Vol.2, No.3, 53-60 (2013) Open Journal of Regenerativ e Medicine
http://dx.doi.org/10.4236/ojrm.2013.23009
Combination of simvastatin and hydroxyapatite fiber
induces bone augmentation
Shang Gao1*, Makoto Shiota1, Masaki Fujii1, Kang Chen1, Masahiro Shimogishi1,
Masashi Sato2, Shohei Kasugai1
1Department of Oral Implantology and Regenerative Dental Medicine, Tokyo Medical and Dental University, Tokyo, Japan;
*Corresponding Author:
2Department of Oral and Maxillofacial Surgery, Tokyo Medical and Dental University, Tokyo, Japan
Received 8 May 2013; revised 10 June 2013; accepted 13 July 2013
Copyright © 2013 Shang Gao et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
This study evaluated the capability of hydroxy-
apatite fiber (HAF) as a carrier and the bone
formation by blending simvastatin. The mixture
of HAF and simvastatin (0.15, 0.45, 0.75 mg) was
placed in 1 ml of tris-buffer and the release of
simvastatin from HAF was calculated per 24
hours for 10 day s. Bilat eral 5 mm-d iameter and 3
mm-hight Teflon chambers were fixed on cal-
varia of adult Japanese white rabbits and filled
with 40 mg HAF which cont aining simv ast atin (0,
0.15, 0.45, 0.75 mg). The animals were sacrificed
at 4 and 8 weeks and calculated radiologically
by Micro-CT. After dyeing by toluidine blue the
samples were analyzed histologically. In all of
the study groups approximately 25% of simvas-
tatin was released until 10 days. The new bone
volume ratio measured by Micro-CT of 4 and 8
weeks group was (22.4%, 21.3%, 41.6%, 26.3%)
and (20.2%, 11.7%, 42.1%, 31.2%) in different
doses respectively. The 0.45 mg group showed
significant ly higher new bone volume r atio than 0
mg group and 0.15 mg group. The histological
measurement and observations also supported
these results. In conclusion, the HAF could be
used as a carrier for simvastatin. Combinations
of HAF and simvastatin have the potentiality to
stimulate new bone formation and approxi-
mately 0.45 mg simvastatin in 40 mg HAF is the
optimal dose in rabbit chamber model.
Keyw ords: Biomaterial; Bone Substitutes; Bone
Formation; Drug Delivery; Growth Factors
1. INTRODUCTION
With the increasing popularity of dental implants, the
lack of sufficient amount of jaw bone has become a sig-
nificant limitation. To overcome this constraint, resear-
chers started focusing on bone regeneration in the con-
text of dental implant surgery [1-4]. Over the past two
decades, hydroxyapatite has proven to be an excellent
bone substitute for its biocompatibility and space main-
tainability [5,6]. More recently, commercialization of
hydroxyapatite bone substitute has been made possible,
with most of the products being available as granular
particles or blocks [7].
In clinical practice, however, these products have
shown poor operational performance, for some clinicians
opt to mix hydroxyapatite with venous blood or platelet-
rich plasma [8,9] or use fiber-type hydroxyapatite. How-
ever, most of the initial attempts for obtaining pure hy-
droxyapatite fiber (HAF) have failed, as the adhesive or
coating materials used drastically reduced its purity
[10,11]. In recent years, pure HAF has been developed
successfully, and its capacity to induce bone formation
has been demonstrated [12,13]. Besides its effects on
bone growth, HAF is also expected to serve as a carrier
by holding substances within its three-dimensional struc-
ture and slowly releasing them—just like a drug-delivery
system [14].
