Keywords alendronate - vitamin D - menopause - femur - rats
Introduction
Osteoporosis is a disease marked by loss of mineralized bone mass, making it fragile
and vulnerable to fractures. Anatomically, there is a decrease in cortical thickness
and porosity, a reduction in the number and size of spongy bone trabeculae, and an
enlargement of the medullary spaces.[1 ]
[2 ] Currently, the world is experiencing an expansion of the incidence of osteoporosis
in both genders, especially in women > 50 years old, as a result of the continuous
aging of the population.[3 ]
Worldwide, osteoporosis is a disease that affects > 200 million people. Even though
it is a disease that can affect both genders, postmenopausal women are the main risk
group, with an estimated prevalence of 30% in Western countries.[4 ] For the year 2050, it is estimated that 70% of the hip fractures that will occur
in Africa, Asia, and Latin America will be related to osteoporosis; therefore, osteoporosis
of great clinical importance.[5 ]
Several effective therapies are competent to produce an attenuation in the risk of
fractures, especially in postmenopausal women. Nitrogen bisphosphonates (BFs) are
the most recommended class of drugs for the treatment of postmenopausal osteoporosis.
They act by inhibiting bone resorption with few side effects.[1 ] Among the most used BFs, sodium alendronate (ALN) stands out.[1 ]
[6 ]
Vitamin D (VD) is a steroid hormone that has varied biological actions in different
target tissues.[7 ] There is already significant evidence that low serum calcium and VD levels accelerate
bone loss.[8 ]
The biomechanical competence of bone is related not only to the amount of bone present,
but also to its microstructure.[9 ] Thus, bone histomorphometry is one of the conventional methods to analyze its microarchitecture,
which allows, in a safe way, to qualify and quantify bone structures.[10 ] Thus, it plays a prominent role in the study of certain metabolic disorders and
their treatments.
Thus, the aim of the present study was to verify how the combined administration of
ALN and VD acts on bone microarchitecture in the treatment groups of Wistar rats with
glucocorticoid-induced osteoporosis.
Materials and Methods
All procedures were approved by the Ethics Committee on the Use of Animals (CEUA)
of the Universidade Federal de Pernambuco (UFPE, in the Portuguese acronym), Recife,
state of Pernambuco, Brazil (Protocol No. 034/2020).
Experimental procedure
An experimental, double-blind animal study (evaluator and pathologist) was carried
out. In the present work, 32 female Wistar rats (Rattus norvegicus albinus) between 8 and 10 weeks old and weighing between 300 and 400g from the Physiology
Bioterium of the UFPE were used.
The animals were housed in individual polypropylene cages with metal lids, kept in
rooms with a controlled ambient temperature of 22°C, with luminosity of 60 lux, kept
in a light/dark cycle of 12 hours controlled by time sensor, on a shaving bed, fed
with feed for rats and water ad libitum .
After a period of 60 days, the animals were randomly divided into 5 groups, as shown
in [Table 1. ] Groups G2, G3, G4, and G5 had osteoporosis induced with the use of intramuscular
dexamethasone, while the animals of group G1 were kept without induction of osteoporosis
to establish the parameter of the negative control group.
Table 1
Group
Number of animals
Group description
Induction of osteoporosis
G1
6
Distilled water orally
No
G2
6
Distilled water orally
Yes
G3
7
Oral sodium alendronate
Yes
G4
7
Vitamin D3 orally
Yes
G5
6
Sodium alendronate and Vitamin D3 orally
Yes
The process of induction of osteoporosis was performed with the administration of
glucocorticoid dexamethasone intramuscularly at a weekly dose of 7.5 mg/kg of body
weight for 5 weeks. At the end of the 5th week of the administration of dexamethasone, the animals were submitted to pharmacotherapeutic
treatment.
The daily volume of therapeutic drugs administered to all groups was 0.03mL of vehicle.
Sodium alendronate administration alone occurred at a dosage of 0.2mg/kg daily for
45 days. Vitamin D3 was administered at a dose of 500 μL (10,000IU/500μL) once a week during the 8-week
supplementation period. The same period and dosage were used for weekly administration
in the combination of the two drugs. The control group animals received distilled
water for the same period and in the same amount as the experimental groups. The medications
were administered orally (gavage method) by appropriate cannulas.
Histomorphometry
After completing the therapeutic regimen, the rats were euthanized with anesthetic
deepening based on xylasin hydrochloride at 2% associated with ketamine hydrochloride
10% for removal of the right and left femurs using a scalpel blade no. 11 and 15 to
perform histological analyses, which were stored in 10% formaldehyde in properly labeled
containers, according to the sample group of each animal.
After collection of femoral bones, 64 specimens were obtained, fixed in 10% formaldehyde,
and kept for a period of 48 hours, necessary for fixation, until the moment of preparation.
