Bone 52 (2013) 1–8

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Bone
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Original Full Length Article

Vitamin D3 and insulin combined treatment promotes titanium implant

osseointegration in diabetes mellitus rats
Ying-ying Wu a, b, 1, Tao Yu c, 1, Xiao-yong Yang d, Feng Li a, b, Li Ma a, b, Yang Yang a, b, Xiao-guang Liu e,
Yong-yue Wang a, b, Ping Gong a, b,⁎
a
State Key Laboratory of Oral Disease, Sichuan University, No. 14, Sec. 3, Renminnan Road, Chengdu 610041, Sichuan, People's Republic of China
b
Department of Implantology, West China School of Stomatology, Sichuan University, No. 14, Sec. 3, Renminnan Road, Chengdu 610041, Sichuan, People's Republic of China
c
Department of Head and Neck Oncology, Sichuan Cancer Hospital, No. 55, Sec. 4, Renminnan Road, Chengdu 610041, Sichuan, People's Republic of China
d
Department of Thyroid and Neck Oncology, Tianjin Medical University Cancer Institute and Hospital, Huanhuxi Road, Hexi District, Tianjin 300060, People's Republic of China
e
Department of Biomaterials Center, Sichuan University, No. 24, Sec. 1, Yihuan Road, Chengdu 610065, Sichuan, People's Republic of China

a r t i c l e i n f o a b s t r a c t

Article history: This study investigated the effect of insulin and vitamin D3 (VD3) treatment on implant osseointegration in
Received 26 June 2012 diabetic mellitus (DM) rats. DM was induced by administration of streptozotocin in rats, which received implants
Revised 1 September 2012 insertion in the femur. Then animals were subjected to different treatment and divided to the following group:
Accepted 3 September 2012
control, diabetic, insulin-treated diabetic, VD3-treated diabetic, insulin and VD3 combination-treated diabetic
Available online 14 September 2012
rats. The glucose levels and weight of rats were periodically evaluated, and serum 25(OH)D3 levels in rats
Edited by: Rene Rizzoli were measured at the end of the experiment. Animals were sacrificed at 12 weeks after surgery, the peri-
implant trabecular microstructure, implant fixation and implant osseointegration were measured by micro-
Keywords: scopic computerized tomography (micro-CT) evaluation, push-out test and histomorphometric analysis. Diabetic
Diabetes mellitus rats displayed significantly higher blood glucose level, lower body weight, lower serum 25(OH)D3 levels, and less
Implant implant osseointegration than controls. Insulin treatment showed restorative effect on body weight and serum
Insulin 25(OH)D3 levels of diabetic rats, but the blood glucose level in diabetic rats were still substantially higher com-
Vitamin D3 pared to controls after 14 days therapy of insulin. Combined treatment restored hyperglycemia in diabetic rats
to be normal, and reversed the impaired osseointegration capacity of implants, with the bone volume ratio and
percent osseointegration increased by 1.37-fold and 1.6-fold in micro-CT evaluation, the maximal push-out
force and ultimate shear strength by 1.3-fold and 2.1-fold in push-out test, and the bone-to-implant contact
and bone area ratio increased by 2.57-fold and 1.44-fold in histomorphometric analysis. Monotreatment also
enhanced implant fixation, but less. These results indicated that insulin and VD3 combined treatment may be
an effective approach to enhance implant fixation in diabetic rats, but whether the results could be extrapolated
to human needs further study.
© 2012 Elsevier Inc. All rights reserved.

Introduction edentulous or partially edentulous patients. However, the majority of

