Micronutrients-incorporated calcium phosphate particles with protective effect on osteoporotic bone tissue

Abstract

Background

Supplementation of individual micronutrient is inadequate for maintaining bone function because single micronutrient can not contribute significantly a positive remodeling balance.

Objective

We developed the highly integrated, stably dietary multi-micronutrients with good bioavailability and low adverse effect on the improvement of bone consolidation in osteoporosis.

Methods

The trace element-codoped calcium phosphate (teCaP) particles were prepared in the modified body fluid and carefully evaluated. Rats, aged 3 months, were ovariectomized and when 6 month intervened with the conditioned, low, moderate, and high teCaP diets.

Results

The teCaP particles showed highly dissolvable in stomach juice-mimicing acidic solutions. Three months after intervention, the body weight increase showed remarkable differences among the low teCaP diet (∼52 g), moderate teCaP diet (∼34 g) and high teCaP diet (∼23 g) group. In particular, the intake of moderate teCaP greatly improved the retention of trace elements in femural bone for better protection against the skeletal weakening, and resulted in a significant increase of bone mineral density (104.06%) in comparison with the conventional high calcium plus vitamin D3 diet (Control group).

Conclusions

These investigations improve our understanding of micronutrient retention on bone consolidation in osteoporotic bone tissue, and also provide new mild wet-chemical approach to prepare potent nutritionally effective edible complements to synergistically relieve bone degeneration and prevent osteoporosis.

Introduction

Osteoporosis represents one of the growing global health problems, particularly in elderly female. This multifactorial pathology involves genetic and environmental factors and exhibits a gradual deterioration of bone mineral, a process that can span over several decades of life (1). Bone mass and density is affected by many factors including hormonal status, nutrition, exercise, adequate sun exposure and life style. Among these factors, nutrition intervention is considered to be crucial in order to prevent such destructive prognosis (2).

The oral route is considered to be a convenient means of nutrient administration, leading to higher patient compliance. The widely applied nutrition intervention to strengthen bone mineral density (BMD) is oral intake of calcium-rich food. Unfortunately, supplementation of individual nutrient is inadequate for maintaining bone function because single agent can not contribute significantly a positive remodeling balance. Chapuy et al. reported a decrease in the risk of hip fractures among elderly women when they were supplemented with vitamin D3 and calcium (3). However, it remains controversial because more recent investigation demonstrates that high calcium intake increases fracture risk (4) and accelerated other complication (5). Strontium ranelate has the ability to reduce the incidence of osteoporotic fracture (6), whereas this trace mineral-working drug does not address other micronutrients necessary for long-term nutrients balance. It is known that the live bone tissues contain a variety of trace minerals, which likely play important biological roles. Typical minerals in bone are 4-8 % of C03, ~0.4-0.9 % of sodium and magnesium, and approximately less than 0.1 % of other trace elements (TEs) (7). Among the inorganic electrolytes involved in bone and the surrounding body fluids, the essential TEs are expected to play a critical role in the maintenance of hard tissue function due to their conditionally essential and nutritional effects. Hence, a well- balanced supplementation of the key TEs designed to deliver at the reference nutrient intake should be encouraged.

Recent researches on the bioactivity of apatite materials have focused on improving bone defect repair by doping TEs such as strontium, silicon, zinc and magnesium (8). Strontium can enhance osteoblast proliferation and inhibit osteoclastic activity (9). Silicon increases type I collagen synthesis, promotes young bone mineralization and inhibits bone loss (10, 11). In normal or ovariectomized (OVX) rats, silicon and strontium supplements at low doses result in increase in bone mass and strength by inhibiting bone resorption and augmenting bone formation (11, 12). Zinc is involved in many metalloenzymes and zinc-deficiency increases the risk of decrease of bone density and osteoporosis (13, 14). The content of magnesium in biological apatite is basically larger than that of other TEs, and it is reported that magensium deficiency gives rise to decreased bone mass (15). Epidemiologic data suggest that loss of such micronutrients is a significant bone health concern (16), while limited studies are available for the synergistic effects of multi-micronutrients. Accordingly, the interdisciplinary knowledge from biomaterial sciences, nutrition and the emerging biotechnology should be integrated into nutrition research to greatly improve nutrient processing and formulation (17).