The 64 specimens were divided as follows: the right femurs were submitted to histological
evaluation, while the left femurs were macroscopically evaluated. The preparation
followed the routine patterns for histological study in all right femurs. After the
fixation period, the 32 femurs were decalcified with a solution of nitric acid (HNO3 ) at 5%, changed daily, for 5 days.
After decalcification, the samples were washed in distilled water and placed in the
histotechnical processor Leica and subsequent inclusion in paraffin Paraplast. With
the aid of a microtome (Hestion), all blocks containing the femoral fragments were
sectioned longitudinally at a thickness of 4 μm and placed on slides previously greased
with Mayer albumin and kept in a regulated oven at 37°C for 24 hours for drying and
gluing.
Subsequently, they were cordoned by hematoxylin and eosin (H&E) according to the methodology
of Junqueira et al.[11 ] The samples were then analyzed under a light microscope and the sections were photographed
in a Nikon 50E Trinocular Biological Microscope with VT 480 videomicroscopy and image
analyzer. All stages of the histological procedure were performed at the Graduate
Laboratory in Translational Health of the UFPE.
For the study of the compact bone, cross-sections of the diaphysis of the right femur
of each animal were used. In these sections, cortical bone thickness was analyzed
by acquiring images of the medial part of the diaphysis. Four measurements were made
in each histological section, prioritizing the upper, lower, and lateral regions of
each section.[12 ] To determine the thickness, cortical bone was measured from the periosteal surface
to the endosteal surface using the properly calibrated IMAGE-Pro Plus program ([Figure 1A ]). From this, the mean cortical thickness for each bone was calculated.
Fig. 1 (A ) Measurement of cortical thickness (μm) in femur under final increase of 40X. (B ) Automatic measurement of the delineated medullary area (μm2 ).
In the measurement of the area of the medullary cavity of the bones, the automatic
measurement performed by the image analysis system IMAGE-Pro Plus, previously calibrated
([Figure 1B ]), and with the functions determined by a specific macro, was used.
For the calculation of bone diameter, the methodology developed by Parfitt et al.
was used.[13 ] In this evaluation, the bone is approximated to a cylinder. Thus, with the measurement
of the medullary area, it is possible to calculate the medullary diameter so that,
together with the cortical thickness value, it is possible to arrive at the estimate
of the bone diameter ([Figure 2 ]).
Fig. 2 Histomorphometry of the bone diameter of the femoral diasis of the rats. Average ± standard
deviation. Presence of statistically significant difference (p < 0.05) between pairs marked with the same superscript symbol.
Statistical analysis
All statistical analyses were performed with the Minitab software, version 19. The
data were reported as mean with standard deviation (SD). The variables were also tested
for their normality through the Shapiro-Wilk test, obtaining a parametric distribution.
For the analysis of the results, the one-way analysis of variance (ANOVA) test was
used, followed by the Bonferroni post-test. Statistical significance was defined for
a p-value < 0.05. All p-values shown are two-tailed.
Results
In all analyses performed ([Figures 3 ], [4 ] and [5 ]), a statistically significant difference was found (p < 0.05; one-way ANOVA test) between the tested groups. Analyzing [Figure 2 ] the femoral diaphysis of the rats, a significantly higher value can be observed
in the cortical thickness (μm) of groups G1 (648.22 ± 77.51), G3 (643.15 ± 59.03),
and G5 (654.57 ± 79.00) compared with that of group G2 (515.53 ± 76.38).
Fig. 3 Cross-sectional representation of femoral diameter for bone diameter calculation.
Adapted from Parfitt et al.[13 ].
Fig. 4 Histomorphometry of cortical thickness of the femoral diasis of the rats. Average ± standard
deviation. Presence of statistically significant difference (p < 0.05) between pairs marked with the same superscript symbol.
Fig. 5 Histomorphometry of the area of the medullary cavity of the femoral diasis of the
rats. Average ± standard deviation. Presence of statistically significant difference
(p < 0.05) between pairs marked with the same superscript symbol.
In the measurement of the medullary area (μm2 ) ([Figure 4 ]), the group that underwent monotherapy with ALN (G3) (365.18 ± 49.99) was statistically
different only when compared with group G2 (484.02 ± 46.36), which presented the lowest
value.
Regarding the analysis of the femoral diameter of the rats ([Figure 5 ]), a significantly higher value was observed in groups G1 (1309.54 ± 154.89), G3
(1298.4) 5 ± 117.65), and G5 (654.57 ± 79.00) compared with the group with osteoporosis
and without treatment (G2) (1321.97 ± 158.40).
