In-vivo efficacy of calcium-phosphate based synthetic-bone-mineral on bone loss resulting from estrogen and mineral deficiencies

Abstract

Osteopenia and osteoporosis affect over 40 million US adults 50 year and older. Both diseases are strongly influenced by estrogen and nutritional-mineral deficiencies. This study investigates the efficacy of orally delivered synthetic-bone-mineral (SBM), a newly developed calcium-phosphate based biomaterial, on reversing bone loss induced by these two critical deficiencies. Thirty 3-month-old female rats were randomly allocated to either control—sham surgery on normal diet; or one of the four experimental groups: sham surgery on a low mineral diet (LMD), ovariectomized (OVX) on LMD, OVX on LMD with SBM with/without fluoride (F). The rats were sacrificed after 6 months, at 9-month-old. After 6 months, although all groups lost bone mineral density relative to controls, the supplemented OVX rats showed higher bone mineral density than their un-supplemented counterparts. The 2 SBM supplemented groups improved bone loading capacity by 28.1% and 35.4% compared to the OVX LMD group. Bones from supplemented rats exhibited higher inorganic/organic ratios. Addition of F did not have a significant influence on bone loss. Our findings suggest that SBM supplement is effective in maintaining and offsetting the deleterious effects of estrogen and/or mineral deficiencies on bone density, microarchitecture and strength.

INTRODUCTION

Healthy bone mass accumulation is influenced by the interdependency of several interconnected factors; bonetropic minerals (Calcium, phosphate, magnesium, zinc, potassium) and nutrients (proteins), vitamin D, endocrine factors (sex steroids, calcitriol, IGF-I), heredity, mechanical forces (physical activity, body weight).^1–4 Estrogen has been well known to cause endosteal bone mineral accrual in females while periosteal in males, thus affecting the cortical thickness.⁵ Evidence suggests estrogen deficiency leads to a dramatic increase in new remodeling units, by each unit of time⁶, primarily causing elevation in osteoclast formation and a complex of pro-inflammatory cells on the bone surface being remodeled, leading to increased bone loss.^7,8 Adult women are hence more prone to osteoporotic fractures than men. Estrogen withdrawal also leads to a decrease in intestinal calcium absorption, efficiency and intake requirement,^9,10 thus not surprisingly studies recommend 700 – 800 mg RDA of calcium in adults.¹¹

Zinc, magnesium and other trace minerals have been known to maintain bone quality through their involvement in collagen and other protein synthesis, regulating calcitonin secretion, alkaline phosphatase activity and reducing osteoclastic activity.^12–14 A narrow therapeutic/toxicity window exists for F⁻ ions, wherein therapeutic concentrations of F⁻ ions have shown to decrease osteoclastic resorption and increase osteoblast phenotype expression alongside increasing BMD, by forming less soluble and larger crystallite flouroapatite.^15–17 It has been made evident that nutritional/mineral rehabilitation can critically recover the BMD in the cachectic state beyond what is achieved with estrogen treatment alone.¹⁸

OVX rats are routinely utilized as valuable models for post-menopausal osteoporosis studies,¹⁹ developing significant cancellous bone loss when fed a normal diet²⁰ and similar in case of dietary calcium deficiency.²¹ However, both cancellous and cortical bone loss have been noted in models effected by combined estrogen and mineral deficiencies within a month.²² While many studies have focused separately on the effects of estrogen deficiency, calcium and vitamin D deficiencies on osteoporosis along with management, the multi-factorial nature of the disease demands more focus on a diet-combined ovariectomy model and its management. A review of studies for current FDA approved osteoporosis treatment found that 80% of studies had been performed under conditions where a certain minimum calcium and vitamin D intake was ensured²³. Thus one may not conclude that mineral deficient patients would have the same outcome with these prescription medications.²⁴ It is imperative to be aware that adequate dietary mineral and Vitamin D intake be present to allow an overall effective treatment with osteoporosis pharmacologics.²⁴