Growth factors, such as platelet-derived growth factor
(PDGF), insulin-like growth factor (IGF), fibroblast
growth factor (FGF), and bone morphogenetic protein
(BMP)-2, have been reported to promote bone regenera-
tion [15-18]. However, 2 issues limit their use in clinical
practice: most of these growth factors are xenogeneic,
and they are expensive. As an alternative, statins—wi-
dely known as competitive inhibitors of 3-hydroxy-3-
methylglutaryl coenzyme A (HMG-CoA) reductase that
lower cholesterol levels [19]—have also been shown to
increase BMP-2-induced bone formation [20,21]. Our
previous study using simvastatin yielded similar results
[22]. This effect appears to depend on the concentration

S. Gao et al. / Open Journal of Regenerative Medicine 2 (2013) 53-60
54
of the drug, but high concentrations of simvastatin are
unwanted because they provoke inflammation to the
mandibular bone [23].
The aims of the present study were 1) to investigate
the carrier properties of HAF by measuring the release of
simvastatin from HAF/simvastatin compound materials;
and 2) to measure bone regeneration induced by different
blends of simvastatin combined with HAF.
2. MATERIALS AND METHODS
2.1. Sample Preparation
We used a previously described HAF compound (Fujii
S., United State Patent No. 4,659,617) that was 5 - 15 μm
in diameter [12,13]. Energy dispersive X-ray analysis
confirmed its purity; its three-dimensional structure is
shown in Figure 1. Simvastatin (Ohara Pharmaceutical
Co. Ltd., Koka, Shiga, Japan) was dissolved in ethanol.
The solution was dropped onto HAF under sterile condi-
tions, and then allowed to dry completely in a laminar
flow hood for 24 h. We prepared 4 groups of samples,
each containing 40 mg HAF and 0 mg, 0.15 mg, 0.45 mg,
and 0.75 mg simvastatin, respectively.
2.2. Measurement of Simvastatin Release
The release of simvastatin from HAF was measured
by using an ultraviolet—visible spectrophotometer (Na-
nodrop ND-1000; NanoDrop Technologies, Wilmington,
DE, USA). The Nanodrop was calibrated using 8 stan-
dards of simvastatin solution at ambient temperature. The
absorbance was measured at 238 nm, and a standard
curve for calculation of simvastatin concentrations was
generated from the absorbance values. The samples were
placed in 1 ml of 0.1 M Tris buffer solution (pH 7.4), and
shaken on a Taitec Personal 11 Shaker (Taitec Corp.,
Tokyo, Japan) at 100 rpm at ambient temperature. The
amount of simvastatin released into the buffer was scored
every 24 h for 10 days. The cumulative concentration
was calculated by using the previously determined stan-
Figure 1. 3D-structure of HAF by SEM (mag-
nification ×800).
dard curve.
2.3. Surgical Procedures for the Rabbit
Cranial Chamber Model
The animal experimental protocol was approved by
the Institutional Committee of Animal Care and Use at
Tokyo Medical and Dental University. Twenty Japanese
white rabbits weighing 2.5 - 3.0 kg were used. The ani-
mals were systemically anesthetized with an intramuscu-
lar injection of ketamine (50 mg/kg Ketalar; Sankyo,
Tokyo, Japan) and thiopental sodium (25 mg/kg Rabonal;
Tanabe, Tokyo, Japan). The surgical area was shaved and
prepared aseptically with povidone-iodine (Isodine Sur-
gical Scrub; Meiji, Tokyo, Japan) for surgery. Before
surgery, 1.8 ml of a local anesthetic (2% xylocaine: epi-
nephrine 1:80,000; Dentsply Sankin, Tokyo, Japan) was
injected into the surgical site. Skin incision and dissec-
tion were carried out coronally, and periosteum incision
and dissection were performed sagittally between the
parietal and the frontal bone. After the periosteum was
elevated, polytetrafluoroethylene chambers (hollow cyl-
inders of 5.0 mm in diameter and 3.0 mm in height with
an outer brim) were fixed with stainless steel screws
(FKG Dentaire, Chaux-de-Fonds, Switzerland) to the
parietal bone on the right and left sides. The chambers
were filled with samples, which were selected randomly
(Figure 2). The skin flaps were sutured with 4-0 nylon.