Diabetes mellitus (DM) is a metabolic disorder characterized by
hyperglycemia associated with a wide range of disorders, such as
retinopathy, nephropathy, cardiovascular disease, osteoporosis, im-
paired wound and bone healing, and increased susceptibility to peri-
odontal disease [1,2]. Considering the prevalence of DM among the
world population and the severe periodontal disease and tooth loss
in these patients, diabetic patients who seek rehabilitative therapies
should be regarded as frequent patients in dental practice [3]. Dental
implants have become a predictable treatment option for the healthy
⁎ Corresponding author at: Department of Implantology, West China School of
Stomatology, Sichuan University, No. 14, Sec. 3, Renminnan Road, Chengdu 610041,
Sichuan, People's Republic of China. Fax: + 86 28 85582167.
E-mail address: dentistgong@163.com (P. Gong).
1
Ying-ying Wu and Tao Yu contributed equally to this work.
studies suggested that DM could negatively interfere with the process
of dental implant osseointegration, even result in implant loosening
and failure due to incomplete and delayed bone formation around
the implant [4]. Studies demonstrated that implant survival rates
could be enhanced in DM patients when blood plasma glucose level
is under control [1,5]. Others accept the fact that insulin therapy is
able to neutralize the effects of diabetes on bone healing, but still
with a poorer implants osseointegration compared with healthy sub-
jects [3,4,6]. McCraken et al. also demonstrated that bone tissue around
implants in insulin-treated rats was not as well organized as healthy
controls [7]. Therefore, enhancement in the osseointegration of dental
implants in DM patients is a challenge to clinicians.
Vitamin D3 (VD3) is either formed in the skin by the absorption of
ultraviolet light, or obtained from nutritional sources, such as eggs,
fish oils and fortified milk [8]. The actions of VD3 are mediated through
binding to the vitamin D receptor (VDR), a member of the nuclear

8756-3282/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bone.2012.09.005
2 Y. Wu et al. / Bone 52 (2013) 1–8
steroid hormone receptor family, and acts as a ligand-activated tran-
scription factor to regulate the expression of target genes. The classic
role of the VD3 endocrine system is to stimulate calcium absorption
in the intestine, thus maintaining normal calcium homeostasis and
indirectly regulating bone mineralization [2,9]. 1,25(OH)2D3, the
biologically active form of VD3, is recognized as a regulator of both
osteoblast mediated bone formation and osteoclast mediated bone re-
sorption [10–12]. Besides these, numerous other disease associations
have been reported with VD3 deficiency, including cardiovascular dis-
ease, common obesity, and DM. An increased prevalence of diabetes
has been described in VD3-deficient individuals [13,14]. According
to clinical studies, it is observed that DM patients have significantly
lower circulating 1,25(OH)2D3 concentration compared to healthy
people [15–17]. In addition, the existing studies provide compelling
evidence that VD3 may play a functional role in the preservation of
glucose tolerance through affecting insulin secretion and insulin sen-
sitivity [18–22]. Zeitz et al. demonstrated that mice with mutations
in the VDR have impaired insulin secretion and lower glucose toler-
ance compared with those with functional VDR [20]. Existing evidence
suggests that VD3 has potential benefits with respect to osteoporosis
and DM. However, the molecular mechanisms by which the VD3 defi-
ciency and DM are related are not well known [23].
Our previous study was designed to find out the potential mecha-