In this study, we hypothesize that while the combined OVX and LMD model would lead to an increase in bone loss and decrease in bone strength, SBM would help preserve and potentially restore the BMD, bone mineral content (BMC), bone microarchitecture and strength in-vivo. In addition, the efficacy of SBM supplementation with or without F has also been examined. SBM is calcium-phosphate based biomaterial consisting of carbonate apatite, similar to bone apatite incorporating Mg²⁺, Zn²⁺, Mn²⁺ and F‾ ions.²⁵ It has been shown to be safe and effective in preventing bone loss in both senile male and female, OVX or LMD rats as an oral supplement,^26–28 injected as slurry subcutaneously^29,30 and most recently as a bone loss prevention and preservation agent in estrogen and mineral deficient sheep model.³¹

MATERIALS AND METHODS

Experimental design

Thirty, 3-month-old female Sprague-Dawley rats (Charles River Laboratories, USA) were obtained for this study. They were first subjected to either bilateral ovariectomy (n = 22) or sham operations (n = 8) and then randomly assigned to one of the five groups. Group 1 (G1) consisted of 4 sham rats on a normal diet and Group 2 (G2) consisted of 4 sham rats on LMD. Group 3 (G3) was 6 OVX rats on the LMD diet. Group 4 (G4) was 8 OVX rats on LMD + SBM with F and Group 5 (G5) was 8 OVX rats on LMD + SBM without F. Since body weight is strongly associated with bone mass, rats were stratified by body weight prior to random assignment to one of the five treatment groups. This resulted in very similar mean body weights (238.166–244.375 g) across the five groups. Rats were housed in pairs, pair fed, given non-fluoridated water ad libitum and weight recorded weekly. Overall diet consumed by the rats, initial weight and weight gained by them was consistent across groups, as seen in Table 1.

Table 1.

Animal weight gain (A) and diet consumed (B) across groups, over 6-month study period.

For the first week, all animals were acclimatized to their new environment and were fed normal pellet diet. G1 was continued on a normal diet throughout the study and G2 and G3 were then switched to LMD throughout. G4 and G5 were fed LMD for 3 months followed by 3 months of a LMD supplemented with the respective SBM (with F and without F). The mineral composition (of total diet) of interest in LMD was Ca- 0.10%, P- 0.11%, Mg- 0.05%, Zn- 35 ppm, Mn- 11 ppm, Mo- 0.15%, Se- 0.19%, F- 0 ppm. The test diets were modified from LMD with addition of 1% SBM (with F and without F). In the test diets, the mineral composition was- Ca- 0.70%, P- 0.97%, Mg- 0.13%, Zn- 408 ppm, Mn- 157 ppm, Mo- 0.82 ppm, Se- 0.30 ppm. G4 diet had an addition of F- 39.5 ppm. The diet with or without SBM were processed to pellet form (Purina Test Diet, Indiana) prior to animal feed. All other nutrients (proteins, vitamins) were added per healthy recommended doses for a rat’s diet. The animal protocol was approved by the NYU institutional animal care and use committee (IUCAC) and NIH guidelines for the care and use of laboratory animals (NIH Publication #85–23 Rev. 1985) had been implemented.

After a period of 6 months, the animals were sacrificed by CO₂ inhalation; the rats were exposed to a gas mixture of 30% CO₂ and 70% O₂ for 2 minutes followed by 100% CO₂ for 4 minutes at a flow rate of 25psi (Euthanex System, USA.) until no sign of breathing was detected. Necropsy was performed on all rats and showed no signs of pathological abnormalities and calcifications in the kidney, liver or heart. Thereafter, their femurs and L5 vertebrae were isolated after soft tissues resection. Bones for mechanical strength measurements (femurs) were wrapped immediately in saline-soaked gauze and frozen until the experiment was performed. Bones for other analyses were immersed in 70% ethanol and stored.

