Introduction and aim of the work
Immobilization or disuse is associated with decrease in bone mass and osteopenia and in some cases leads to osteoporosis with increased risk of fractures . Osteoporosis is one of the major health problems afflicting our modern world, especially disuse osteoporosis, and occurs commonly in bedridden patients, a population of patients that is rapidly increasing because of aging-associated diseases .
Adequate Ca intake is an essential component of any preventive regimen. Sufficient vitamin D supply is essential for optimal dietary Ca absorption . It was reported that administration of vitamin D3 alone without Ca did not lead to a reduction in the incidence of hip fracture in elderly individuals, whereas cosupplementation of vitamin D3 and Ca was associated with a reduction in the incidence of hip fracture .
It has been proposed that vitamin K has a role in bone metabolism, and low vitamin K intake has been related to low bone density . It was reported that vitamin K, which was originally recognized as a factor required for normal blood coagulation, had begun to receive more attention for its role in bone metabolism. Vitamin K-dependent proteins were discovered to be involved in bone metabolism for the first time in the mid-1970s, when serious bone malformation was observed in children born to women who were treated with vitamin K antagonists during the first trimester of pregnancy .
Few studies were concerned with the use of vitamin K in the treatment of immobilization osteoporosis. Until now there is no published work correlating scanning electron microscopic (SEM) findings with the effect of dietary supplements, especially of vitamin K, on disuse osteoporosis.
Aim of the work
The aim of this study was to investigate the effects of dietary supplements (Ca, vitamin D, and vitamin K) on SEM findings in rats with disuse osteoporosis.
We hypothesize that dietary supplements could improve the bone quality in rats with disuse osteoporosis.
Materials and methods
The experiment was conducted on 30 albino rats with an average weight of 200g.
Inclusion criterion: Only adult male rats were included in the study.
Exclusion criterion: Senile or female rats were excluded.
Animals were housed in wire mesh cages and fed a standard chow diet in addition to tap water. The experiment was conducted in the Medical Research Center in Ain Shams University during the period from January 2007 to January 2008. The animals were housed for 7 days before beginning the experiment for acclimatization, and then they were randomly divided into the following groups:
Group I (the control group): This group included five rats that received the ordinary balanced diet.
Group II: This group included five rats that were immobilized by casting their right lower limb for 4 weeks. Four to five layers of Paris cast (Gypsona, was obtained from Afri Medical Egypt, Cairo under licence of BSN Medical Limited London, UK.), reaching above the knee, were used to immobilize the right knee joint in extension. The right ankle joint was immobilized in planter flexion to avoid weight bearing on this limb. The cast was checked daily and changed if necessary .
Group III: Group III was divided into four subgroups according to the given dietary regimen:
Subgroup IIIa: This subgroup included five rats that were immobilized by casting their right lower limb for 4 weeks. They also received Ca citrate orally (0.09mg/g body weight daily) [8,9] concomitant with casting their right lower limb.
Ca citrate was given in the form of Ca Pharco tablets, which were manufactured by Pharco Pharmaceuticals (Alexandria, Egypt). They consisted of 950mg Ca citrate tablets, containing 200mg of elemental Ca.
Subgroup IIIb: This subgroup included five rats that were immobilized by casting their right lower limb for 4 weeks. They received vitamin D orally (0.5μg/kg daily)  concomitant with casting of their right lower limb. Vitamin D was in the form of 1α (OH)D3 and was termed one-alpha or alfacalcidol. We used one-alpha drops that provided 2μg/ml of alfacalcidol. It was manufactured by Minapharma under license from Leo Pharmaceutical Products (Ballerup, Denmark).
Subgroup IIIc: This subgroup included five rats that were immobilized by casting their right lower limb for 4 weeks. They also received Ca and vitamin D (in the same previous doses), concomitant with casting of their right lower limb.
Subgroup IIId: This subgroup included five rats that were immobilized by casting their right lower limb for 4 weeks. They concomitantly received vitamin K orally (0.0009mg/g daily [8,11]) during casting of their right lower limb.
Vitamin K1 (phytomendione) was obtained from konakion MM Paediatric ampoules. Konakion was a product manufactured by Roche under license from F. Hoffmann-La Roche Ltd (Basel, Switzerland) by CENEXI SAS (Fontenay-sous-Bios, France).
At the end of the experiment, animals were sacrificed by ether inhalation anesthesia. The distal metaphysises of the immobilized femur were dissected and processed as following.
Preparation of decalcified material
The excised parts were cut transversely and fixed in neutral buffered formaldehyde for 2 days. They were then decalcified using the chelating agent EDTA. The time required for decalcification was 4 weeks. The solution was changed daily. An ample volume of decalcifying solution should be used, at least 30–50 times the volume of tissue .
The decalcified specimens were processed and sections were cut at the metaphysis at a thickness of 7μm and stained with H&E for morphometric study.
Preparation for scanning electron microscopy 
The distal end of the immobilized femur was selected, cut longitudinally, irrigated with saline to remove the bone marrow, and then processed for SEM in the Electron Microscopic Unite in the Anatomy Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt.