Here we developed the TEs-codoped calcium phosphate (teCaP) particles in biomimetic solutions and examined the association of intake of low-, moderate-, and high-calcium diets conditioned with teCaP with TE retention and bone consolidation in osteoporotic animal models. Simulated body fluid (SBF) is a well-known inorganic solution in Biomaterial science which contains nearly equal inorganic ions as human blood plasma and have been widely applied to clarify the similarity between biological processes in vitro and in vivo (18). Not only is the strategy carried through without involving body-unfriendly additives, but also the fruit juice organic molelcules can modulate the crystallization and dissolution of CaP (19). Additional insights into bone mass improvement were gleaned through oral administration in OVX rats. Our findings suggest this multi-micronutrient manufacture that work in biomimetic mineralization medium can be used in a variety of micronutrient-mineral hybrid systems, for oral administration with drink or food to prevent and treat osteoporosis.

Materials and Methods

Chemicals and materials

The inorganic reagents were used in as-received conditions (BBI, Canada). The fresh fruit juice (F%) including apple (Yantai, China), orange (Jaxing, China), and grape (Tulufan, China) were washed with deionized water. After washing the fruit in deionized water and drying its surface in a light stream of N2, the fresh clear pure juices were made with an electric juice extractor (JuYang Co., China) and filtered with 0.80-Ym pore membrane.

Preparation of fruit juice-modified SBF (mSBF)

SBF was prepared and buffered to pH 7.4 as described previously (18). Then, solutions (50 mM) of SrCl₂, ZnCl₂, and Na₂SiO₃ were added to SBF, followed by a 0.22-Ym pore membrane filtering (thereby denoted as mSBF). More mSBFs were prepared by droping pure apple, orange, and grape juices with 0.05-1.0% (vS) juices and denoted as mSBF-a, mSBF-o and mSBF-g, respectively (see Tab. 1). For easier abbreviation, we adopted the following nomenclature: mSBF-a-3, mSBF-a-6 and mSBF-a-9 corresponded to the element ratios of SrHEa, ZntEa and Si/P molar ratio of 3% (or 0.3%), 6% (or 0.6%) and 9% (or 0.9%) TEs in the aqueous media.

Table 1.

Main inorganic ion concentrations (mmol>L-1) in mSBF*

Mediudl	Ca++	HPO	Nad	Kd	SO°	Cd	O o nD	Md3°	Sr™	dhm	SiOn°
Blood plasma	2.5	1.0	142.0	5.0	0.5	103.0	27.0	1.5	0	0	0
SBF	2.5	1.0	142.0	5.0	0.5	147.8	4.2	1.5	0	0	0
mSBF-a-6	2.5	1.0	142.1	5.0	0.5	148.1	4.2	1.5	0.15	0.015	0.06
mSBF-o-6	2.5	1.0	142.1	5.0	0.5	148.1	4.2	1.5	0.15	0.015	0.06
mSBF-g-6	2.5	1.0	142.1	5.0	0.5	148.1	4.2	1.5	0.15	0.015	0.06

□ The mineral ions in 100% fresh juices were not calculated in these reaction media.

Preparation of teCaP multinutrients

In a typical procedure, 2000 mL of mSBF-a-6 containing 1.0 vol% apple juice was kept at ~75PC with magnetic stirring. After 8-h ageing period, the white precipitates were filtered, washed with diluted HCl (pH ~3.8) and deionized water for three times, and dried overnight. More teCaP powders were synthesized in the mSBF by changing juice concentrations (from 0.05 to 1.0 vol%) under the same temperature. We therefore define the apple, orange, and grape juice-mediated teCaPs as teCaP-a, teCaP-o and teCaP-g, respectively. The teCaP precipitates were also prepared in F%free mSBF while the other reaction conditions remained the same. The samples were examined by X-ray diffraction (XRD; Rigaku D/max-rA), Fourier transform infrared (FTIR; Nicolet), scanning electron microscopy (SEM; HITACHI), and thermograwimeteric and differential thermal analysis (TG-DTA; TG/DTG6200). The surface chemistry and elemental contents of teCaPs powders were quantified using X-ray photoelectron spectroscopy (XPS; Thermo-VG) with monochromatic Al &X resource. The electron take-off angle was fixed at 45P and the vacuum pressure was maintained below 10^-9 torr.

In vitro dissolution experiment

Dissolution studies for teCaP-a (50 mg 30 mL) were conducted in triplicate in pure fruit juices and two kinds of acetate buffers at pH 4.5R0.1 (denoted as buffer-4.5) and 2.0R0.1 (denoted as buffer-2) for a period of 96 h, respectively. After incubation at 37PC for required time in a shaker, 2 mL suspension was removed and centrifuged for inductively coupled plasma (ICP; Thermo) elemental analysis.