Discussion
Within the spectrum of pharmacological treatments of osteoporosis, therapeutic agents
can be divided into two major classes: antiresorptive compounds and bone formation
stimulants.[14 ] The former reduce osteoclastic activity, which forms gaps in the surface of the
bones, allowing them to be filled by a new matrix before the remodeling cycle restarts.
The second compounds, also called anabolic agents, intensify the action of osteoblasts,
which, in each remodeling cycle, increases the deposition of osteoid matrix.[14 ]
[15 ]
Among the drugs with antiresorptive action, we can mention BFs, calcitonin, estrogens,
and selective modulators of estrogen receptors. Fluoride, parathyroid hormone, and
teriparatide are examples of anabolic agents.[15 ]
Bisphosphonates form a class of chemicals that act as inhibitors of bone resorption.[16 ] Sodium alendronate is a powerful second-generation BF, which initially fixates on
the bone matrix and is later assimilated by osteoclasts to, then, inhibit its action.
It inhibits farnesyl diphosphate synthesis by blocking the signaling pathway of mevalonate
in osteoclasts, also inhibiting the activation factors thereof, such as receptor activator
of nuclear factor kappa-Β ligand (RANKL), which is the main mediator of osteoclastic
differentiation, activation, and proliferation.[17 ] However, there are concerns regarding adverse effects related to chronic use of
BFs, such as musculoskeletal pain, atypical fracture of the femur, osteonecrosis of
the jaw, and severe suppression of bone remodeling.[18 ]
Vitamin D plays a crucial role in a multitude of physiological functions, such as
in modulating calcium homeostasis and skeletal phosphate; it exerts a significant
influence on the growth and differentiation of various tissues; it has immunomodulatory
functions; and it also acts on bone mineralization, muscle functions, and balance.[19 ]
[20 ]
Vitamin D deficiency is common in patients with osteoporosis and hip fractures. Inadequate
VD levels are considered one of the potential factors for failure of drug treatment
of osteoporosis (significant loss of bone mineral density and fractures).[16 ]
1.25 dihydroxycholecalciferol, or calcitriol, is the active metabolite of VD and is
responsible for regulating the expression of genes encoding several proteins, including
calcium and bone matrix transporters. In addition, VD modulates genes involved in
the protein cycle that decrease proliferation and increase cell differentiation, such
as osteoclastic precursors. This property may explain the action of VD on bone resorption,
on intestinal calcium transport, and on the skin.[21 ]
[22 ]
According to Ferreira Junior et al.,[23 ] the histomorphometric study of bone is an extremely valuable method for the dynamic
evaluation of the bone remodeling process and to determine the extent of bone loss
and of bone tissue formation, being able to identify osteometabolic changes such as
osteoporosis.
Dexamethasone-induced osteoporosis is characterized by two phases: a rapid phase in
which bone mineral density (BMD) is reduced, probably due to excessive bone resorption,
via osteoclasts, and a late, progressive, phase in which BMD decreases due to impairment
in bone formation.[24 ] Analyzing the results obtained for positive control for osteoporosis (G2), it is
possible to verify that the osteoporotic induction process was successful, since,
when compared with the negative control (G1; without osteoporosis), there was a reduction
in cortical thickness and an increase in medullary spaces, both measurements with
a statistically significant difference in relation to the negative control group (G1).
These characteristics, along with other measurements not explored in the present study,
are marks of osteoporosis.[25 ]
The results of the present study also demonstrated that, when analyzing bone cortical
thickness, both ALN monotherapy and combination therapy with ALN + VD were able to
preserve bone structure. However, there was no difference when commencing these therapeutic
regimens. Isolated VD therapy has not been shown to be statistically capable of preserving
bone mass in osteoporotic states. This same evaluation can be applied to bone diameter
analysis.
Regarding the other histomorphometric measurement evaluated, the area of the medullary
cavity, it was observed that only therapy with ALN was able to demonstrate an important
effect on the preservation of bone mass. Thus, there was less effect on trabecular
bones when neither the combination therapy focused on this study nor the isolated
treatment with VD was used. This result differs from others found in the literature,
which demonstrated that VD alone was able to significantly increase bone mass in rats.[26 ]
[27 ]
[28 ] However, it is noteworthy that, although there was no statistical difference in
the paired evaluation, in all global analyses of the three variables (cortical thickness,
medullary area, and bone diameter), the p -values were statistically significant.
Conclusion
Together, the data presented in the present study demonstrate that concomitant treatment
with daily ALN and weekly VD is effective in the prevention of glucocorticoid-induced
bone loss. However, there was no difference between the therapy tested and treatment
only with ALN. Since prolonged use of BFs, such as ALN, can cause serious adverse
effects, its association with VD may be clinically a good choice to replace ALN, since
histomorphometric analyses showed similarities in bone mass preservation results and
VD, due to its properties, may possibly avoid and/or minimize these problems associated
with ALN therapy.
