Animals and induction of osteoporosis

All experiments were performed incompliance with the relevant laws, faculty guidelines, and the Research Ethical Committee. Faculty of Medicine, Assiut University, approved the experiments, IRBno: IRB17101923. Forty-eight female adult albino rats weighing 200–250 g were obtained from the animal house of the Faculty of Medicine, Assiut University. The rats were 2.5–3 months of age before the induction of osteoporosis by dexamethasone. Rats were housed in the animal house at room temperature and maintained at 22–24 °C. Animals were fed ad libitum on a commercial pellet ration and kept under normal 12 h light/dark cycle. Osteoporosis was induced by the administration of dexamethasone sodium phosphate 7 mg/kg b.w. (Amriya Pharma, Cairo, Egypt), injected intramuscularly once a week for 3 consecutive weeks [18]. Urethane was the anesthetic agent used by an IP injection, in a dose of 1.4 g/kg.

Experimental design

Animals were divided into 6 groups, 8 rats each: Group-I kept untreated and were administered milk and considered as a control group. Group-II kept as untreated-osteoporosed control group where osteoporosis was induced by I.M. injection of dexamethasone in a dose of 7 mg/kg b.w., as described [18]. Group-III or alendronate treated group. According to the body surface area table [19], the rat dose in mg/kg was calculated from the human dose of 70 mg/kg, by multiplying the human dose by 0.018. Accordingly, the rat dose was equal to 1.26 mg alendronate per 200 g rat. One tablet of alendronate 70 mg (Aesica pharmaceuticals GmbH) was ground and dissolved in 55.5 ml distilled water. Osteoporosed animals were administered alendronate orally by a stomach tube in 0.5 ml of the alendronate stock solution per 100 g b.w. of rat, for 8 weeks. Therefore, the final dose in v/w was 0.5 ml/100 g b.w. for each rat. Group-IV or olive oil treated group where osteoporosed animals received olive oil (Isis Company, Cairo, Egypt) daily in a dose of 1 ml/100 g b.w., orally by a stomach tube for 8 weeks [20]. Group-V or Lepidium sativum treated group where osteoporosed animals received ground Lepidium sativum seeds (Local commercial, Cairo, Egypt) suspended in warm milk which was freshly prepared daily in a dose of 0.12 g/100 g b.w., orally for 8 weeks. This dose was a trial dose in this experimental design. The dose was 12.6 g of ground seeds suspended in full cream commercial milk. Group-VI or “combination treated” group, where osteoporosed animals received a combination of olive oil and Lepidium sativum for 8 weeks using the same previously mentioned doses, respectively.

Sample collection, preparation, and storage

Blood samples were collected from each rat of 8 rats per group at the time of their sacrifice. Blood was received in centrifuge tubes. The samples were then centrifuged at 4000 rpm for 10 min at room temperature. Serum was then separated and stored in Eppendorf tubes and kept at − 20 for biochemical analyses.

Biochemical analyses

Calcium and inorganic phosphorous were measured using commercial diagnostic kits provided by BioSystems S.A. (Costa Brava, Barcelona, Spain) following the standard protocol. The instrument automatically calculated the concentration of Ca2+ in mg/dl (mmol/l). The concentration of phosphorous in blood was expressed as mg/dl (mmol/l). The Rat Osteocalcin/Bone Gla Protein (OT/BGP) ELISA Kit (Sinogeneclon Biotech Co., Ltd., China) was used for estimation of osteocalcin in rat serum following the manufacturer’s instructions. Briefly, osteocalcin was first incubated with the biotinylated antibody in a streptavidin-coated microtiter well then washed and incubated with the HRP conjugated antibody. Following another wash, the enzyme antibody bound to the well was incubated with a substrate solution in a timed reaction and then measured in a spectrophotometric microtiter plate reader. A standard curve was generated and the concentration of osteocalcin in the samples is determined directly from this curve.

Histopathology and immunohistochemistry

For bone tissue collection, the leg of each animal was disarticulated at the hip, knee, and ankle. Femurs were then removed and immediately placed in 10% formalin. The femur was then cleaned of soft tissue around. Bone (femur) specimens were trimmed and placed in processing cassettes, decalcified for 48 h in a stirred glass beaker containing a solution of 100 ml distilled water and 5 ml concentrated nitric acid, then washed in tap water for 8 h, and transferred to 70% ethanol. Bone specimens were processed and embedded in paraffin and sectioned at 5 µm for conventional histopathological examination. Sections were stained with hematoxylin–eosin (H&E) and visualized by Olympus microscope to study the histopathological changes.

For immunohistochemical staining, tissue sections were deparaffinized in xylene, rehydrated in descending series of alcohol, and incubated in citrate buffer (pH 6.0) solution at 80 °C for 15 min. The sections were then placed in 3% H2O2 solution for 15 min to block the endogenous peroxidase activity and subsequently incubated at room temperature for 60 min with osteopontin mouse monoclonal antibody (Vector laboratories, CA, USA). The signal was developed by diaminobenzidine to visualize the immunocomplexes. The developed signals were examined using Olympus light microscopy (Olympus, Waltham, MA) and photomicrographs of at least three representative fields were taken by ToupCam digital camera.

Immunohistochemical evaluations

The immunohistochemically stained slides of bony tissue were examined by using light microscopy. The staining results were scored semiquantitatively according to the Remmele immunoreactive score (IRS). The osteopontin protein expression score was calculated as the product of the percentage of positively stained cells which was divided into five grades of 0–4 (0%, < 10%, 10–50%, 51–80%, and > 80%) and multiplied by the intensity of the immunohistochemical reaction scaled from 0 to 3. Then the obtained IRS was interpreted as 0 to 1 = no expression (negative); 2 to 3 = weak expression; 4 to 8 = moderate expression; and 9 to 12 = strong expression [21].

Statistical analysis

The data were presented as mean ± standard error of the mean (SEM). The software SPSS version 20.0 (SPSS Inc., Chicago, IL) was used in statistical analyses and significance of the data was considered at p-value of less than 0.05. The t-test was used to compare dexamethasone-induced versus normal rats and ANOVA for comparison of multiple treated groups.