Animal models and maintenance

Animal received human care, and the study protocol complied with Zhejiang University guideline for care and use of laboratory animals. The animals were 3-month-age female Sprague-Dawley rats of average weight 220 g at the beginning of the study. The animals were OVXed by the well-accepted procedure to develop trabecular bone osteoporosis. They were kept in stainless steel cages under standard conditions with free access to food and drinking water. Water consumption and body weight were measured every month.

Diets and feeding

Three months after OVX, animals were randomly divided in four groups and assessed for diet intervention (n=10[group): (1) animals received the high-calcium diet (15 mg Ca[fl:g diet) fortified with calcium carbonate & vitamin D3 tablet (CaltrateD; denoted as CTL group); (2) low-calcium diet (3 mg Ca[B;g diet) conditionalized with teCaP-a (denoted as L-ex group); (3) moderate-calcium diet (9 mg Ca/Jg diet) conditionalized with teCaP-a (denoted as M-ex group); (4) high-calcium diet (15 mg Ca[B;g diet) conditionalized with teCaP-a (denoted as H-ex group). The other diet components were given at recommended levels for rodent diet according to AIN-93M guidelines (20). Animals were fed by group, and the diet was refreshed every two days. The actual levels achieved and additional diet specifications were listed in Table 2.

Table 2.

The main inorganic ion nutrient contents (Yg/kg diet) in diet intervention

□rou+	Ca	Md	Sr	dta	Si
CTL	15000	1500	34	90	15
L-ex	3000	300	295	85	30
M-ex	9000	900	885	255	90
H-ex	15000	1500	1475	425	150

Bone specimen analysis

At the final stage of experiment, animals were anesthetized with sodium pentobarbital and sacrificed by exsanguination. Faeces was obtained by large intestine puncture and dried at 250PC for 24 h. Lumbar spine (L3) and femurs were cleaned free of connected soft tissues and immediately weighed. Areal BMD at the lumbar spine (L3) and entire femur (ndB) were assessed by Dual-energy X-ray absorptiometry (DEXA; Norland Medical System) for three times. The femur specimens were dried at 180PC for 12 h in a furnace and followed by separated transversely with stainless steel saw to obtain about 8 mm long femoral stem. The samples were then pressed by mechanical machine (Instron) for compression strength determination. For elemental analysis, the femoral head and neck were calcinated at 600PC for 4 h. The calcinated bone samples and faeces were crushed with an agate inside a 100 class laminar-flow hood and then dissolved in 10% HCl solution for ICP analysis.

Statistical analysis

The data were expressed by mean standard deviation (SD). Statistical analysis of data was accomplished by one-way ANOVA and Scheffe’s F-test. The differences at p<0.05 were considered to be statistically significant.

Resutls

Morphological observation, phase determination and in vitro dissolution

SEM images showed that the teCaPs obtained from the F% free mSBF showed globular particles of 200~500 nm in diameter (Fig. 1a). However, the teCaP-a, teCaP-o, and teCaP-g particles, precipitated in the 1.0% F%containing mSBFs exhibited similar aggregated morphological features with domains of tens of naonometers in size (Fig. 1b-d). The XRD patterns showed that the powders were composites of hydroxyapatite (HA) and tricalcium magnesium phosphate (TCMP), and the phase composition ratios varied slightly according to Rietveld refinement method analysis (Fig. 1a-d, insets). With the increase of TE concentrations in mSBF, the co-doping ratio of strontium and zinc in precipitates increased significantly, but the silicon content was stable (0.13—0.28%). Magnesium could also be doped into the prcipitates as the secondary phase (i.e. TCMP) (Fig. 1e). The XPS analysis for the sample surface layer (tens of nm in thickness) revealed that the teCaP-a, teCaP-o, and teCaP-g were composed of organic component (C: 9.4—16.0%), CaP (Ca: 12.1-13.3 at%, P: 9.4—12.7 at%) and TEs (Sr: 0.7-0.9 at%, Zn: 0.23-0.29 at%, Si: 0.17—0.37 at%), with (Ca+Sr+Zn+Mg)/(P+Si) ratio of 1.48~ 1.56; the surface layer of the particles had a considerable amounts of magnesium (Mg: 1.42—1.97 at%; Mg/Ca >0.11). Moreover, TG-DTA analysis demonstrated that the weight loss of teCaP-a samples could be assigned sevaral steps such as loss of water (~180PC), decomposition of organic components (200—550PC), dissociation of CO2 (550—800PC) and OH group (800—1000PC), indicating the presence of FJderived organic molecules in the solid phases.