Preparation of SBM

The experimental SBM was prepared by a modified hydrolysis method²⁵. Briefly, the mixture of dicalcium phosphate dihydrate, CaHPO₄∙2H₂O, Mg and Zn chlorides (MgCl₂ and ZnCl₂ respectively) were hydrolyzed in double deionized water containing dissolved potassium carbonate and sodium fluoride (for SBM with F only). Reaction temperature was 95°C; pH unadjusted and reaction time 16 hours, then rinsed with double deionized water and dried at 100°C in low temperature oven. Higher concentrations of Mg, Zn and F were added to the SBM, than in normal rat bone mineral, to take advantage of their positive effects on bone remodeling.¹⁷

Characterization of SBM and bone

The SBM preparations and retrieved bone were characterized using X-ray diffraction (XRD) (Philips X’Pert), Inductive coupled plasma mass spectrometry (ICP) (Thermo Jarrell Ash, Trace Scan Advantage) and thermal analysis; DSC-TGA (SDT Q600, Texas Instruments).

XRD was utilized to determine the crystallite size of the SBM and bone mineral using a curved crystal monochromator, operating at 45 KV and 45 mA, scanning in the range of 20–40°, 0.02°(2θ) step size at 3 secs/step. Crystallite size along the c-axis direction was determined from the broadening at half height (β1/2) of the [002] diffraction peak (at around 2θ = 25.8°) using the Scherrer formula: t = 0.9λ / (β1/2 cosθ), where ‘t’ is crystallite size, λ (wavelength) = 1.545Å, θ = diffraction angle, β1/2 is the difference of sample and β1/2 = (B2 - b2)1/2, B2 = observed broadening from the XRD pattern, b2 = instrumental broadening (0.001 radian).

DSC-TGA instrument was used to quantify organic, carbonate and mineral content from weight loss (%) in the sample at different ranges of temperature. 10 mg bone powder was heated from room temperature (25°C) to 950°C at a rate of 20°C/min.

Composition of Ca, P, Mg and Zn in the bone and SBM was determined using the ICP instrument. 10 mg of powdered sample, ashed at 600°C, was dissolved in 1:1 HCl, and made up to 100 ml (with double deionized water) in a volumetric flask. The specimen in solution and standard solutions (Fisher Scientific) were pumped through argon plasma excited by 2 KW 27.12 MHz radio frequency generators. The characteristic wavelengths for the elements determined were: Ca: 317.9 Å; P: 213.6 Å; Mg: 279.5 Å and Zn: 202.5 Å.

Assessment of BMD and BMC

DXA was used to measure BMC (g) and BMD (g/cm²). For the DXA (pDXA, Norland) measurements, the “small subject” mode was used for scanning. The scan field size used for the femur and L5 were 1.5×4.22cm, 1.5×1.5cm, scan speeds were 5 and 10 mm/s and scan times were 7 and 4 min, respectively.

The bone microarchitecture of the femur and vertebrae (stored in 70% alcohol) were determined using μCT (CT40 Scanco Medical, Bassersdorf, Switzerland). Bones in 70% alcohol were placed in the μCT specimen holder and sealed with parafilm tape to prevent drying. 150 slices (medium resolution at 20 μm per slice) for the femur and 280 slices for the spine were obtained using 55 kVp energy and 145 μA current. The integration time used was 150 ms and total scan time for each sample was around 70 min. The 3D structural parameters obtained were Bone volume/total volume (BV/TV), trabecular thickness (Tb.Th), and trabecular separation (Th.Sp). Porosity was calculated as (1-BV/TV).

Bone morphology study

Qualitative analysis of the L5 bones was performed using a scanning electron microscope as an adjunct to the μCT data. The L5 bones were cleaned of extraneous soft tissues, sectioned at the center of the body of the spine using a low-speed saw (IsometBuehler, USA) under water cooling, sonicated with 3 drops NaOCl in 10 ml deionized water for 2 minutes, followed by double deionized water sonication for another two minutes repeated two times and air dried. The bones were gold coated using sputter coating (Emitech K 650) for SEM observation (Hitachi S3500N, Japan) operating at 10 kVp.

