Introduction and Rochester trials (8, 9). However another study


A close relationship between diabetes
mellitus (DM) and fragility fracture was demonstrated in both Type 1 diabetes
mellitus (T1DM) and Type 2 DM (T2DM). The increase in fracture risk was
associated with the decrease in bone mineral density (BMD) in T1DM, whereas BMD
is usually normal or increases in T2DM (1, 2). It was shown that the femur
fracture risk increased twofold in T2DM (3). Additionally, it was set forth
that the biomechanical quality of the bone changed in T2DM owing to the presence
of no relationship between fracture risk and BMD. Studies indicated that the
bone quality was impaired, which was related to the decreased bone strength in diabetic rats (1). Changes in the stiffness of diaphyseal femur,
energy absorption capacity, and tensional strength were reported (4, 5).
Nevertheless, the entire biomechanical quality and especially the changes in
intrinsic properties of the bone are unclear (1).

We Will Write a Custom Essay Specifically
For You For Only $13.90/page!

order now

Metformin is widely used as the first-line
antidiabetic drug of choice in T2DM patients (6). There are conflicting results
in literature about the influence of metformin on fracture risk in T2DM. ADOPT
trial showed no effect of metformin on fracture risk in T2DM (7).
However another study indicated that metformin reduced the fracture risk (8).
Glyburide is an insulin secretagogue of sulphonylurea group. Glyburide
treatment was shown not to have an effect on bone mass in ADOPT and Rochester
trials (8, 9). However another study showed that glyburide treatment reduced
osteoblastic marker levels (10).

Combination treatments are widely used in
T2DM. Glucovance is an oral antidiabetic agent which includes metformin and
glyburide (11). Influence of Glucovance treatment on intrinsic and extrinsic
biomechanical properties of diabetic bones was not evaluated until today.
Maximum fracture strength which reflects the structural integrity of the tissue
best and maximum deformation amount which is used for determining the
structural properties of the tissue are the most frequently used important
biomechanical parameters. In this study, we investigated the effect of
Glucovance treatment on biomechanical properties of the bone in rats with
streptozotocin (STZ)-induced DM.

Materials and Methods


total of 28 male Wistar-Albino rats (12-week-old; 210-300 g) were housed in an
air-conditioned room at a constant temperature of 22± 2ºC with 12:12
h light/dark cycle and fed a standard diet and water ad libitum. Following
1-week of acclimatization prior to experimental procedures, the rats were
assigned randomly and equally (seven rats per group) to 4 experimental groups
including control (C; no treatment; n=7), sham Sh; distilled water (orally by
gavage, for 8 weeks); n=7, diabetes DM; streptozotosin (45 mg/kg,
single i.p injection); n=7 and diabetes + Glucovance treatment DM+G;
streptozotosin (45 mg/kg, single i.p injection) + Glucovance (Glucovance,
500/5 mg/kg/day/rat, orally by gavage, for 8 weeks)

All experiments and protocols described in the present study were performed
in accordance with the Guide for the Care and Use of Laboratory Animals, as
adopted by National Institutes of Health (U.S.) and also approved by the
Medical Faculty Experimentation Ethics Committee of Mersin University.

Induction of DM

was induced by single intraperitoneal injection of 45 mg/kg STZ (STZ;
Sigma-Aldrich, Interlab Corporotion, Istanbul, Turkey) dissolved in 0.01M
sodium citrate (pH adjusted to 4.5) under ether anesthesia. After 3 days
following STZ administration, blood was collected from tail vein and samples
were analyzed for blood glucose by using a glucometer (Aqua-Check, Roche,
Basel, Switzerland). Animals with fasting blood glucose levels (BGLs) of
>250 mg/dL were considered as diabetic rats (12).