During the observation period, all animals were given
water and a standard feed ad libitum. Animals were sac-
rificed at 4 weeks or 8 weeks with a lethal dose of thio-
pental sodium. The entire cranial bone was harvested and
fixed for 10 days in neutral 10% formalin.
2.4. Micro-Computed Tomography
(Micro-CT) Analysis
The samples were scanned by a micro-CT scanner
(a) (b)
(c) (d)
Figure 2. The animal surgical procedures. (a): Periosteum inci-
sion and elevating; (b): The chambers were fixed on both sides
of rabbits; (c): The chambers were filled with samples; (d):
Sutured with 4-0 nylon.

S. Gao et al. / Open Journal of Regenerative Medicine 2 (2013) 53-60 55
(SMX-90CT; Shimadzu Science East Corporation, Tokyo,
Japan) with a voxel size of 60 μm/pixel, and quantified
by using the Tri/3D-Bon software (Ratoc System Engi-
neering Co. Ltd., Tokyo, Japan). The percentage of new
bone volume occupying the total chamber volume was
determined.
2.5. Histological Processing
After micro-CT analysis, the specimens were dehy-
drated in ascending grades of ethanol, following infiltra-
tion, and then embedded in methacrylate-based resin
(Technovit 7200 VLC; Heraeus Kulzer, Wehrheim, Ger-
many). The sections were cut and ground to a thickness
of about 100 m. The sections were finally stained with
0.1% toluidine blue. Histological observation was per-
formed under a light microscope. The percentage of new
bone in total chamber volume was measured with an
image software (Photoshop CS6 extended, Adobe Sys-
tems complex, California, USA). The measurement was
performed by a co-author.
2.6. Statistical Analysis
For statistical analysis, the data were tested by one-
way analysis of variance (ANOVA). The data were fur-
ther analyzed by Fisher’s least significant difference test
and Bonferroni multiple comparison test. All statistical
analyses were performed using a commercial computer
program (SPSS v. 11.5; SPSS Inc., Chicago, IL, USA). A
value of p < 0.05 was considered to be statistically sig-
nificant.
3. RESULTS
3.1. Simvastatin Was Released from HAF in
2 Phases
Approximately 10% of absorbed simvastatin was re-
leased after 24 h. After this initial burst release, gradual
and stable release of simvastatin was observed for 10
days (Figure 3). A similar release pattern was observed
regardless of the initial concentration of simvastatin.
3.2. Micro-CT Analysis
Four weeks after surgical intervention, the percent-
age of new bone in the total chamber volume was
22.36% ± 4.98%, 21.30% ± 7.70%, 41.58% ± 6.03%,
and 26.32% ± 6.19% in the groups treated with differ-
ent simvastatin/HAF ratios (0/40 mg, 0.15/40 mg,
0.45/40 mg, and 0.75/40 mg, respectively). Significant
differences were recognized between the 0/40 mg and
the 0.45/40 mg groups (p = 0.047), and between the
0.15/40 mg and the 0.45/40 mg groups (p = 0.037).
After 8 weeks, the percentage of new bone was 20.22%
± 5.53%, 11.72% ± 3.53%, 42.14% ± 4.43%, and
31.22% ± 8.58%, respectively. Significant differences
were recognized between the 0/40 mg and the 0.45/40
mg groups (p = 0.17), between the 0.15/40 mg and the
0.45/40 mg groups (p = 0.002), and between the
0.15/40 mg and the 0.75/40 mg groups (p = 0.031)
(Figure 4).
3.3. Histology
The main histological findings are presented in Fig-
ures 5-8. Four weeks after surgical intervention, the
inner space of HAF-filled chambers was maintained
clear in each group (Figure 5), i.e., there was no con-
nective tissue invagination from the top of the cham-
bers. However, some blood cells were observed. We
also noted that the HAF fragment was oriented in dif-
ferent directions from sample to sample. The newly
formed bone was found towards the bottom of the
chamber, close to the host bone, in both control and
simvastatin/HAF 0.15/40 mg groups. On the other hand,
in the 0.45/40 mg group, the newly formed bone was
found approximately at middle-height level between
top of the chamber and host bone; in the 0.75/40 mg
group, the newly formed bone progressed up to ap-
proximately one-quarter of the total height of the
chamber. The new bone connected with the host bone
Figure 3. The release curve of simvastatin from HAF. Data
were shown as mean ± SD, n = 6. After an approximately 10%
initial burst release, stable release was observed for 10 days.