Biomechanical testing

The femurs that were wrapped in wet gauze and frozen right after they were dissected from the rats were slowly defrosted and soaked in a saline solution for 15 minutes to prevent the specimens from drying out. The rat femurs were cleaned of any remaining soft tissues, particularly in areas where a load would be applied.

Bone strength was determined using a three-point bend test on a universal testing machine (Instron series 5560-Table Top Load Frame) at a loading rate of 1 mm/min. A fixture composed of two parts was used to hold the specimens in place during the test. The lower part of the fixture consisted of two cylinders separated by 16 mm that support the posterior aspect of the femur. One support was placed distal to the lesser trochanter and the other support, proximal to the distal metaphysis. The upper part of the fixture provided the load midway between the bottom two supports. An initial stabilization load of 1–2N was applied to each sample before the actual loading. While the tests were performed, Bluehill software was used for real time data collection, including the load, displacement, and time. All the mechanical tests were performed until fracture occurred. The strength was calculated using the following equation: σ = F*L*R/π(R⁴-r⁴) where σ is the strength, F is the load, L is span support distance, R is the outer radius of femoral mid-shaft where the fracture occurred, r is the inner radius of femoral mid-shaft where the fracture occurred.

Statistical analysis

One-way analysis of variance (AVOVA) and Tukey post-hoc tests were used to compare the effect of treatment groups on BMD, BMC, bone microarchitecture and strength in the femur and L5 vertebra, for a pre-determined significance level 0.05 and 95% confidence interval. Because of much greater precision in the measurements in the control group (G1), and the need for homogeneous variances in the ANOVA, G1 observations were not included in the ANOVA, and this exclusion is indicated by use of a horizontal reference line in the Figures. One can interpret significant differences from the other groups by non-overlap with the higher end of their error bars. All analysis used IBM SPSS software (v22; IBM Corp., Armonk, NY).

RESULTS

Characterization of rat bone and SBM

The XRD spectra for bones exposed to the SBM preparations (with F and without) were similar to normal rat bone, while crystallite size was higher in G4 bones than in other groups (Table 2). The inorganic/organic ratio was higher in OVX with supplemented rat bones than in OVX with LMD bones. LMD by itself also produced lower ratios than normal diet (Table 3). Since the detection limit of our ICP is less than 0.1 ppm, any difference greater than 0.3 ppm can be considered statistically significant. In light of this analysis, it can be concluded that G1 has the highest mineral content among all groups, whereas G3 has the lowest (Table 2).

Table 2.

Composition of ashed rat bone by weight % (parts per million) and crystal size (Å) of bone apatite.

Group	Ca	P	Mg	Zn	Crystal size
G1	27.71	16.76	1.10	2.90	263
G2	26.08	16.04	0.95	0.08	210
G3	25.60	12.21	0.50	0.05	178
G4 & G5	26.44	15.03	2.05	1.32	272 & 257

Table 3.

Mean (SD) weight % (wt%) of water, organic, carbonate and inorganic content in rat bone across four study groups.

Group	wt% water	wt% organic	wt% CO3	wt% mineral (+CO3)
G1	7.0±0.20	23.45±0.50	9.3±0.48	69.55±0.36
G2	8.03±0.53	27.13±0.55	6.46±1.68	64.84±0.16
G3	7.75±0.78	31.12±0.54	5.15±0.84	61.11±1.02
G4	7.21±0.63	28.82±0.96	8.10±0.58	63.96±0.92
G5	6.85±0.33	28.13±0.87	7.13±0.51	65.01±0.98

Bone density and mineral content

As shown in Figure 1 (A, C), the BMD of the femur and L5 averaged .25 and .27 g/cm², respectively, in G1. In G2 23.7% BMD reductions were seen in the femur and 34.5% in L5, compared to G1. In L5 (Figure 1, C), G3 was associated with a further significant reduction in BMD - 45.4% compared to G1 (and other groups - p<0.001 compared to G4, G5 and p<0.05 compared to G2). This change was offset by SBM supplementation in G4 and G5, where BMD losses were limited to 23.5% of G1 (G4 and G5 > G3, p< 0.001). Similar effects may be noted in the BMC of L5 (Figure 1, D). (All % differences were obtained by comparison of the means).