Figure 1.

SEM images of precipitates from mSBF in the absence (a) and presence (b, c, d) of 1.0 vol.% F%; ICP analysis for the TE contents in teCaP-a prepared in presence of different TE concentrations in mSBF-a (e); TE ions (average value) released in acidic buffer-4.5 (f) and buffer-2.0 (g) from teCaP-a, and calcium and phosphorous ions (average value) released in F% (h) and acidic buffer (i) from teCaP-a. insets in (a)~(d) represent the XRD patterns of the samples. insert in (g) represents the teCaP-a dissolution mimicing in the stomach juice. Bars in (a)~(d) represent 200 nm The mineral elements were determined in faeces after 3 months diet intervention. As shown in Figure 2c, the experimental groups contained various mineral ions, but only the H-ex group had significantly higher calcium and phosphorus contents than other groups. The trace mineral ions were detected and strontium was not in consistent with its contents in diets, and M-ex and H-ex groups showed a significantly statistical difference (p<0.05).

In vitro dissolution of the representative teCaP-a samples in buffers (buffer-4.5, buffer-2.0) and pure fruit juices, was determined by ICP. The TE ions could be readily released from the particles within the initial 4 h and followed by a slow release. Particularly, considerable amount of magnesium and strontium (24~32 ppm) and physiological favorable silicon and zinc (3~4 ppm) was released into buffer-2.0 in the initial 2 h, implying that the particles were stomach juice-soluble (Fig. 1f, g). Calcium and phosphate also produced a rapid dissolution in the acidic pure FJmedia (Fig. 1h, i).

In vivo test

The development of trabecular osteoporosis in femur of OVX rats has been confirmed in our previous studies (21). There were no serious adverse events with the exception of a rat death after 42-day of low-calcium diet (L-ex group) judged to be unrelated to its interventions. After the diet intervention, there were no significant differences among the groups, except that the L-ex group had significant increase in average body weight (52.6 g) in comparison with the other groups (S35.2 g) (Fig. 2a). However, the low-calcium (L-ex group) and high-calcium (CTL group) diets were disadvantageous for improving BMD in femur (Fig. 2b). The moderate- and high-calcium (M-ex, H-ex groups) diets incorporated with TEs could enhance BMD in femur by 4.82~6.49% in comparison with that of CTL (pdD.05). But the lumbar spine BMD did not change significantly except for L-ex group.

Figure 2.

Body weight increase (a), BMD (b) of the different OVX rat groups and main mineral element contents in the faece (c) after the 3-month diet intervention stage (□pCD.05, nUB)

Mineral element retention

As for the trace mineral ion distribution in the femoral head and stem (Fig. 3a, b), the strontium and silicon contents were well associated with the diet conditions, except for a slightly less silicon content in femoral head of H-ex than that of M-ex group. interestingly, M-ex group showed significantly higher strontium, magnesium and zinc contents than the CTL group in the femural head and stem (pHD.05), except for silicon without significant difference. Indeed, magnesium was over ten-fold of the other trace minerals, but a low-calcium intake (L-ex) influenced magnesium absorption in femoral bone. Moreover, the CaUP ratios in femoral head and neck increased considerabley with an increase of calcium dose in the diets, but the CaUP ratios in femoral stem were significantly lower than the CTL group after 3 months diet intervention (p<0.05) (Fig. 3c).

Figure 3.

Ca/P atom ratio in femoral head and neck and femoral stem of calcined femoral bone (a), and TEs contents in femoral head (b) and femoral stem (c) of calcined femur after the 3-month diet intervention (*pC0.05, n=6)

Bone strength of femoral bone stem

The mechanical strength of as-dried femoral stem was evaluated by applying a compressive load (Fig. 4). The representative stress pattern showed an initial linear elastic region, followed by a steady-state stress or failure. The CTL group revealed a significant stress fluctuation, with microstructure collapse before fracture. The M-ex and H-ex groups showed a significantly higher stress (over 200 N) than L-ex group (Q00 N). In particular, the M-ex group exhibited the highest compressive strength (33.3R2.1 MPa), increased by 32.65% in comparison with CTL group, suggesting the teCaP-conditioned, moderate-calcium diet may significantly consolidate the osteoporotic femoral mineral.