nism of how VD3 affect insulin sensitivity, and results suggested that
1,25(OH)2D3 positively influences the osteoblast differentiation and
insulin sensitivity by activating an endocrine regulatory loop through
which insulin signaling promotes bone acquisition and simultaneous-
ly stimulates undercarboxylated osteocalcin secretion, which in turn,
act as a circulating hormone to regulate insulin production and sensi-
tivity [24]. All the evidence suggests that VD3 might be beneficial
not only for diabetes, but also, for osteoporosis by promoting bone
formation. So this study evaluated a novel strategy for improving im-
plant osseointegration in diabetic rats using the dual action of VD3 on
bone formation and glucose homeostasis.
Materials and methods
Animals
All animal care and experiments were conducted in accordance
with international standards on animal welfare and being compliant
with the Animal Research Committee of the University. Male adult
Wistar rats (supplied by the Experimental Animal Center of Sichuan
University) between 10 and 11 weeks old with 180–240 g body weight
were selected and studied in this study. They were housed in separate
cages with climate-controlled condition (25 °C, 55% humidity, 12 h
Fig. 1. Implants were placed into the femur. The left row showed that an implant was placed into the implant bed made by a rotary drill, and the right row exhibited the represen-
tative radiograph of femur with implant.
light alternating with 12 h darkness). Rats had free access to standard
food and water ad libitum.
Inducement of diabetes
After 1 week of adaptation to the laboratory environment, rats
were divided into different groups. Diabetic rats were created by a
single intravenous injection of freshly prepared streptozotocin (STZ)
(Sigma, St. Louis, MO) dissolved in 0.1 M citrated buffer (PH 4.9) at
a dose of 40 mg/kg. Rats with blood glucose levels over 300 mg/dl
at 1 week after injection were used for experiments [25,26].
Implant preparation
The rod-shaped implants with 10 mm in length and 1 mm in di-
ameter used in this study were made of commercial pure titanium
and supplied by Prof. Liu (National Engineering Research Center of
Biomaterials, Sichuan University, China). All implants were machined
and grit-lasted with 25 μm aluminum oxide (Al2O3) particles, and
then were sequentially washed with NaOH at 40 vol.% and deionised
water in ultrasonic bath. Before the implant surgery, all implants
were sterilized in autoclave.
Implant surgery
Animals were anesthetized by intraperitoneal injections of 10%
chloral hydrate (3.3 ml/kg). All surgical procedures were performed
under sterile conditions. Incisions of approximately 10 mm in length
were bilaterally performed at the medial side of the knee joint, and
the extensor mechanism with the knee joint was dislocated laterally.
With the knee in flexion, an implant bed was made through each
intercondylar notch with a rotary drill into the medullary canal of
the femur via distal femoral metaphysic, and an implant was placed
into the femur until the implant end was below the articular surface
(Fig. 1). Then the extensor mechanism was reconstructed, soft tissues
were sutured in separate layers and all the animals received intra-
muscular antibiotic injection for three post-operative days. Animals
were allowed for free movement without any restriction. The repre-
sentative radiograph of the femur with implant was also shown in
Fig. 1.
Treatment
Three days after implantation, animals were divided into the follow-
ing groups: control, diabetic, insulin-treated diabetic, VD3-treated dia-
betic, and insulin and VD3 combination (insulin +VD3)-treated diabetic
 Y. Wu et al. / Bone 52 (2013) 1–8 3

