Effect of silicon supplement on osteopenia induced by ovariectomy in rats

2000 Springer-Verlag New York Inc.
Effect of Silicon Supplement on Osteopenia Induced by Ovariectomy
in Rats
H. Rico, J. L. Gallego-Lago, E. R. Herna´ndez, L. F. Villa, A. Sanchez-Atrio, C. Seco, J. J. Ge´rvas
Departamento de Medicina, Universidad de Alcala´, 28801, Madrid, Spain Received: 15 February 1999 / Accepted: 25 June 1999 Abstract. The effect of silicon (Si) supplement on prevent-
Similar to the examples above, supplements of 50 mg ing bone mass loss induced by ovariectomy (OVX) in rats Si/100 g of diet stimulates rat growth and increases body was investigated. Three groups of 15, 100-day-old female weight [4]. Carlisle [5] observed that by supplementing Wistar rats each, with a mean initial weight of ∼260 g per chick diets with Si, as it was done by Schwartz and Milne animal, were selected for the present study. One of the [4], the body weight increased significantly in less than a experimental group consisting of 15 OVX rats was fed a month. Both experiments have proven beyond a doubt that diet supplemented with 500 mg of Si per kg of feed (Si + the effect of Si favors somatic development in experimental OVX). The other two groups consisting of 15 OVX and 15 animals. In 1983, Parfitt [6], while referring to bone and the sham-OVX rats did not receive these supplements. Morpho- risk of excessive bone mass loss synonymous with osteo- metric (weight and length) and densitometric studies with porosis, stated that several trace elements including zinc and dual-energy X-ray absorptiometry were performed on the Si were essential for normal bone growth and development.
whole femur and 5th lumbar vertebra of each animal 30 In this sense, Si seems to be indispensable for the synthesis days after the experiment. The Si + OVX rats did not show of bone marrow, given its osteoformatory effect on the col- a loss of bone mass induced by OVX at axial level (5th lumbar vertebra) or periphery (femur). Nonetheless, a sig- Experimental rat models are considered to be suitable for nificant increase (ANOVA with Bonferroni/Dunn post hocs extrapolation of results to human subjects [8, 9]. The use- test) of longitudinal development of the femur (P < 0.0001) fulness of dual-energy X-ray absorptiometry (DXA) for was patent. These results, obtained through the measure- bone-mass determinations in small animals has been vali- ments of axial and peripheral bones, warrant closer scrutiny dated repeatedly [10] and our team had relied extensively on in connection with the Si inhibitory effect on bone mass loss this technique in previous experimental studies [11, 12].
as well as the stimulatory effect on bone formation. Both More recently [13], we have demonstrated that zinc exerts a actions, namely, inhibition of resorption and stimulation of powerful influence by inhibiting the loss of bone mass in formation, infer that Si may have a potential therapeutic rats subjected to strenuous physical exercise. In view of application in the treatment of involutive osteoporosis.
these assertions, the present study was centered on the veri-fication of the effect of Si on the bone mass loss through Key words: Silicon — Ovariectomy — Bone loss — Rats
— Bone mineral content — Bone mineral density.
Material and Methods
Silicon (Si) is an essential mineral in the animal diet [1] to Three batches (15 animals each) of 100-day-old female Wistar rats such an extent that it is preferentially found in the growth with a mean initial weight of ∼260 g were investigated. All rats regions of the body and consequently it has been considered were fed Mucedola type 4RF21 (Mucedola s.r.l. Milano, Italy) an important element that plays a crucial role in bone cal- feed containing 7.1 g/kg calcium and 5 g/kg phosphorus; the en- cification [2]. In addition to bone, Si deficiency is mani- ergy content of the feed was 3100 kcal/kg. The rats were kept for fested by abnormalities involving articular cartilage and 30 days in the animal laboratory of the University of Alcala´, connective tissue. Chicks from the Si-deficient group had Madrid (Spain). Living conditions (12 hours of light and 12 hoursof dark cycles), mean room temperature (22°C), habitat, and the thinner legs and smaller combs in proportion to body size.
diet were observed in accordance with current guidelines imposed Likewise, their long-bone tibial joints were markedly by the European Union Council. Experimental procedures were smaller and bones contained 34–35% less water than the followed according to the guiding principles on Care and Use of chicks whose diet was supplemented with Si [3].
