the long

term (Lioubavina et al. 1999). Whether the

relatively slender pieces of bone that

formed in the titanium cap model will

become denser after further healing or

will gradually resorb remains to be eluci-

dated. In our study, there was a significant

across-group difference in the percentage of

mineralized bone in the newly generated

tissue at the control sites. The percentage

of mineralized bone in each group increased

gradually with the total dose of IP (No-IP:

22%, Post-IP: 26%, and Pre-IP: 36%).

Furthermore, at the test sites, the greatest

percentage of mineralized bone (34%) oc-

curred in the Pre-IP group. In similar ex-

perimental studies (Lundgren et al. 1995;

ControlNo-IP Post-IP Pre-IP

Test Control Test Control Test0

10

20

30

40

50

(%)

Fig. 5. Percentage areas of mineralized bone at the control and test sites in all groups. Horizontal lines indicate

the mean values of each site.



Minegishi et al. 2002; Slotte & Lundgren

2002; Yamada et al. 2003), the percentage

of mineralized bone ranged from 22% to

34% at 3 months. In our study, the per-

centage of mineralized bone at both the test

and control sites in the Pre-IP group was

greater than previously reported percen-

tages. These findings indicate that the

daily administration of IP is potentially

useful for improving the quality of newly

generated bone using GBR. The quality of

newly generated bone and the percentage of

mineralized bone may depend on the total

dose of IP.

Perhaps the different results for newly

generated tissue in the formation of new

extra calvarial and mandibular bone are

related to differences in the species and

host bone studied. In addition, the different

materials, volumes, dimensions, and designs

of the experimental devices used in the

studies may have influenced the different

outcomes. In addition, a prolonged healing

period would affect bone morphology and

the quality of newly generated tissue beyond

the skeletal envelope. Within the limitations

of this rabbit experimental model, we con-

clude that the daily intake of IP before or

after GBR inhibits the resorption of augmen-

ted tissue and would be useful for improving

the quality of newly generated bone beyond

the skeletal envelope.

Acknowledgements: We thank

Textcheck for excellent assistance in

preparing this manuscript. Thanks are

also due to Takeda Chemical Industries

Ltd. (Osaka, Japan) for the donation of

Ipriflavone. This study was supported in

part by a 1999 grant from the Sato

Fund, Nihon University School of

Dentistry, and by a 2000–2002 Grant-in-

Aid of Scientific Research (C-2,

#12,672,042) from the Ministry of

Education Science, Sports, and Culture

of Japan.

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