rats. Each group consisted of 6 animals. The insulin-treated diabetic rats

received subcutaneous doses of Neutral Protamine Harguerdon insulin
(Novo Nordisk A/S, Denmark) twice a day (5.5 UI at 20:00 hours and
3.5 UI at 8:00 hours) during the entire period of the experiment [1].
Daily, VD3-treated diabetic rats received 12 μg/kg of the cholecalciferol
(Sigma, St. Louis, MO) dissolved in 0.3 ml of coconut oil [2]. The sup-
plementation was administrated via gavage for a period of 14 days.
12 weeks after implantation surgery, animals were sacrificed, and the
distal femora with implants were harvested for valuation.

Estimation of blood glucose

Blood samples were obtained from each animal using a tail snip on
0 day (before the start of the experiment), the 3rd, 6th, 10th, and
14th day of the treatment, and every 2 weeks since the 14th day of
different treatments to the end of the experiment. Blood glucose
levels were estimated by the glucose-oxidase enzymatic method.

Body weight

The body weight of rats subjected to different treatments was

recorded at the following periods: 0 day (before the start of the
experiment), 1 month, 2 months, and 3 months after the implant
placement.

Microscopic computerized tomography (micro-CT) evaluation Fig. 2. Image of the jig for push-out test.

After sacrifice of animals 12 weeks after the implantation, the femur

with implants (n=6 specimens per group) was scanned on a μCT system
(μ-CT 80 scanner Scanco Medical, Bassersdorf, Switzerland), which
was set to 70 kV, 114 mA, and 700 ms integration time. Multi-level
thresholds procedure was applied to discriminate bone from other tis-
sues. The three-dimensional (3-D) images reconstructed from micro-
tomographic slices were used for qualitative and quantitative evaluation,
with the constrained 3-D Gaussian filter (σ=1.2, support=1) for part
suppression of the noise in the volumes [27]. The volume of interest
(VOI) was defined as bone tissues from 2.0 mm above the growth
plate to proximal 50 slices, extending with a radius of 200 μm from the
implant surface. After segmentation, the bone volume per total volume
(BV/TV), the mean trabecular thickness (Tb.Th), the mean trabecular
number (Tb.N), the mean trabecular separation (Tb.Sp), and the mean
connective density (Conn.D) were assessed within the VOI zone. The
%OI was calculated as the ratio between bone and total voxels in direct
contact with the implant.
Push-out test
Immediately after micro-CT evaluation, push-out test was performed
on these specimens (n=6 specimens per group) using a universal
material testing system (Instron 5566; Instron, Norwood, MA, USA). A
custom designed holder was used to maintain the downward compres-
sion to centre the implant and align it vertically (Fig. 2). About 2 mm of
the implant end in the femur metaphysic was exposed by epiphyseal
separation, and the compression speed was 1 mm/min. The maximal
force and interfacial shear strength were calculated by the displacement
versus force.
Histomorphometric analysis
axis of implant using a rotary diamond saw (SP1600, Leica, Germany),
and ground by Leica SP2600 (Germany). Thus, we obtained a 50-μm-
thick undecalcified section, which was stained in 1% toluidine blue.
Hitomorphometric analysis was performed on sections by means of
a Leica DMI 6000 B micro-system (Germany). Bone-to-implant con-
tact (BIC, the linear percentage of the interface with direct bone-to-
implant contact to total interface of the implant in the cancellous
bone) and bone area ratio (BA, the area percentage of bone tissue
to the whole area) were measured approximately 2 mm above the
epiphyseal plate, and BA was measured within the area extending
200 μm from the implant surface [29].
Serum value of 25(OH)D3
12 weeks after implantation surgery, approximately 200 μl of
blood was obtained from each animal using a tail snip before sacrifice
(n = 6 specimens per group). Serum was obtained by centrifugation
at 14,000 ×g for 10 min at 4 °C from blood. Values of 25(OH)D3 in
the serum of rats were analyzed by enzyme-linked immunosorbent
assay (ELISA) strictly following the manufacturer's protocol (ELISA
kit, R&D System, USA). Briefly, 100 μl of grades standard solution or
sample was added into the precoated 96-well plates, and the plate
was sealed with cover and incubated at 37 °C for 60 min. Then remove
the cover, discard the plate content, and wash the plate for several
times. Add antibodies and substrate in order into the wells and the re-
action were stopped by stop solution after 15 min. The OD absorbance
was read at 450 nm in a microplate reader within 30 min.

Animals were sacrificed 12 weeks after implantation, the femur
with implants were removed immediately, cleaned of soft tissue,
and maintained in a 4% neutral formalin buffered solution for 2 days
(n = 6 specimens per group). Then these specimens were washed,
dehydrated through a graded series of ethanol solutions, and embed-
ded in methylmethacrylate without decalcification [28]. Subsequent-
ly, the embedded specimens were sawn perpendicular to the long
Statistical analysis
Data were expressed as mean ± SD (standard deviation), and stat-
ically analyzed using one-way ANOVA followed by Newman–Keuls
post hoc tests. Differences at probability of less than 0.05 were consid-
ered statistically significant. All data were analyzed with SPSS 15.0
software.
4 Y. Wu et al. / Bone 52 (2013) 1–8

Results Table 2
Weight (g) for the rats subjected to different therapies during the experiment.