Animals as approved and overseen by the appointed institutionalanimal care committee. Sample size was calculated in a pilot studyafter determining the variability of densitometric measurements.
The standard deviation (SD) of bone mineral density (BMD) was 10 and the hypothesized difference among the groups was 15 units.
Table 1. Group characteristics of three groups of rats
a P < 0.0001 vs othersb P < 0.005 vs sham-OVX according to ANOVA with Bonferroni/Dunn post hocs test Alpha risk was found to be 0.05 and beta risk was 0.20 for a determined by six separate measurements done on three rat femurs two-sided control. The number of animals in each group was es- and 5th vertebras at intervals of 3–4 days were 0.8% and 0.7%, tablished according to the formula nc ס ne ס 2(Za + Zb)2s2/D2, respectively. The instrument was calibrated daily.
where nc ס number of animals in the control group; ne ס number of animals in the experimental group; s ס SD; D ס difference to The rats were randomly integrated into three groups based on their body weight. Those with highest and lowest weight werealternatively incorporated into one of the groups, so that at the end Descriptive statistics are presented as mean ± SD. The nor- of the randomization the mean body weight of each group was mal distribution of data was confirmed by calculating skew easily comparable. All of the 45 animals were anesthetized intra- and kurtosis before applying standard tests. The studied peritoneally with ketamine hydrochloride (10 mg/kg) and parameters (continuous variables) in each group (nominal acepromazine (3 mg/kg), their abdomens were shaved, and the variables) were compared using analysis of variance skin was cleaned with 70% ethanol and povidoneidone (Betadine) (ANOVA) and covariance to determine the effects of nomi- solution. A longitudinal midline incision was made in the subum- nal variables; data were analyzed by ANOVA with a post bilical region to expose the rectus abdominous muscle and the hoc test of differences among groups using the Bonferroni/ abdominal cavity. The urinary bladder was retracted and the uter-ine horns were identified. The ovarian arteries were ligated and Dunn test. A minimum P-value of 0.005 was the necessary bilateral ovariectomy (OVX) was performed in 30 animals; in the condition for statistical significance. Data were processed remaining 15 rats the ovaries were exposed but not excised (sham- on a Macintosh computer using the StatView 4.02 statistical OVX). In all the other animals the abdominal musculature was package (Abacus Concepts, Berkeley, CA, USA).
sutured and the skin was closed with staples.
The experimental groups of OVX rats were fed the common diet supplemented with an additional 50 mg Si/100 g of diet (500 ppm) as sodium metasilicate (Na2SiO3-9H2O) (OVX + Si group).
A group of 15 OVX rats and the group of 15 sham-OVX rats didnot receive the above supplements. The water consumption, body Group characteristics (number, baseline, and final body weight, and food intake were measured twice a week in sham- weight, anthropometric data, etc.) are summarized in Table OVX rats with the same amount of food given to ovariectomized 1. As it can be observed, the initial weight did not differ animals (pair feeding) to avoid hyperphagia and overweight asso- among the three rat groups, but it had increased significantly in the OVX and Si+OVX groups at the end of the studywhen compared with the sham-OVX (P < 0.0001) group.
The femur length (mm) and its weight had decreased in the Morphometric and Densitometric Studies OVX group contrary to the other two groups (P < 0.0001).