Figure 1:

BMD and BMC in the femur (A, B) and L5 (C, D) across study groups. Control is presented as a horizontal reference line. Bars show mean ± 2 standard errors of the mean (SE).

By contrast, in the femur (Figure 1A), G3 did not lead to a significant additional loss of BMD over G2 alone, and mineral replacement effects on BMD were not as strong offsetting effects of OVX and LMD, although G5 > G3 (p=0.023).

Similar effects may be noted for BMC of the femur (Figure 1, B) although outliers hinder clear interpretation of effects in G4 (p<0.05 with outlier only). The BMC baseline (G1) for femur and L5 were 0.515 and 0.143 g. The loss of BMC in G3 accounts for 26.2% and is not significantly different than in G2 – 22.3%. SBM supplementation did not majorly improve BMC in the femur.

Overall, for both femur and L5 bones, SBM with and without fluoride produced statistically similar effects on decreasing bone loss. Further, OVX with LMD has a greater effect on loss of mineral density and content of vertebra (p<0.05) than long bones, while SBM is closely associated with reserving the mineral depleting effects of OVX with LMD.

Mechanical properties of Femur

In continuation with the pattern of femoral mineral content and density loss in G2 and G3, critical fracture load – the load bearing capacity before critical fracture; showed similar trends. Although, significant improvements in load bearing was noticed in both supplemented groups (G4, G5) compared to G2 and G3. As seen in Figure 2, the baseline (G1) critical fracture load was 137.927 N. Overall, the loss of load bearing capacity in G2 and G3 compared to the baseline accounted for 28.2% and 30.4%. Tukey post hoc tests revealed a significantly higher load bearing capacity in G4 and G5 (in G4; p = 0.002 and G5; p < 0.0001) compared to G3. This load bearing was improved by 28.1% in G4 and 35.4% in G5, compared to G3. Comparative to G2, load bearing was also improved in G4 (p = 0.013), G5 (p = 0.001) by an increase of 24.2% in G4 and 31.3% in G5. Significantly, this improvement of load bearing in G4 was only 10% lower than baseline and 5% lower in G5.

Figure 2:

Results of the three-point bending flexural test in the femur across study groups with control as reference line. A - Critical fracture load, B - Strength of femur. All values depicted in the bar graph are represented by mean and error bars - standard error mean (SE).

For bone strength the overall reduction compared to G1 (255.164 MPa) was 16.9% for G2 and 27.9% for G3. Supplementation improved strength by 29.3% in G4 (p = 0.035) compared to G3; while being comparable to the sham group. Compared to G1 only 6.7% and 17.3% difference in strength was seen in G4 and G5 rats, the improvement in strength in G4 was 11.3% higher than G5. Amongst the non-supplemented groups, 13.2% lowered strength was seen in G3, compared to G2. Furthermore, significant improvement in elastic modulus was seen in G4 (p = 0.015) and G5 (p = 0.037) rats compared to G3 and thickness of femur was markedly greater in G5 (p = 0.044).