Blood glucose level Animal status 0 day 1 month 2 months 3 months

Control 197 ± 16 297 ± 13* 368 ± 24* 416 ± 19*

Blood glucose levels of all experiment animals were presented in
Table 1 and Supplementary Table 1. Blood glucose levels in rats before
STZ administration were all within the normal range. Diabetic rats
showed significantly greater glucose level than controls since STZ ad-
ministration. During the initial 14 days of experiment, both of insulin
treatment and VD3 treatment showed positive effects on decreasing
the glucose level in diabetic rats (p b 0.05) (Table 1). After 1 month
of therapy, high blood glucose levels in diabetic rats were found to
be normal in mono-insulin treated or combination-treated rats. In
mono-VD3 treated rats, the blood glucose level decreased significant-
ly, but still remained high compared with those in controls (p b 0.05)
(Supplementary Table 1).
Body weight
Changes on body weight were monitored throughout the experi-
ment (Table 2). All animals had a significant gain in weight over the
time period except the untreated diabetic rats. STZ administration in-
duced loss of weight in diabetic rats, which were significantly lower
than controls (pb 0.05). Combined treatment increased the body weight
in diabetic rats to the highest level compared to monotreatment
(pb 0.05).
Micro-CT evaluation
3D micro-CT images depicted the bone–implant interface and tra-
becular topography among rats subjected to different treatments
(Fig. 3). Combined treatment has the strongest effects on improving im-
plant osseointegration and peri-implant trabecular microstructure.
Quantitative evaluation gave more detailed information on %OI and
trabecular parameters around implants, which was shown in Table 3.
Combined treatment significantly increased the BV/TV by 1.37-fold,
the %OI by 1.6-fold, the mean Tb.Th by 0.64-fold, the Tb.N by 1.38‐
fold, and the Conn.D by 1.41‐fold and decreased the Tb.SP by 41.5% in
comparison to untreated diabetic rats (pb 0.05). Monotreatment also
revealed similar effects but to a more moderate extent.
Push-out test
Results of push-out test were expressed as maximal push-out force
and ultimate shear strength in Fig. 4, which were similar to the results
of micro-CT evaluation. After a 12-week healing period, combined
treatment exerted the strongest effects on improving values of the
two biomechanical indices. The maximal push-out force increased
by 1.3-fold, and the ultimate shear strength by 2.1-fold compared to
untreated diabetic rats (p b 0.05). And there were no significant differ-
ences about these indices between combination treated diabetic rats
and controls (p > 0.05). Monotreatment also significantly increased
values of the two biomechanical indices, but not more than combined
treatment.
Diabetic 218 ± 22 241 ± 14 257 ± 19 233 ± 17
Insulin-treated diabetic 204 ± 18 274 ± 18* 319 ± 17* 345 ± 17*
VD3-treated diabetic 220 ± 19 252 ± 22 288 ± 20* 319 ± 13*,a
Insulin + VD3-treated diabetic 205 ± 23 291 ± 24* 347 ± 22*,a 404 ± 19*,a,b
Data are expressed as mean±SD; n=6 specimens/group. *: pb 0.05, for diabetic rats vs.
others; a: pb 0.05, for VD3-treated or insulin+VD3-treated diabetic rats vs. insulin- treated
diabetic rats; b: pb 0.05, for insulin+VD3-treated diabetic rats vs. VD3-treated diabetic
rats.
Histomorphometric analysis
The undecalcified sections showed the details of implant osseo-
integration and peri-implant bone mass (Figs. 5A–E). Results from
histomorphometric analysis were presented as BIC and BA (Figs. 5F
and G). Diabetic rats developed BIC of 18.4%, significantly less than
controls, which had 62.1% BIC (p b 0.05). BA associated with implants
placed in untreated diabetic rats was also dramatically less than that in
controls. Insulin treatment significantly increased histomorphometric
parameters of implant in diabetic rats, with the BIC increased by
1.51-fold and the BA by 0.61-fold compared to untreated diabetic rats.
However, these parameters were lower than those of combination
treated diabetic rats, with the BIC increased by 2.57-fold and the BA
by 1.44-fold compared to untreated diabetic rats (pb 0.05). The param-
eters of BIC and BA in the combination treated diabetic rats were similar
to that in controls, indicating that implant osseointegration of diabetic
rats was restored to near normal levels by combined treatment. There
was no significant difference of BA between the monoVD3-treated dia-
betic rats and untreated diabetic rats (p>0.05).
Serum value of 25(OH)D3
25(OH)D3 serves as the indicator of VD3 nutritional status, so the
values of 25(OH)D3 in rats were measured by ELISA kit. Results in
Fig. 6 showed that serum 25(OH)D3 level in untreated diabetic rats
significantly decreased compared to controls, but it was elevated to
normal range by insulin treatment. Administration of VD3 resulted
in a significantly higher serum 25(OH)D3 level than that in controls,
and combined treatment enhanced value to the highest level among
the rats subjected to different treatment (p b 0.05).
Discussion
In our study, the hyperglycemia of rats was induced by adminis-
tration of STZ, the facts of increased blood glucose level and decreased
body weight are similar with previous reports as a result of the
destruction of insulin secreting pancreatic β-cells by STZ [30]. The in-
fluence of DM on dental implants has been studied in recent years,
because this systemic disease may reduce bone mineral content and
negatively interfere with the process of implant fixation. Previous