Measurements were taken on H&E-stained sections using an automatic analysis system with Leica Image Analysis software (Leica Q 500MCO analyzer Program, Germany) installed on a personal computer. This image analysis was carried out in the Histology Department, Faculty of Medicine, Ain Shams University.
The following parameters were quantitated  and expressed in micrometer:
- Cortical bone thickness (McTh): It was measured by drawing a vertical line from just beneath the periosteum to the endosteum.
- Trabecular bone thickness (MtTh): Bone trabeculae were measured at their midpoint away from their branching areas.
Five H&E-stained sections were obtained from five blocks from five different animals in each group. The mean values were estimated. The SD was calculated and statistical analysis was carried out using SPSS statistical program version 17. Data were evaluated using one-way analysis of variance. Data were considered significant if P was less than 0.05.
Scanning electron microscopy results
Group I: SEM examination of the longitudinal sections of the rat femur showed the outer cortical bone and inner cancellous bone trabeculae (Fig. 1a). The cancellous bone was seen in the metaphysis as numerous thick branching and anastomosing bone trabeculae that appeared to be of uniform thickness (Fig. 1b). The Haversian canal was seen surrounded by osteocyte lacunae in a concentric manner (Fig. 1c). Osteocyte lacunae with the openings of their canaleculi were also observed (Fig. 1d).
Group II: SEM examination showed a thin cortex (Fig. 2a). Thin, irregular, discontinuous spike-like bone trabeculae were seen with loss of uniform thickness (Fig. 2b). Bone trabeculae were frequently seen fractured (Fig. 2c). An apparent increased diameter of the Haversian canal was noticed compared with the control group (Fig. 2d). Multiple small cavities were seen in the outer part of the compact bone (Fig. 3a). At higher magnifications, bone-like trabeculae were seen in the compact bone (Fig. 3b). Multiple cracks were seen in the endosteal surface of the compact bone (Fig. 3c). Osteoclasts were frequently seen attached to the endosteal surface of the bone, and their ruffled border was seen facing the bone surface (Fig. 3d).
Subgroup IIIa: SEM examination showed few cancellous bone trabeculae at the metaphysis (Fig. 4a). Decreased thickness of the cortical bone could be seen (Fig. 4b). An apparent increased diameter of the Haversian canal was also noticed as compared with the control group (Fig. 4c). Examination of the compact bone also revealed the presence of multiple cavities (Fig. 4d).
Subgroup IIIb: SEM examination revealed few cancellous bone trabeculae at the metaphysis compared with the control group. Broken tarbeculae were seen. Cracks could also be observed in some bone trabeculae (Fig. 5).
Subgroup IIIc: SEM examination showed increased cortical thickness as compared with group II. Branching bone trabeculae were also seen in the metaphysis (Fig. 6a). Bone trabeculae appeared in localized areas as a sheet-like structure (Fig. 6b).
Subgroup IIId: SEM examination showed extensive branching and anastomosing bone trabeculae in the metaphysis as compared with the control group (Fig. 7).
The histomorphometric results
The McTh in group II showed a significant decrease when compared with the control group and subgroups IIIa, IIIb, IIIc, and IIId.
The McTh in subgroup IIIa showed a significant decrease when compared with the control group and subgroups IIIb, IIIc, and IIId. Significant increase was noticed when compared with group II.
The McTh in subgroup IIIb showed a significant decrease when compared with the control group and subgroups IIIc and IIId. Significant increase was noticed when compared with group II and subgroup IIIa.
The McTh in subgroup IIIc showed a significant increase when compared with group II and subgroups IIIa and IIIb. Nonsignificant decrease was noticed when compared with the control group and subgroup IIId.
The McTh in subgroup IIId showed a significant increase when compared with group II and subgroups IIIa and IIIb. Nonsignificant change was noticed when compared with the control group and subgroup IIIc.
The MtTh in group II showed a significant decrease when compared with the control group and subgroups IIIa, IIIb, IIIc, and IIId.
The MtTh in subgroup IIIa showed a significant decrease when compared with the control group and subgroups IIIb, IIIc, and IIId. Significant increase was noticed when compared with group II.
The MtTh in subgroup IIIb showed a significant decrease when compared with the control group and subgroups IIIc and IIId. Significant increase was noticed when compared with group II and subgroup IIIa.
The MtTh in subgroup IIIc showed a significant increase when compared with group II and subgroups IIIa and IIIb. Significant decrease was also noticed when compared with the control group and subgroup IIId.
The MtTh in subgroup IIId showed a significant increase when compared with group II and subgroups IIIa, IIIb, and IIIc. Nonsignificant change was noticed when compared with the control group.
Immobilization is one of the most important causes of osteoporosis, especially after cast application and in elderly individuals who might be bedridden or suffering from joint diseases. Most previous studies used bisphosphonate as primary treatment for osteoporosis, but evidence from recent studies has shown the adverse effects of its long-term use .