Microarchitecture study

Compared to G3, the bone quality and microarchitecture in G4 and G5 rats were markedly improved. Figure 3 shows preservation of L5 trabecular bone microarchitecture in G4 and G5 compared to G3. Quantification of 3D cortical structures in the femur and trabecular structures in L5, as seen in Table 4, revealed a trend of bone microarchitecture improvement (p<0.05; one-way anova) in G4 and G5. In the femur, as expected, G3 had the lowest BV/TV – a 74.4% loss compared to the reference (G1). While the supplemental groups were able to retain more BV/TV compared to G3, they were at least 55% lower than the reference bone volume. In comparison to G3, the Tukey post hoc test in G4 femurs further revealed significant improvement in bone volume (p = 0.007) and Tb. Th (p = 0.005) and in G5 for Tb.Th (p = 0.004). In the L5, 30% reduction in BV/TV was observed in G3 compared to G1 while in G4 and G5 23.7% and 23.4% differences respectively were observed compared to G1. Similarly, a 14.2 % decrease in Tb.Th was seen in G3 compared to G1 while 0.9% and 6.1% differences respectively were seen in G4 and G5 compared to G1. Overall, while G3 had the most reduction in bone microarchitecture, although contrarily these effects were inconsistent in G2. Although, G4 and to some extent G5 were able to retain more of the bone microarchitecture in comparison to G3. Figure 4 shows the morphological improvement in L5 bone microarchitecture as seen through SEM in G4 and G5 compared to G3 and G2.

Figure 3:

Representative μCT 3D reconstruction images of the femur comparing the microarchitectural changes across four study groups. The figure clearly elucidates the significant preservation of bone in G4 and G5 compared to G3. A - G2: Sham on LMD, B - G3: OVX on LMD, C - G4: OVX with SBM with F and D - G5: OVX with SBM without F.

Table 4.

Three-dimensionally computed analysis of the femur and L5 across the four study groups.

μCT 3D analysis	G1 (control)	G2	G3	G4	G5
BV/TV (%)
Femur	36.7±0.02	20.9±4.0*a	9.4±1.0	16.2±4.4*a	13.7±3.1*b
L5	32.8 0.7	36.3±.3.3*	25.2±4.9	32.0±3.8*	32.0±8.2*
Tb.Th (mm)
Femur	0.13±0.013	0.08±0.005*	0.10±0.013	0.12±0.01*b	0.12±0.01*b
L5	0.104±0.01	0.096±0.004	0.091±0.004	0.102±0.006	0.098±0.014
Tb.Sp (μm)
Femur	0.49±0.21	0.50±0.16*a	1.12±0.26	0.94±0.19*b	1.05±0.17*b
L5	0.251±0.019	0.27±0.02	0.35±0.08	0.29±0.03	0.27±0.04

Values are expressed as mean ± SD;

^*One way anova (p<0.05), compared to G3;

^aTukey posthoc (p<0.05) compared to G3 and

^bcompared to G2. Reference data was not incorporated in the statistical analysis. BV/TV: bone volume/total volume, Tb.Th: trabecular thickness, and Th.Sp: trabecular separation.

Figure 4:

Photomicrographs of L5 vertebrae under the SEM at 10.0 kV and 1 mm resolution (left) and 100 μm resolution (right) showing qualitatively improved microarchitecture in G4 and G5 compared to G3; A - G2: Sham on LMD, B - G3: OVX on LMD, C - G4: OVX with SBM with F and D - G5: OVX with SBM without F.

DISCUSSION

Our present findings demonstrate that estrogen deficiency coupled with LMD leads to an accelerated loss of bone density, microarchitecture and strength compared to the sham operated LMD group. Furthermore, SBM prevented bone loss and improved bone biomechanical properties in lumbar vertebra and femur of an OVX + LMD model, per our hypothesis.

The weight gain in the rats across study groups was consistent even though the sham LMD rats consumed markedly more food than the other groups. This could have led to higher mineral/nutrient consumption in these rats compared to the OVX and LMD rats. This phenomenon is demonstrated throughout the study, contrary to the expectation of sham LMD rats displaying low BMD, microarchitecture and strength. This evaluation is also seen in the Ca, P, Mg and Zn composition of ashed rat bones across groups. OVX and LMD rats lost most mineral, in particular phosphate, Mg and Zn content compared to the sham MD rats. While the supplemented group rats were able to improve the mineral content of the bone comparable to control. Crystal size can determine the dissolution of bone apatite crystals in-vivo and while the OVX and sham LMD rat bone crystals had the smallest crystal sizes, the supplemented animal bones had improved crystallinity. Similarly, the inorganic/organic ratio that was expectedly decreased in the OVX and LMD rats, was improved in the supplemented rat bones. The combined effect of OVX and LMD has a more profound effect on the BMD, BMC, architecture and strength; affecting the overall bone health, compared to only LMD group. Overall, these analytical tests imply a positive trend in bone mass and microarchitecture accrual in supplemented rat bones in comparison to the OVX and LMD rats.