Table 1
Blood glucose (mg/dl) level in experimental rats.

Animal status 0 day 3rd day 6th day 10th day 14th day

Control 112.3 ± 12 116.5 ± 13* 108.6 ± 9* 122.3 ± 14* 127.7 ± 15*

Diabetic 115.6 ± 8 365.1 ± 22 397.3 ± 16 376.8 ± 17 383.3 ± 15
Insulin-treated diabetic 107.3 ± 12 326.8 ± 14 282.6 ± 17 247.9 ± 14* 175.5 ± 13*
VD3-treated diabetic 122.7 ± 11 347.4 ± 13 311.0 ± 15 272.5 ± 12* 234.4 ± 11*
Insulin + VD3-treated diabetic 119.3 ± 13 335.6 ± 19 255.4 ± 14* 208.5 ± 9*,a,b 164.6 ± 10*,b

Data are expressed as mean ± SD; n = 6 specimens/group. *: p b 0.05, for diabetic rats vs. others; a: p b 0.05, for VD3-treated or insulin + VD3-treated diabetic rats vs. insulin-treated
diabetic rats; b: p b 0.05, for insulin + VD3-treated diabetic rats vs. VD3-treated diabetic rats.
 Y. Wu et al. / Bone 52 (2013) 1–8 5

Fig. 3. Micro-CT images of the distal femur with implants 12 weeks after implantation. The left column showed transverse 3-D images through cross- sectional plane of implants,
and the right column exhibited coronary 3-D images through the central portion of the long axis of implants.

Table 3
Quantitative results of the micro-CT evaluation 12 weeks after implantation.

Parameters Groups

Control Diabetic Insulin-treated diabetic VD3-treated diabetic Insulin + VD3-treated diabetic

BV/TV (%) 44.97 ± 4.3* 18.10 ± 2.6 29.58 ± 3.4* 20.91 ± 4.0a,b 42.81 ± 7.6*,a
% OI 60.87 ± 4.3* 20.38 ± 2.6 44.09 ± 3.6* 35.38 ± 1.9*,a,b 64.26 ± 6.1*
Tb.Th (μm) 0.24 ± 0.02* 0.14 ± 0.01 0.18 ± 0.02* 0.17 ± 0.03*,b 0.23 ± 0.01*,a
Tb. N (mm−1) 3.46 ± 0.27* 1.4 ± 0.11 2.58 ± 0.13* 2.07 ± 0.25*,a,b 3.33 ± 0.28*
Conn. D (mm−3) 34.24 ± 2.9* 14.36 ± 0.64 21.08 ± 1.28* 24.3 ± 3.0*,b 34.64 ± 3.7*,a
Tb. Sp (μm) 0.21 ± 0.02* 0.53 ± 0.04 0.4 ± 0.04* 0.32 ± 0.03* 0.31 ± 0.03*,a