Regardless of the difference concentrations of simvastatin, the
release pattern was similar.

S. Gao et al. / Open Journal of Regenerative Medicine 2 (2013) 53-60
56
Figure 4. Micro-CT analysis, 4 weeks: The percentage of new bone in the total chamber volume was 22.36% ± 4.98%, 21.30% ±
7.70%, 41.58% ± 6.03%, and 26.32% ± 6.19% in the groups treated with different simvastatin/HAF ratios. 8 weeks: The volume
was 20.22% ± 5.53%, 11.72% ± 3.53%, 42.14% ± 4.43%, and 31.22% ± 8.58%, respectively (n = 5, *p < 0.05).
(a) (b)
(a) (b)
(c) (d)
(c) (d)
Figure 6. Four weeks after surgical intervention ((a): 0/40 mg
group (b): 0.15/40 mg group (c): 0.45/40 mg group (d): 0.75/40
mg group). The HAF fragment (F) was oriented in different
directions. The staining and morphological appearance of new
bone (NB) was different from the host bone. Some blood cells
(BC) was observed between the NB and F. (Undecalcified
samples, stained with toluidine blue).
Figure 5. Four weeks after surgical intervention ((a): 0/40 mg
group (b): 0.15/40 mg group (c): 0.45/40 mg group (d): 0.75/40
mg group). The diameter of chamber was 5.0 mm. There was
no connective tissue invagination from the top of the chambers.
0/40 mg and 0.15/40 mg groups: Towards the bottom of the
chamber, the newly formed bone was found. 0.45/40 mg group:
The newly formed bone was found at middle-height level be-
tween top of the chamber and host bone. 0.75/40 mg group:
The new bone progressed up to one-quarter of the total cham-
ber’s height (Undecalcified samples, stained with toluidine
blue).
nective tissue (Figure 7). However, the space between
the HAF fragment—which was oriented in different
directions from sample to sample—and the new bone
was filled with blood cells and fat cells. In contrast to
the samples dissected at 4 weeks after surgery, samples
dissected at 8 weeks after surgery clearly showed the
trabecular bone structure. Moreover, newly formed
bone stained more similarly to host bone. The amount
and relative position of new bone in the chambers were
similar to those found at 4 weeks. The newly formed
bone bridged with the host bone and showed a “moth-
and grew toward the HAF. It was possible to differen-
tiate new bone from host bone based on differences in
staining and on morphological appearance, i.e., new
bone presented a “moth-eaten-like appearance” (Fig-
ure 6).
Eight weeks after surgical intervention, the inner
space of the chambers was still clear and free of con-
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S. Gao et al. / Open Journal of Regenerative Medicine 2 (2013) 53-60 57
eaten-like appearance” (Figure 8). Inflammation was
not observed at any of the 2 time points analyzed.
The results of histological measurement were similar
to the micro-CT’s (Figure 9). For four weeks, the per-
centage of new bone in the total chamber volume was
22.30% ± 9.52%, 21.36% ± 5.24%, 41.38% ± 11.18%,
and 19.51% ± 3.80% in the groups treated with differ-
ent simvastatin/HAF ratios (0/40 mg, 0.15/40 mg, 0.45/
40 mg, and 0.75/40 mg, respectively). Significant dif-
ferences were recognized between the 0/40 mg and the
0.45/40 mg groups (p = 0.010), the 0.15/40 mg and the
0.45/40 mg groups (p = 0.007), and between the 0.45
mg/40 mg and the 0.75 mg/40 mg groups (p = 0.003).