Figure 4.

Representative compressive stress-strain curves of the femoral stems. Below the graph are summarized the compressive strength (MPa) obtained from the curves (means R SD). Insert in (b) represents the femoral stem in universal compressive testing machinebone mineral. This means that trace mineral absorption and apatite mineral maturation may contribute to the bone fracture resistance. In summary, these results confirm that the dietary calcium may affect the body weight, but the multi-micronutrient administration more readily regulate the BMD and bone strength in osteoporosis (33). Although the micronutrients contributed synergistically to bone resorption resistance in OVX rats, it is unclear to what extent the organic components from F% reflects the bone turnover or age-related changes in the animal model. We will further evaluate the synergistic efficacy of organic micronutrients and the safety of the composites.

Discussion

This study is aimed to develop a simple, reliable way to produce in situ micronutrients-integrated dietary additives for relieving bone degeneration and decrease bone fracture in elderly population. Wet-chemical treatment of mSBFs shows a facile approach to produce the CaP-based multinutrients via intentionally adding fresh F% and appropriate amounts of TE ions (i.e. zinc, strontium, silicon) into the standard SBF. With this approach, the multi-micronutrients can be integrated into the Ca/P-rich particles to minimize use of inert materials, substantially balance the synergistic absorption and bioavailability, and suppress premature burst release in stomach acid. In particular, the experimental investigation revealed that the teCaP-a is highly soluble in stomach juice-mimicking medium in vitro and the oral administration of teCaP-a multinutrients may significantly improve the bone mass in oVX rats, in comparison with the commercial calcium carbonate/vitamin D3 agent.

Osteoporosis has been recognized as the most frequent chronic condition occurring in postmenopausal women and, optimization of bone health by appropriate dietary and lifestyle practices is a key factor in management of the disorder. Hitherto production of nutritional fortificants via integrating micronutrients in calcium-rich minerals for dietary prevention is a valuable pursuit but encountered with many difficulties. Most approaches attempted were intaking of adequate calcium and vitamin D, rather than comprehensive ones (i.e., calcium and multi-micronutrients). A modest, beneficial effect on multi-micronutrient intervention is limited (22, 23). Calcium carbonate has higher elemental calcium (40%) than other edible calcium salts. However, calcium intake without accompanying other nutrients would abet other complications. CaP and calcium carbonate appear to be equally effective in supporting bone building, so that the CaP additives pose no health risk (24). Indeed, the biological apatite contains carbonate, sodium and trace minerals. The critical levels of trace minerals are considered to play pivotal roles in bone remodeling (25). Thus, the present study is inspired by the composition of biomineral to produce nutritionally active teCaP in mSBF, as further discussed below.

The morphology and phase analyses validated our previous findings that the fruit juice-derived organic molecules influenced CaP growth (19). The TG-DTA analysis suggested that the organic molecules were present in the obtained teCaP-a. As the molecular size of these organic molecules is not enough to be taken into CaP crystals, the presence of these small molecules among the obtained precipitates is mainly caused by the adsorption on teCaP-a particles. Previous investigations have shown that the proteins and amino acids, which have a high negative charge, inhibited the crystal growth of HA (26). Comparing the irregular nano-sized aggregates and spherical particles precipitated in the presence and absence of F%, there is evidence of interaction occurred between organic molecules and CaP crystal faces. Since organic components increased with increasing F%concentration in mSBF (from 0.05% to 1.0%), a clear distinction between organic components volatilization among teCaP-a particles could be measured by TGA. It is well investigated that the natural grape, orange, and apple juices are attractive sources of organic micronutrients and are of high pharmacological significance for chronic diseases (27., 28., 29.). Although the organic micronutrient species and contents were not considered in this study, the apparent carbon contents in the surface layer by XPS anaylsis increased in teCaP-a, teCaP-o, teCaP-g (i.e. 9.4—16.0%) significantly compared with teCaP (5.8 at%) from in F%free mSBF.