BMD is considered a gold standard for the diagnosis of osteoporosis. In our study, as a direct trend to the analytical results discussed above, OVX rats with LMD had the lowest BMD and BMC compared to the reference. This trend was similar in both the femur and L5, although the effect was more marked in the trabecular bone (L5) compared to the cortical bone (femur). Similarly, the loss in BMD was higher in sham L5, compared to the femur even though the difference in loss between the two groups was higher in L5. In contrast, SBM was able to prevent bone loss and improve the BMD in both L5 and femur. SBM was effective in preventing bone loss in both trabecular and cortical bones, compared to the reference in a range of 27–33%. This prevention of bone loss was marked in an OVX and LMD model, compared to LMD rats this effect was not significant.

Estrogen deficiency induced osteoporosis is contributed by other changes as well. The impairment in bone microarchitecture and its biomechanical properties are more evident in the OVX and LMD group when compared to the sham LMD group where mineral deficiency is the key to bone loss. In our study, we observed that bone microarchitecture was severely affected by this combination of estrogen and mineral deficiencies compared to only mineral deficiency, in particular the cortical bone (femur). In the trabecular bone, this trend of significant bone loss was more noticeable in overall bone volume. Again, the sham LMD rats did not lose as much bone as anticipated compared to OVX. Introduction of SBM supplement to the LMD diet in OVX rats significantly preserved the microarchitecture, in particular the Tb.Th and these effects were seen in both cortical and trabecular bones, even though more significant in the former. The rat model used in this study represents a combination of estrogen and mineral deficiency induced low peak bone mass osteoporosis. Rats reach a “mature” skeletal size by 210 days (30×7).³² If skeletally immature rats are involved, then a low peak bone mass is achieved, a fact that is considered to be a high risk factor for human osteoporotic fractures. This trait is why the skeletally immature rat is an appropriate animal model in the research of endocrine, nutritional and environmental factors, all of which can influence peak bone mass. The skeletally mature rat is an appropriate animal model for the research of postmenopausal and immobilization osteoporosis.³³ It is important to distinguish the current study model from humans on basis that all rat groups here were fed a nutritionally complete non-mineral diet throughout, while a mineral based supplement was provided to the treatment groups only during the latter half of the study.

Strength of the bones is a direct component of the BMD and microarchitecture. Following the trend of decreased BMD, BMC and microarchitecture of femoral bone in OVX-LMD rats, the strength is of these bones is markedly reduced as well. Although the strength of the sham-LMD bones is decreased as well, the effect of LMD is not very clear. In this study, we are also able to exhibit the efficacy of SBM in improving the strength and biomechanical properties of the femur compared to a combined estrogen and mineral deficiency led bone strength loss. Low bone mass is the single most accurate predictor of increased fracture risk. While OVX and LMD lead to loss of bone mass, SBM was able to offset the lost bone. Reduced BMD is proportional to increased fracture risk and bone remodels in response to habitual and normal loads. The principal stresses are concentrated in compressive trabeculae during gait while in tension during fall.³⁴ When the environmental loads on a bone are changed by trauma, pathology or change in life pattern, functional remodeling reorients the trabeculae so that they align with the new principal stress trajectories. Osteoporotic fractures are associated with loss of bone mass and trabecular interconnectivity.³⁵ Porosity and mineral content is another important aspect in determining bone mechanics.³⁶