After 8 weeks, the percentage of new bone was 22.45%
± 4.04%, 18.21% ± 5.44%, 41.11% ± 11.79%, and
33.24% ± 2.58%, respectively. Significant differences
were recognized between the 0/40 mg and the 0.45/40
mg groups (p = 0.004), between the 0.15/40 mg and the
0.45/40 mg groups (p = 0.001), and between the
0.15/40 mg and the 0.75/40 mg groups (p = 0.020).
4. DISCUSSION
In this study, we confirmed the role of simvastatin in
bone formation and regeneration. Simvastatin and other
statins have been widely used for lowering serum levels
of cholesterol in hypercholesterolemia, hyperlipidemia,
and arteriosclerosis [19]. In recent years, several studies
have shown that statins enhance BMP-2 and vascular
endothelial growth factor (VEGF) gene expression in
osteoblasts, suggesting their bone-promoting effect [21,
24,25]. Animal studies involving systemic or local ad-
ministration of statins have also yielded positive results
[23,26-28]. However, systemic administration of statins
for bone formation requires a much more higher dose
(a) (b)
(c) (d)
Figure 7. Eight weeks after surgical intervention ((a): 0/40 mg
group (b): 0.15/40 mg group (c): 0.45/40 mg group (d): 0.75/40
mg group). The diameter of chamber was 5.0 mm. The inner
space of chamber was clear and free of connective tissue. The
new bone volume of each group was similar with 4 weeks but
was stained more similarly to the host bone (Undecalcified
samples, stained with toluidine blue).
(a) (b)
(c) (d)
Figure 8. Eight weeks after surgical intervention ((a): 0/40 mg
group (b): 0.15/40 mg group (c): 0.45/40 mg group (d): 0.75/40
mg group). The HAF fragment (F) was oriented in different
directions. Blood cells (BC) and fat cells (FC) were observed
between the new bone (NB) and F. The NB clearly showed the
trabecular structure, and bridged with the host bone showed a
“moth-eaten-like appearance” (Undecalcified samples, stained
with toluidine blue).
than for lowering cholesterol, and safety has not yet been
clearly reported [29,30]. On the other hand, local ad-
ministration can be performed with smaller doses, but it
requires multiple or continuous injections [31]. In re-
sponse to this drawback, local application of low-dose
statin with a slow-release drug-delivery system has been
suggested [32,33].
In this study, we showed that HAF can serve as a sub-
stance carrier by demonstrating the stable release of sim-
vastatin from HAF/simvastatin compound materials. This
is in agreement with the report by Oda et al. [14], who
suggested HAF as a potential component of a drug-de-
livery system based on its biocompatibility, biode-grad-
ability, and cotton-like fibrous 3D structure. Importantly,
the effects of HAF in bone formation have been demon-
strated by using the rabbit cranium chamber model [12]
and the post-extraction tooth socket model [13]. Kimura
et al. [12] demonstrated that the combination of HAF
with autogenous bone was effective in vertical bone
augmentation, and Machida et al. [13] indicated that
HAF could not only allow but also promote bone healing
of the socket after tooth extraction. Therefore, we used
simvastatin as growth-factor-like substance and HAF as
scaffold in the present study. Here, an in vitro study
showed that simvastatin is released gradually over time
after an initial burst release from HAF. This release pat-
tern was considered to be similar to that observed in sev-
eral studies using other biomaterials, including alpha-
tricalcium phosphate (TCP) [34], collagen sponge [35],
electrospun fiber material [36], as the drug-delivery sys-
tem. The bone-promoting effect of simvastatin correlated
well with concentration [37]; thus, the initial burst re-
lease was thought to be a critical phase [34]. Addition-

S. Gao et al. / Open Journal of Regenerative Medicine 2 (2013) 53-60
58
Figure 9. Histological analysis, 4 weeks: The percentage of new bone in the total chamber volume was 22.30% ± 9.52%, 21.36% ±
5.24%, 41.38% ± 11.18%, and 19.51% ± 3.80% in the groups treated with different simvastatin/HAF ratios. 8 weeks: The volume
was 22.45% ± 4.04%, 18.21% ± 5.44%, 41.11% ± 11.79%, and 33.24% ± 2.58%, respectively (n = 5, *p < 0.05).
ally the release pattern always showed a constant curve
regardless of concentration, indicating that the correla-
tion coefficient of the release volume might be similar.