Data are expressed as mean ± SD; n = 6 specimens/group. BV/TV: ratio of bone tissue volume to total tissue volume; %OI: ratios between bone and total voxels in direct contact
with the implant; Tb.Th: the mean trabecular thickness; Tb.N: the mean trabecular number; Conn.D: the mean connectivity density; Tb.Sp: the mean trabecular separation.
*: p b 0.05, for diabetic rats vs. others; a: p b 0.05, for VD3-treated or insulin + VD3-treated diabetic rats vs. insulin-treated diabetic rats; b: p b 0.05, for insulin + VD3-treated diabetic
rats vs. VD3-treated diabetic rats.
6 Y. Wu et al. / Bone 52 (2013) 1–8
Fig. 4. Histograms of mechanical push-out test parameters 12 weeks after implantation. Data are expressed as mean ± SD; n= 6 specimens/group. *: p b 0.05, for diabetic rats vs.
others; a: p b 0.05, for VD3-treated or insulin + VD3-treated diabetic rats vs. insulin-treated diabetic rats; b: p b 0.05, for insulin + VD3-treated diabetic rats vs. VD3-treated diabetic
rats.
experiments have confirmed that DM can impair bone healing and
remolding in the limbs, cranium, and jaw. Additionally, the inflamma-
tory response around dental implants was greater in diabetic subjects,
which would influence implant osseointegration [31,32]. Results of
our analysis demonstrated that implant osseointegration, peri-implant
trabecular microstructure and implant fixation significantly decreased
in untreated diabetic rats.
Previous studies in diabetic patients have focused mainly on control-
ling blood glucose by insulin treatment to improve implant osseo-
integration [1,3,6,7]. Verhaeghe et al. demonstrated that although
most parameters of bone growth and formation in insulin-dependent
BB rats were restored to normal by insulin treatment, the mineral appo-
sitional rate was lower in the insulin treated BB rats than that of controls
[33]. Other studies also accept the fact that normal glucose level
obtained by insulin therapy might not restore all alterations induced
by DM [3]. In this study, body weight and serum 25(OH)D3 levels in
diabetic rats were elevated to normal range by insulin treatment. How-
ever, the high blood glucose levels in diabetic rats were not restored
to normal level until 1 month by mono-insulin treatment. A study by
McCracken observed that the diabetic rats which received a subcutane-
ous slow-release insulin implant maintained blood glucose levels
similar to controls at 2, 7, and 14 days [7]. An explanation for these dif-
ferences may originate in the inadequate insulin doses in this study. Re-
sults from push-out test also showed that insulin increased implant
fixation in diabetic rats 12 weeks after implantation, with the maximal
push-out force and ultimate shear strength increased by 78% and 47%
in push-out test. However, the results from micro-CT evaluation and
histomorphometric analysis indicated that peri-implant trabecular mi-
crostructure of insulin treated diabetic rats was not as well organized
as controls and implant osseointegration was also lower compared
to controls. This finding is in agreement with the study by Fiorellini
et al., who showed that although insulin treatment improves the total
quantity of bone formation in diabetic subjects, the bone–implant-
contact was significantly less than healthy controls [34].
VD3 has been extensively studied for its dual action of promoting
bone remodeling and maintaining glucose homeostasis, but few evi-
dence of influence of VD3 on implant osseointegration in diabetic
rats was detected. In our study, serum 25(OH)D3 level in untreated
diabetic rats decreased significant compared to controls, and admin-
istration of VD3 resulted in a higher serum 25(OH)D3 level than that
in controls. However, the OI% in micro-CT evaluation and bone area
ratio in histomophometric analysis in diabetic rats were not signifi-
cantly improved by mono-VD3 treatment. Verhaeghe et al. pointed
out that despite exogenous 1,25(OH)2D3 results in a normal in vivo re-
sponse in the duodenum and kidney in BB rats, the diabetes-induced
defect in bone matrix formation was not restored [35]. This may be
because of the uncontrolled hyperglycemia in mono-VD3 treated dia-
betic rats. Del Pino-Montes et al. demonstrated that an increase in
bone mineral density (BMD) in both cortical and trabecular bone
could be seen in the diabetic-treated reversed rats after 1,25(OH)2D3
therapy, but these increases in BMD did not appear in animals still
remaining diabetic [36]. So maybe VD3 would play its largest role in
bone remodeling and glucose homeostasis in diabetic subjects when
blood glucose level was under control. Our study based on systemic
use of insulin and VD3 aimed to investigate the effect of combined
treatment on implant osseointegration in diabetic rats and to find
out whether the effect is additive. Results demonstrated that hyper-
glycemia and body weight in diabetic rats were restored to normal
level by insulin and VD3 combined treatment, and the impaired osseo-
integration capacity of implants in diabetic rats was also reversed by
the combined treatment. In micro-CT evaluation, the VOI was defined
approximately 2 mm above the growth plate and 200 μm from the
implant surface to identify the peri-implant trabecular microstructure.
In histomorphometric analysis, undecalcified sections obtained from
site about 2 mm above the epiphyseal plate distal were evidence
of new bone formation. As a result, increased bone volume ration,
improved trabecular parameters and implant osseointegration were
observed in combination treated diabetic rats. In addition, push-out
test further demonstrated enhanced implant fixation in combination
treated diabetic rats. The strongest relationship between micro-CT
and push-out parameters was revealed in BV/TV and the maximal
push-out fore, which indicated that increased bone formation on
bone–implant interface played an important role in early implant
stability [37]. All the results demonstrated that VD3 contributed to
the promoted new bone formation and then the improved implant
osseointegration in diabetic rats. Del Pino-Montes et al. explained
that maybe VD3 administration can halt the pancreatic damage caused
by STZ, and then enhance both insulin synthesis and secretion, which
improves bone production and mineralization in diabetic-reversed
subjects [36]. Other studies also indicated that VD3 exerts its effects
 Y. Wu et al. / Bone 52 (2013) 1–8 7