Bone evaluations

eight weeks, the study was terminated. At termination, the left femur of each
animal was harvested under Ketamine (Ketalar, Eczac?bas?, Istanbul, Turkey)
anesthesia (50 mg/kg, i.m.) and stored at ?20 °C until mechanical testing.

biomechanical measurements and parameters were established from the protocols
which were previously described by Gürgül et al (13) and Burr (14). Tensile test was performed at the left femur to
measure the ultimate tensile strength (N; maximum load), displacement (mm),
stiffness (N/mm), energy absorption capacity (mJ), ultimate stress (MPa),
ultimate strain (mm/mm), elastic modulus (GPa), and toughness (MPa) of bone by
using a custom-made biomaterial testing machine (MAY BTS03, Commat Ltd, Ankara,
Turkey). The data were collected by using BIOPAC
MP100 Acquisition System (BIOPAC Systems, Santa Barbara, CA, USA) and analyzed
by Acqknowledge Version 3.5.7 software (BIOPAC Systems, Santa Barbara, CA,
USA). Cross-sectional area (mm2) of the
femoral shaft was measured by computerized tomography (Toshiba Aquilion 64
Slice CT, Toshiba American Medical Systems, Tustin, CA, USA) and analyzed using
Vitrea software (Version 2.0, Minnetonka, MN, USA).

mineral density (BMD; g/cm2) was measured at mid-diaphysis femoral
region by dual-energy X-ray absorptiometry (DEXA; Norland 45 XR, Norland
Scientific Instruments, Fort Atkinson, WI, USA) and the analysis was performed
by using Illuminatus-based DEXA software (Version 4.2.0, Visual MED, Charlotte,

Statistical Analysis

analysis was made using SPSS software (Version 11.5.1, Lead Technologies Inc.,
Chicago, IL, USA) and Statistica software (Version 6.1, StatSoft Inc.,
Tulsa, OK, USA). After checking for normal
distribution with Shapiro–Wilks test, data were analyzed by one-way ANOVA or
ANOVA for repeated measurements followed by Tukey’s multiple comparison tests
as applicable. The data were expressed as “mean ± standard deviation (SD)”
and/or percent (%). p0.05), it was shorter than Sh
group (p0.05, for each).
BMD was signficantly greater in DM rats compared to C and Sh group (p0.05).

Mechanic parameters of diaphyseal femur

Mechanic parameters of diaphyseal femur are summarized
in Table 3. Displacement decreased significantly in DM (p0.05). Ultimate stress was significantly lower in DM
compared to C, Sh and DM+G rats (p0.05), it was
shorter than Sh group (p0.05, for each). BMD was significantly greater in DM rats compared to C
and Sh group (p0.05).

Mechanic parameters of diaphyseal femur

Mechanic parameters of diaphyseal femur
are summarized in Table 3. Displacement (mm) decreased significantly in DM
Ultimate stress (Mpa) was significantly lower in DM compared to C, Sh and DM+G
rats (p0.05 for each). These results suggest that Glucovance treatment may play
a role in BMD restoration.

The exact mechanisms of the effect of T2DM on bone
fragility are not known. Recently, the relationship between the duration of
diabetes ad fracture was evaluated in T2DM and bone strength was seen to be
better in the early stages of the disease (16, 17). Type 1 collagen is the most
frequently found collagen in bone. Collagen matrix plays an important role in
mechanic power of the bone through forming cross links between molecules. There
are two types of collagen cross links in the bone as enzymatic cross link and
glycation or oxidation-induced advanced glycation endproducts (AGEs). Bone
strength decreases independently from BMD in diabetic bones due to AGEs
formation in case of hyperglycemia and oxidative stress (18, 19). In recent
studies, enzymatic and AGEs cross links were shown to effect toughness,
stiffness and elastic modulus of the bone (18, 20-22).