SBF is known to be a metastable buffer solution like human plasma (18). It is saturated with respect to the formation and precipitation of an amorphous CaP phase owing to its relatively high Ca/P ratio of 2.5. The small variations in the temperature of SBF solutions are known to cause the spontaneous and in situ precipitation of different CaPs as a function of solution pH. Usually, treatment of biomaterials in SBF at human body temperature form biomimetic carbonated HA layer, but other studies also report the magnesium-substituted tricalcium phosphate (i.e. TCMP) produces in the modified SBF (30). Therefore, magnesium is readily substituted in the CaP structure as a secondary phase of TCMP (Ca_3-xMg_x(P0₄)₂) in the present study. TCP is widely used in artifical bone cements and toothpaste. In addition, it is also serves as a texturizer, bakery improver and anti-clumping agent and dietary or mineral supplement to food and feed (7). It is reasonable to postulate that the TCMP in the teCaP particles is highly desirable for potent nutritional efficacy. Furthermore, the teCaP-a particles are able to produce tailorable TE contents if more or less TE precursors are added in mSBF in advance (Fig. 1e). It is shown that the teCaP-a dissolution and TE release rate are mainly dependent on the pH value (Fig. 1f, g). The release rate of calcium, phosphorus, and trace minerals was significantly increased by decreasing pH within the initial 4 h (Fig. 1h, i). This time stage was usually the gastric residence time of food (31). Buffered, acidic solutions with pH 1.0~2.0, to mimic the strong acidic environment of stomach juice, were commonly used in comparative analyses of the absorption and bioavailability of low soluble nutrients. Although the solution composition does not correspond to the stomach juice, it is successfully used for nutrient studies since it facilitates the initial dissolution.

Overall, the nutritional effects of teCaP-a are evaluated compositionally, structurally, mechanically, and by ICP, DEXA and mechanical tests after 3 months of oral administration in OVX rat model. M-ex group (moderate calcium diet integrating with multi-micronutrients) showed greater mean compressive strengths than CTL group (1.32 times greater) and H-ex group (high calcium diet fortified with teCaP-a; 1.09 times greater) at 3 months. Mechanical failure of femoral stem consistently occurred within the bone, even in the case of L-ex group, but microcracks occurred firstly in CTL group (Fig. 4). These results suggest that the moderate-calcium diet conditioned with teCaP-a is highly favorable for improving the femoral bone strength in osteoporosis.

Several attempts have been made to improve the simultaneous intake of Ca and micronutrients from food supplements (19, 32). Despite the apparently positive results for the body weight in calcium carbonateQitamin D3 fortified diet, analysis of bone formation showed that calcium carbonateC3itamin D3 had lower BMD value than the moderate calcium diet conditionalized with teCaP-a (Fig. 4). Our data suggested that the teCaP-a multinutrients had the potential to improve the femoral BMD of OVX rats, as seen in the osteoporotic rat femoral marrow space by injecting the TEs-multidoped CaP analogs (21). Typically, the moderate calcium diet conditionalized with teCaP-a induced a marked increase in bone formation as shown by the marked elevation of femoral trace mineral contents (Fig. 3). Importantly, the excreted (trace) mineral elements in faece were not associated with the diet composition because strontium was absorbed by rat compared with other mineral elements in faece. This implies that the teCaP-a would be nearly fully dissolved during digestion in the stomach juice of osteoporotic rats. It is reasonable to conclude that strontium is highly absorbed in the rat body, because it substitutes for calcium to complex with phosphate group in the bone mineral. This means that trace mineral absorption and apatite mineral maturation may contribute to the bone fracture resistance. in summary, these results confirm that the dietary calcium may affect the body weight, but the multimicronutrient administration more readily regulate the BMD and bone strength in osteoporosis (33). although the micronutrients contributed synergistically to bone resorption resistance in oVX rats, it is unclear to what extent the organic components from FJs reflects the bone turnover or age-related changes in the animal model. We will further evaluate the synergistic efficacy of organic micronutrients and the safety of the composites.

Concision

These findings demonstrated the biomimetic mineralization reactions in vitro offers an expedient way to prepare micronutrients-integrated dietary supplements for bone consolidation, and the TEs-[F%-added SBF showed promise for atomically codoped teCaP production via a wet-chemical treatment. This new multinutrient demonstrated superior dissolution in simulated stomach juices. It is also demonstrated that the osteoporotic femur is systemically regulated by TE retention and can be reversed by dietary supplementation. Encouraged by the results of this study, we intend to investigate these multi-micronutrient supplements in large animals in order to promote the translation of this research into clinical practice. We envision that the nutritionally effective trace element-codoped CaP could be combined with an appropriate treatment for osteoporosis, thus leading to a substantial improvement in the quality of life of these patients.

Statement of conflict of interest

No potential conflict of interest was reported by the authors.