Although SBM was able to preserve bone and biomechanical properties, the effects of adding F to SBM as a bone loss prevention agent seems questionable due to insignificant difference in results of the two treatment groups, thus may require further testing. In the results of the current and previous studies we conducted, F seemed to increase the bone apatite crystallinity. The rationale to add F to SBM was to utilize its effects on enhancing crystallinity and reducing bone apatite solubility, when incorporated within ‘normal’ limits.^37–39 A narrow therapeutic over toxicity window complicates the utilization of F, even while studies have found association between F intake, osteoclastogenesis and increase in circulating parathyroid hormone among its other effects.⁴⁰ Regardless, the combined effects of other minerals incorporated in SBM have been consistent in preventing bone loss throughout the study. The results are consistent with previous literature reporting positive individual effects of these minerals. A meta-analysis study on effects of Ca intake over hip fracture risk concluded its neutral effects while suggesting a focus on optimal combination and possible correction of phosphate deficiency by using Ca-P supplements.⁴¹ Studies suggest that a Ca-P salt can exert positive effects on bone acquisition and maintenance.¹ Mg deficiency leads to bone loss in what might include a phosphate-induced release of inflammatory cytokines and impaired 1,25-dihydroxyvitamin D and parathyroid hormone. Low Mg alters the structure of apatite crystals and is associated with reduced levels of PTH and thus decrease vitamin D.¹³ Mg in association with potassium has been seen to quantitatively improve bone health and reduce the risk of hip fracture in both men and women.⁴² Zn is also involved in the bone turnover process by regulating calcitonin secretion in turn reducing osteoclastic activity, collagen synthesis and alkaline phosphatase activity. Zn has been shown to have a stimulatory effect on osteoblastic bone formation and mineralization. Zn inhibits osteoclastic resorption due to inhibiting osteoclast-like cell formation from bone marrow cells and stimulating apoptotic cell death of mature osteoclasts, while also suppressing receptor activator of nuclear factor ligand (RANKL) induced osteoclastogenesis. It has been suggested that Zn accumulated in bone may activate alkaline phosphatase and collagen synthesis in osteoblasts. Zn has also been known to enhance the anabolic effects of vitamin D3.^4,14,43 Mn is also an important osteotropic element. Apart from the stimulation of the synthesis of bone matrix, it also has an effect on general calcification. It is important for skeletal integrity in adults. Mn is needed for the biosynthesis of mucopolysaccharides in bone matrix formation and is a cofactor for several enzymes in bone tissue. Mn deficient animals have alterations in IGF metabolism, growth, and bone. Mn supplementation, along with Ca and Zn resulted in greater gain in bone compared to Ca alone in postmenopausal women over a 2-year period.^3,44 Findings released on World Osteoporosis Day by the International Osteoporosis Foundation (IOF) show that 89% of those who used the IOF Ca Calculator - which is based on Institute of Medicine (IOM) recommendations – were Ca deficient. The Ca Calculator results were based on 6,908 users from 83 countries.⁴⁵ Through this study we report the preventive and bone preservation efficacy of a combinational mineral preparation in OVX, even while the base diet provided other vital bonetropic nutrients (proteins, vitamins). While osteoporosis is caused by an imbalance between bone formation and bone resorption, the efficacy of SBM preparation in presence of estrogen deficiency may be explained in terms of the individual and combined effects of the Ca, P, Mg, Zn and Mn ions on bone cell activities (bone formation and bone resorption) when released from the SBM or when incorporated in the newly formed bone.

Future studies will evaluate the bone loss and strength recovery efficacy of SBM on a larger animal model while also evaluating dosage and biomaterial safety.

CONCLUSIONS

In conclusion, we confirm some of our research hypotheses. Compared to sham-LMD, OVX-LMD group had higher bone mass loss and lowered strength while the SBM supplemented OVX group was able to preserve bone and its biomechanical properties, vital to preventing osteoporotic fractures. The inorganic/organic ratio was lower in OVX-LMD group compared to SBM treated OVX or sham groups. Also between the two treatment groups, the presence of F in the SBM supplement provided no significant difference in bone loss prevention or improving strength compared to non-supplemented groups.