Biomechanical properties such as ultimate stress,
ultimate strain, elastic modulus and toughness are the main parameters used for
evaluating the fragility of the bone. These parameters show the biomechanical
integrity of the bone. It is accepted that crystallinity increase,
mineralization defects (hypo or hyper-mineralization) and/or collagen
deformation increase indicate the combined changes in these parameters (1, 14,
23, 24). In this study, all these parameters were detected to decrease in DM
rats. That ultimate stress increased and reached the levels of control group in
Glucovance treatment-receiving diabetic rats compared to the ones which do not
receive suggests that this treatment has a partially beneficial effect on
biomechanical integrity of the bone.

Biomechanical properties such as displacement, maximum
load, stiffness and absorbed energy are the structural parameters of the bone.
These parameters are closely related with the quality and strength of the bone
(1). Bone strength and displacement were shown to decrease and stiffness was
shown to increase in T2DM rats (1, 25). In our study, maximum load, energy
absorption capacity and displacement were shown to decrease and stiffness was
shown to increase in DM rats and this result indicates that bone quality is
impaired and femur strength is decreased in DM. Maximum load is the power which
is applied until the bone is broken.(14) Presence of a significant
increase in maximum load in Glucovance-receiving diabetic rats compared to DM
suggest that this treatment has a healing effect on bone quality and femur

Geometric properties are important factors for
evaluating the quality  and strength of
the bone (13, 26-28). In this study, there was not a significant difference
between groups with regard to cross-sectional area of femur. Femur length was
detected to significantly reduce in DM compared to C and Sh rats. There was not
a significant difference between Glucovance-receiving diabetic rats and C group
with regard to femur length.

In conclusion, an improvement in ultimate stress and
maximum load, the impaired biomechanical parameters of the bone, following
Glucovance treatment suggests that this treatment may have a partial improvement
in increase of the quality of the diabetic bone and in decrease of fragility in
STZ-induced diabetic rat model.

Conflict of interest: The authors declare that
there is no conflict of interest that could be perceived as prejudicing the
impartiality of the research reported.

Financial Disclosure: None.


1. Erdal N,
Gürgül S, Kavak S, Yildiz A, Emre M. Deterioration of Bone Quality by
Streptozotocin (STZ)-Induced Type 2 Diabetes Mellitus in Rats. Biol Trace Elem Res. 2011;140(3):342-353.

Leecka-Czernik B. Bone as a target of type 2 dm treatment. Curr Opin Investig

3. Janghorbani M, Van Dam RM, Willett WC, Hu FB. Systematic review of type 1 and type 2 diabetes
mellitus and risk of fracture. Am J Epidemiol. 2007;166(5):495-505.

4. Dixit PK, Ekstrom RA. Decreased breaking
strength of diabetic rat bone and its improvement by insulin treatment. Calcif
Tissue Int 1980;32(3):195-199

5. Einhorn TA, Boskey AL, Gundberg CM, Vigorita
VJ, Devlin VJ, Beyer MM. The mineral and mechanical properties of bone in
chronic experimental diabetes. J Orthop Res 1988;6(3): 317-323.

6. Halimi
S. Management of type 2 diabetes: new or previous agents,
how to choose? Presse Med. 2013;42(5):861-870.

7. Kahn SE, Zinman B, Lachin JM, Haffner SM, Herman WH, Holman RR, et al. Diabetes Outcome Progression Trial (ADOPT) Study Group. Diabetes Care, Rosiglitazone-associated fractures in
type 2 diabetes. Diabetes Care. 2008;31(5):845-851

8. Melton LJ 3rd, Leibson CL, Achenbach SJ, Therneau TM, Khosla S. Fracture risk in type 2 diabetes: update of a
population. J Bone Miner Res. 2008;23(8):1334-1342

9. Kahn SE, Haffner SM, Heise MA, Herman WH, Holman RR, Jones NP, et al. ADOPT Study Group. Glycemic durability of rosiglitazone, metformin, or
glyburide monotheraphy. N Engl J Med. 2006;355(23):2427-2443.