In the current study, SEM examination of immobilized rats revealed decreased thickness of the outer cortical bone and tapered, spindle-shaped broken trabeculae. This was in agreement with the findings of other authors . In the current study, widening of the Haversian canals was also noticed. Similar findings were also observed by others who reported that in severe cases of osteoporosis the Haversian systems were so enlarged that the cortex mimicked cancellous bone . In the present work, multiple cavities were seen in the compact bone of the immobilized group. On higher magnifications, this area of compact bone was seen to be formed of bone-like trabeculae. It was reported that this osteoclastic resorption with subsequent widening of the Haversian canals appeared first in the inner parts of the cortical bone (cancellization), which then gradually emerged over the central part of the cortical bone to be turned into trabeculae (trabeculization) .
The current study showed that the use of Ca alone with immobilization was not effective in preventing the effects of disuse osteoporosis on bone. It was reported that bone loss in women who have been menopausal since the past 5 years were not affected by Ca supplementation . This was against findings of other authors who reported that Ca citrate was effective in averting the bone loss that occurred in early postmenopausal women, but this protection was less marked than that reported by estrogen supplementation or alendronate .
Treatment with vitamin D alone with immobilization in this study did not improve most osteoporotic changes. This was against the findings of some authors who reported that alfacalcidol increased bone mineral density in a dose–dependent manner . In this study, combined treatment with Ca and vitamin D in immobilized rats resulted in an improvement in some osteoporotic changes. A few areas of the cancellous bone showed a sheet-like appearance without forming a three-dimensional network. It was reported that vitamin D increases bone size, and adequate Ca supplementation might be needed to stimulate cortical bone growth and increase cancellous bone mass . The reason for these conflicting results regarding the use of vitamin D might be attributed to differences in the study population, to the dosage of vitamin D used, or the duration of treatment. It was also reported that genetic differences might contribute to the conflicting results among studies using vitamin D in osteoporosis, as the response to plain vitamin D therapy might be influenced by the vitamin D receptor genotype .
In the present work, SEM examination of immobilized rats that received vitamin K revealed an extensive trabecular network that was of uniform thickness. The histomorphometric results also showed a significant increase in the mean trabecular bone thickness when compared with the immobilized group. Some authors found that treatment with vitamin K increased cortical bone mass in children with severe physical disability such as in those suffering from cerebral palsy or hemiplegia in cases of both hemiplegic and nonhemiplegic tibia . It was reported that vitamin K improved bone metabolism and structure, decreased bone loss, and increased bone biomechanical properties. Vitamin K could prevent bone loss in rats with simulated weightlessness .
According to the findings of this study, vitamin K was found to be superior to combined administration of Ca and vitamin D with respect to the effects on the cancellous bone, which was also confirmed by the histomorphometric results. It was reported that the effect of vitamin K on bone density was superior to that of vitamin D . It was also reported that high Ca intake and supplementation of vitamin D during space flight did not affect bone metabolism but prevented an elevation of serum Ca level, whereas vitamin K counteracted the reduction in bone formation . Family physicians should be aware about the importance of encouraging adequate vitamin K intake, particularly among elderly individuals, to prevent increased bone resorption . Regarding the mechanism of action of vitamin K, it was mentioned that vitamin K was essential for the activation of vitamin K-dependent proteins, which were involved in bone metabolism, such as osteocalcin and matrix GIa protein. When osteocalcin was carboxylated, it assumed molecular properties that allowed it to tightly bind hydroxyapatite in the bone, thereby promoting mineralization. Matrix GIa protein played a key role in the inhibition of tissue calcification. As with all vitamin K-dependent proteins, matrix GIa must be carboxylated to function properly .
The absence of X-ray or DXA scans and the limited period of immobilization in this experiment are the main limitations of this study.
To study the effects of different dietary supplements on the prevention of disuse osteoporosis in humans.
It was concluded that immobilization has serious effects on the bone structure especially on cancellous bone trabeculae. Combined administration of Ca and vitamin D with immobilization was seen to be more effective than single-agent therapy. Vitamin K, for the period of this experiment, showed better efficacy than combined administration of both Ca and vitamin D with respect to cancellous bone parameters. It is believed that supplementation with vitamin K could prove beneficial for the prevention of immobilization-induced osteoporosis; however, additional clinical studies are needed to address these effects on humans.
It is recommended to avoid prolonged periods of immobilization or bed rest. It is also advisable to give vitamin K to bedridden patients or elderly individuals who may suffer from joint disease or any condition leading to their immobilization.
The suggested dose of vitamin K according to this study will be about 10mg/day for adults. Additional studies are needed to address the effects of combined treatment with both vitamin K and D.
My deep gratitude goes to Dr Youssef Shoukry, Lecturer of Anatomy and Electron Microscopic Unite, Anatomy Department, Ain Shams University, Cairo, Egypt
Conflicts of interest
There is no conflict of interest to declare.
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Keywords:© 2012 The Egyptian Journal of Histology
Ca; immobilization; osteoporosis; scanning microscopy; vitamins K and D