A similar change was observed for F-BMC mg, V-BMC At the conclusion of the 30-day experiment, the rats were sacri- mg, F-BMD mg/cm2, V-BMD mg/cm2 as well as in verte- ficed by exsanguination from the abdominal aorta after being anes- bral weight and F-BMC/FW mg/g (P < 0.0001 in all). Sub- thetized with 4 mg/100 g body weight of sodium pentothal. Suc- sequently, the only parameters where no differences were cess of ovariectomy was confirmed at necropsy by failure to detectovarian tissue and by observing marked atrophy of the uterine observed were the length or height of the 5th lumbar ver- horns. The rat femurs and 5th lumbar vertebrae were dissected and soft tissue was removed. Femoral and vertebral length were mea-sured with a caliper and bones were weighed on a precision bal-ance. The bone mineral content (BMC) and BMD of the whole Discussion
femur (F-BMC mg and F-BMD mg/cm2) and 5th lumbar vertebra(V-BMC mg and V-BMD mg/cm2) were measured separately.
Our results showed that Si inhibits the loss of bone mass in Because of the influence of weight on bone mass [11], femur BMCwas corrected for the animals final body weight (femur BMC/FW rats subjected to OVX and that it promotes the longitudinal mg/g). As in our earlier studies [11–14], we have relied on DXA growth of long bones, in this case, the femur. On the other (Norland XR-26, Norland Co., Fort Atkinson, Wisconsin, USA).
hand, Si incremented corporal mass by 11.5%. It was shown Our coefficients of variation (CV) for the current measurements, that OVX induces a deficit of longitudinal growth in bones [15] and that Si is very important for it sanctions the lon- in human and animal nutrition, vol. 2. Academic Press, Or- gitudinal development, as claimed by Schwart and Milne [4] and later confirmed by Carlisle [5]. Sontag [16] has 2. Carlisle EM (1970) Silicon: a possible factor in bone calcifi- reported that at 150 days of life, femoral length in female Wistar rats reached a mean of ± 34 mm. In our study the 3. Carlisle EM (1976) In vivo requirements for silicon in articu- longitudinal development of the femur in the Si-OVX group lar cartilage and connective tissue formation in chick. J Nutr was notably enhanced. This phenomenon may be related to the mentioned effect of Si, that it promotes bone growth [4], 4. Schwartz K, Milne DB (1972) Growth-promoting effects of which has also been certified by most recent studies [17].
The mentioned reports showed that an aluminum-silicon 5. Carlisle EM (1972) Silicon—an essential element for the mixture increases the proliferation and differentiation of os- teoblasts. It is an action that can be dependent on the men- 6. Parfitt AM (1983) Dietary risk factor for age-related bone loss tioned mixture, known otherwise as Zeolite A, which in turn exerts effect on TGFb, a citoquine that stimulates bone for- 7. Carlisle EM (1980) A silicon requirement for normal skull mation in vivo [18]. This action is not to be linked to the aluminum by the toxic effect of aluminum on the bone [19].
8. Frost HM, Jee WSS (1992) On the rat model of human os- The increase in the body weight of rats treated with Si teopenias and osteoporoses. Bone Miner 18:227–236 supplement was also patent, which fully coincided with the 9. Wronski TJ, Yen CF, Qi H, Dann LM (1993) Parathyroid reports of other authors [4, 5], even though their claims hormone is more effective than estrogen or bisphosphonates inferred greater proportion, as the one observed by us.
for restoration of lost bone mass in ovariectomized rats. En- Nonetheless, it once again proved the positive effect of Si on the rats’ somatic development. The rats’ weight in this 10. Mitlak BH, Schoenfeld D, Neer RM (1994) Accuracy, preci- study was significantly correlated with bone mass [11].
sion, and utility of spine and whole-skeleton mineral measure-ments by DXA in rats. J Bone Miner Res 9:119–126 When the final bone mass, such as femur BMC (F-BMCmg) was divided by the final rats’ weight (F-BMC/FW mg/ 11. Rico H, Amo C, Revilla M, Arribas I, Gonza´lez-Riola J, Villa LF, Rodrı´guez-Puyol M (1994) Etidronate versus Clodronate g), the results were the same at significant levels, closely in the prevention of postovariectomy bone loss. An experi- matching those observed in the study on femurs. This fact mental study in rats. Clin Exp Rheumatol 12:301–304 alone vouches for the importance that the Si exerts on the 12. Rico H, Alamo C, Revilla M, Villa LF, Alguacil LF (1992) prevention of bone loss induced by OVX.