10. Zinman B, Haffner SM, Herman WH, Holman RR, Lachin JM, Kravitz BG, et al. ADOPT Study Group. Effect of rosiglitazone,
metformin, and glyburide
on bone biomarkers in patients with type 2 diabetes. See
comment in PubMed Commons belowJ Clin Endocrinol

11. Davidson JA, Scheen AJ, Howlett HC. Tolerability profile of metformin/glibenclamide
combination tablets (Glucovance): a new treatment
for the management of type 2 diabetes mellitus. Drug Saf. 2004;27(15):1205-1216.

12. Andrade-Cetto A, Mart´?nez-Zurita E, Wiedenfeld H.
Hypoglycemic effect of Malmea depressa root on streptozotocin diabetic rats. J Ethnopharmacol

13. Gurgul S, Erdal N, Yilmaz SN, Yildiz A, Ankarali
H. Deterioration of bone quality by long-term magnetic field with extremely low
frequency in rats. Bone 2008;42(1):74–80

14. Burr
DB. The contribution of the organic matrix to bone’s material properties.
Bone  2002;31(1):8-11.

15. Erdal N, Gürgül S, Demirel C, Yildiz A. The effect of insulin therapy on biomechanical deterioration
of bone in streptozotocin (STZ)-induced type 1 diabetes mellitus in rats. Diabetes Res Clin Pract. 2012;97(3):461-467

Merlotti D, Gennari L, Dotta F, Lauro D, Nuti R. Mechanisms of impaired bone
strength in type 1 and 2 diabetes. Nutr Metab Cardiovasc Dis. 2010;20(9):683-690.

17. Reid IR. Relationships between fat and bone. Osteoporos Int

18. Saito M, Fujii K, Mori Y, Marumo K. Role of
collagen enzymatic and glycation induced crosslinks as a determinant of bone
quality in spontaneously diabetic WBN/Kob rats. Osteoporos Int

19. Baynes JW. Role of oxidative stress in development
of complications in diabetes. Diabetes 1991;40(4):405–412

20. Saito M, Marumo K. Collagen cross-links as a
determinant of bone quality: a possible explanation for bone fragility in
aging, osteoporosis, and diabetes mellitus. Osteoporos Int 2010;21(2):195–214.

21. Viguet-Carrin S, Roux JP, Arlot ME, Merabet Z, Leeming DJ, Byrjalsen I, et al. Contribution of the advanced glycation end
product pentosidine and of maturation of type I collagen to compressive
biomechanical properties of human lumbar vertebrae. Bone 2006;39(5):1073–1079.

22. Vashishth D, Gibson GJ, Khoury JI, Schaffler MB,
Kimura J, Fyhrie DP. Influence of nonenzymatic glycation on biomechanical
properties of cortical bone. Bone 2001;28(2):195–201.

23. Turner CH. Biomechanics of bone:
determinants of skeletal fragility and bone quality. Osteoporos Int

24. Boyar H, Turan B, Severcan F. FTIR
spectroscopic investigation of mineral structure of streptozotocin induced
diabetic rat femur and tibia. Spectroscopy 2003;17:627-633.

25. Einhorn TA, Boskey AL, Gundberg CM,
Vigorita VJ, Devlin VJ, Beyer MM. The mineral and mechanical properties of bone
in chronic experimental diabetes. J Orthop Res 1988;6(3): 317-323.

26. Martin RB, Boardman DL. The effects of
collagen fiber orientation, porosity, density and mineralization of bovine
cortical bone bending properties. J Biomech 1993;26(9):1047–1054.

27. Ferretti JL, Cointry GR, Capozza RF, Capiglioni R,
Chiappe MA. Analysis of biomechanical effect on bone and on the bone muscle
interactions in small animal models. J Musculoskelet Neuronal Interact

28. Valko M, Leibfritz D, Moncol J, Cronin MTD, Mazur
M, Telser J. Free radicals and antioxidants in normal physiological functions
and human disease. Int J Biochem Cell Biol 2007;39(1):44-84.