Therefore, the release of simvastatin was considered to
be adjusted by altering the amount of simvastatin added
initially.
In the animal experiment, we used the rabbit cranial
chamber model. Traditionally, the cranial bone defect
model [38], tibia bone defect model [39], and post-ex-
traction tooth socket model [40] were used to evaluate
new bone formation in vivo. However, these models are
not always rigorous enough to identify and evaluate the
extent of new bone formation, since in these models
bone can heal without pharmacological treatment [41].
Instead, the rabbit cranial chamber model offers a good
model for new bone formation; in this model, the amount
of new bone can be evaluated conveniently since the size
of the chamber is constant. In this study, 40 mg HAF
were used. This dose had been demonstrated as the opti-
mal dose for bone formation by Fujii et al. (2013) with
using the same animal model.
To quantify the amount of newly formed bone, we
used micro-CT analysis and histological measurement.
Both showed the similar results, that suggested the valid-
ity of estimation method. We observed that 0.45 mg sim-
vastatin induced significantly greater bone formation
than lower amounts added to HAF at 4 weeks and 8
weeks after surgery, respectively. This strongly suggests
that the bone-promoting effect of simvastatin is dose-
dependent, with 0.45/40 mg simvastatin/HAF being the
most effective combination. In previous studies, Nyan et
al. [37] applied 0.1 mg simvastatin with 14 mg alpha-
TCP to rat calvarial defects, and Pradeep et al. [32] per-
formed a randomized trial to treat chronic periodontitis
with approximately 1.2% simvastatin solution. Both
studies showed positive results with simvastatin concen-
trations that were similar to the most effective dose of
our study (0.45/40 mg).
Histological observations supported the above-men-
tioned findings. In addition, staining intensity and mor-
phology of new bone at 8 weeks were more similar to
host bone than at 4 weeks, which suggested the advanced
maturation of new bone within 8 weeks. However, at an
earlier time point, similar staining intensity and mor-
phology of new bone were observed regardless of the
different concentrations of simvastatin, which indicated
that simvastatin could stimulate bone formation but not
bone maturation. In agreement with our observations,
Lee et al. [42] suggested that it was possible for simvas-
tatin to stimulate bone formation but not bone maturation,
and that much of the newly formed bone by simvastatin
was resorbed in the long term.
Some researchers reported that a high dose of simvas-
tatin could evoke inflammatory response in animal stud-
ies [23,37], but no inflammation was found in the present
study. This indicates that our highest dose (0.75 mg)
might not have been high enough to cause inflammatory
reaction. Another curious effect of simvastatin is ectopic
bone formation [14]. In our study model, in which the
chamber was covered by periosteum, some mesenchymal
cells could have had the chance to enter from the top of
the chamber. However, there was no new bone formed at
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S. Gao et al. / Open Journal of Regenerative Medicine 2 (2013) 53-60 59
the top level of the chamber. Instead, newly formed bone
connected with the host bone tightly.
In conclusion, this study revealed that HAF has the
potential to be used as a carrier for simvastatin. Combi-
nations of HAF and simvastatin stimulated new bone
formation in a dose-dependent manner. Under our ex-
perimental conditions, we found 0.45/40 mg simvastatin/
HAF to be the optimal combination dose for bone forma-
tion.
5. CONCLUSION
In conclusion, this study revealed that HAF has the
potential to be used as a carrier for simvastatin. Combi-
nations of HAF and simvastatin stimulated new bone
formation in a dose-dependent manner. Under our ex-
perimental conditions, we found out that 0.45/40 mg
simvastatin/HAF is the optimal combination dose for
bone formation.
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