Effect of adjuvant-induced arthritis on bone mass in rats and It is widely held that experimental oophorectomy of its prevention by non-steroid anti-inflammatory drugs laboratory animals supposedly mimics the sequence of (NSAIDs). Clin Exp Rheumatol 10:595–597 events that take place in human subjects following meno- 13. Seco C, Revilla M, Herna´ndez ER, Gerva´s J, Gonza´lez-Riola pause. In the presently oophorectomized rats, bone mass J, Villa LF, Rico H (1998) Effects of zinc supplementation on loss was rapid. Ovariectomy results in severe cancellous vertebral and femoral bone mass in rats on strenuous treadmill osteopenia of murine long bones and vertebras [15]. A simi- training exercise. J Bone Miner Res 13:508–512 lar phenomenon was observed in our study, where the loss 14. Escribano A, Revilla M, Herna´ndez ER, Seco C, Villa LF, due to OVX was evident in the axial skeleton (vertebra), Rico H (1997) Effects of lead on bone development and bone reaching 30% and 25% in the periphery (femur), respec- mass: a morphometric, densitometric and histomorphometric tively. There is ample evidence that after immediate meno- study in growing rats. Calcif Tissue Int 60:200–203 pause and/or OVX, the loss is greater at the axial skeleton 15. Ousler MJ, Kassem M, Turner R, Riggs BL, Spelberg TC than in the periphery [20], which was possible to confirm in (1996) Regulation of bone cell function by gonadal steroids.
the present study. Nonetheless, the observed significant ef- In: Marcus R, Feldman D, Kelsey J (eds) Osteoporosis. Aca- fect on Si on the bone mass has to do with the prevention of post-OVX losses, which is known to be secondary to an 16. Sontag W (1992) Age-dependent morphometric alterations in the distal femora of male and female rats. Bone 13:297–310 Based on results concerning longitudinal development in 17. Keeting PE, Ousler MJ, Wiegand KE, Bonde SK, Spelsberg the peripheral skeleton, we are in a position to conclude that TC, Riggs BL (1991) Zeolite A increases proliferation, dif- Si has a very important effect on the stimulation of bone ferentiation and TGF-beta production in normal adult human formation. It is also obvious that these results need to be osteoblast-like cells in vitro. J Bone Miner Res 7:1281–1289 related to the recent studies conducted by Hott et al. [21] 18. Mundy GR, Boyce BF, Yoneda T, Bonewald LF, Roodman and Schutze et al. [22] which demonstrate that Si decreases GD (1996) Cytokines and bone remodeling. In: Marcus R, the osteoclast number by 20% and that Si exerts a potent Feldman D, Kelsey J (eds) Osteoporosis. Academic Press, SanDiego, CA, pp 301–313 inhibitory effect on bone resorption while increasing therate of bone formation [21]. These findings coincide with 19. Rico H (1991) Minerals and osteoporosis. Osteoporosis Int Eisinger and Cairet’s studies [23] which show that Si is amore potent bone formation stimulus than other drugs, such 20. Parfitt AM (1996) Skeletal heterogeneity and the purposes of bone remodeling. Implications for the understanding of osteo- as ethidronate, used in the treatment of osteoporosis. The porosis. In: Marcus R, Feldman D, Kelsey J (eds) Osteoporo- double action to which we refer—on one hand, a potent sis. Academic Press, San Diego, CA, pp 315–329 inhibition of the bone resorption and on the other a signifi- 21. Hott M, de Pollak C, Modrowski D, Marie PJ (1993) Short- cant stimulation of bone formation—suggests that Si as- term effects of organic silicon on trabecular bone in mature sumes two crucial functions in the treatment of osteoporo- ovariectomized rats. Calcif Tissue Int 53:174–179 sis. The forthcoming conclusion was amply justified by the 22. Schutze N, Oursler MJ, Nolan J, Riggs BL, Spelsberg TC good results obtained in the present study.
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