Fig. 5. Histological images of the distal femur with implants approximately 2 mm above the epiphyseal plate 12 weeks after implantation (A: Control; B: Diabetic; C: Insulin-treated
diabetic; D: VD3-treated diabetic; E: Insulin+VD3-treated diabetic; toluidine blue staining, original magnification×40); and histograms of the BIC (F) and BA (G) in hitomorphometric
analysis. Data are expressed as mean±SD; n=6 specimens/group. *: pb 0.05, for diabetic rats vs. others; a: pb 0.05, for VD3-treated or insulin+VD3-treated diabetic rats vs. insulin-
treated diabetic rats; b: pb 0.05, for insulin+VD3-treated diabetic rats vs. VD3-treated diabetic rats.

through VDR, which are found in a wide variety and tissues, including
T and B lymphocytes, skeletal muscle and the pancreatic islet β-cells.
And VD3 may play a functional role in maintaining glucose tolerance
through its effects on insulin secretion and sensitivity [2,38,39].
Our results demonstrated the beneficial effects of combined treat-
ment on implant fixation in diabetic rats. However, the observation
time of 12 weeks merely revealed results as the early stages of im-
plant fixation, and longer observation time was needed for evaluation
of the long-term efficiency of insulin and VD3 combined treatment. In
addition, physiological conditions of diabetic rats were different from
that of diabetic patients, so the VD3 administration in DM patients
needs research in further study.
Conclusion
This study took advantage of the dual action of VD3 on promoting
bone remodeling and maintaining glucose homeostasis in diabetic
subjects. Our results indicated that insulin and VD3 combined treat-
ment had additive effects on reversing the impaired osseointegration
capacity of implants in diabetic rats, resulting in greater gains in im-
plant osseointegration and fixation than monotreatment. However,
it should be noticed that results from animal studies might not be
linearly and directly applied to humans, as a result of the genetic dif-
ferences between both species. So the administration of VD3 in DM
patients needs research in future study.
8 Y. Wu et al. / Bone 52 (2013) 1–8
Fig. 6. Histograms of serum 25(OH)D3 level 12 weeks after implantation. Data are ex-
pressed as mean±SD; n=6 specimens/group. *: pb 0.05, for diabetic rats vs. others;
a: pb 0.05, for VD3-treated or insulin+VD3-treated diabetic rats vs. insulin-treated diabetic
rats; b: pb 0.05, for insulin+VD3-treated diabetic rats vs. VD3-treated diabetic rats.
Supplementary data to this article can be found online at http://
dx.doi.org/10.1016/j.bone.2012.09.005.
Acknowledgments
This study was supported by a grant from the National Natural
Science Foundation of China (81170995). We appreciate the assis-
tance of Sixiu Chen, Ning Gi and Ga Liao from the State Key Laboratory
of Oral Disease, Sichuan University.
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