Protective Effects of Eggshell Powder and Eggshell-Derived Calcium Compounds on Glucocorticoid-Induced Osteoporosis in Albinos Wistar Rats

Protective Effects of Eggshell Powder and Eggshell-Derived Calcium Compounds on Glucocorticoid-Induced Osteoporosis in Albinos Wistar Rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Protective Effects of Eggshell Powder and Eggshell-Derived Calcium Compounds on Glucocorticoid-Induced Osteoporosis in Albinos Wistar Rats souad cheraitia, Soumia Keddari, Ahlem Laissouf, Meriem Mokhtar, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9012106/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Glucocorticoid-induced osteoporosis (GIO) is a common complication of prolonged steroid treatment, leading to impaired mineral metabolism and bone micro-architecture. This study evaluates the protective effects of eggshell powder (EPS) and its derivatives, particularly calcium acetate (CA) and nano-calcium lactate (NCal), in a model of methylprednisolone-induced osteoporosis in Wistar rats. The eggshells, collected locally, were characterized chemically and morphologically, as were their CA and NCal derivatives, which were obtained respectively by chemical transformation of the powder with acetic acid and by precipitation from calcium oxide and lactic acid, then characterized by FTIR an XRD, revealing the presence of characteristic functional groups (O-H,C=O,C-O,CO 3 -2 ) and typical crystalline structure. The in vivo study was conducted on ten groups of female rats, including a negative control, a positive control, and eight groups treated with ESP, CA, NCal and CaCO 3 at doses of 20 and 40 mg/kg. Biochemical parameters (calcium, phosphorus, and alkaline phosphatase ), femoral ash content, and histological analysis of the femur were evaluated. The results indicated a significant improvement in mineral metabolism in the treated groups, characterized by an increase in serum calcium and phosphorus concentrations, a reduction in alkaline phosphatase activity, and femoral remineralization confirmed by elevated calcium, phosphorus, and magnesium levels in femoral ash. Histological analysis revealed partial to complete restoration of trabecular bone structure with thick, well-organized trabeculae rich in osteocytes, contrasting with the bone degradation observed in untreated osteoporotic rats. In conclusion, eggshell powder and its derivatives, particularly calcium acetate and calcium nano-lactate, show great potential as a natural source of calcium for the prevention and treatment of glucocorticoid-induced osteoporosis. eggshell glucocorticoids osteoporosis calcium acetate calcium nano-lactate Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Osteoporosis (OP) is a major public health problem worldwide, mainly due to aging populations. It is a generalized metabolic disease of bone tissue [1], [2] . It is defined by a decrease in bone strength, resulting in increased vulnerability to fractures. The mechanical strength of bone depends on both the quantity and quality of bone tissue [3] . Osteoporosis is a common condition that affects both men and women, with a significantly higher incidence in women. Recognized as a major public health issue, it causes approximately 1.3 million bone fractures per year, representing an estimated economic cost of nearly $ 10 billion in the United States [4]. Osteoporosis is divided into two main forms: primary and secondary. Type I primary osteoporosis is linked to the decline in estrogen in women after menopause, while type II osteoporosis, know as senile osteoporosis, is a consequence of physiological aging. Secondary osteoporosis is closely linked to various underlying causes such as vitamin D deficiency, insufficient calcium intake, or iatrogenic factors [5] . Furthermore, glucocorticoid-induced osteoporosis is the most common form of secondary osteoporosis, accounting for nearly 20% of all reported cases of osteoporosis [6], [7] . Glucocorticoids are widely prescribed for the treatment of various inflammatory and autoimmune conditions. However, glucocorticoid-induced osteoporosis (GIO) is one of the most common and severe adverse complications associated with their administration [8] . In healthy adults, bone tissue undergoes continuous remodeling as a result of a closely coordinated physiological process. During this mechanism, osteoclasts ensure the resorption of old bone tissue, while osteoblasts are involved in the formation and mineralization of newly formed bone tissue, thus contributing to the maintenance and structural development of the skeleton [9, 10] . The use of glucocorticoids leads to inhibition of osteoblast activity, stimulation of bone resorption by osteoclasts, reduced intestinal calcium absorption, and secondary hyperparathyroidism. The interaction of these mechanisms results in a rapid decrease in bone mineral density (BMD) [11] . Several therapeutic options are available for the management of secondary osteoporosis. Previous studies have shown that certain nutritional compound, particularly eggshells, have beneficial effects by promoting bone mineralization and stimulating osteoblast activity in patients with osteoporosis [12] . Calcium extracted from eggshells is currently widely used as a low-cost calcium supplement. Recent studies have shown that eggshells are highly effective source of calcium for stimulating bone mineralization in ovariectomized rats [13] . Therefore, the disposal of eggshells without recovery represents an avoidable loss of valuable natural resources [14]. Global egg production has increased dramatically, from 0.75 trillion to 1.63 trillion eggs between 1994 and 2021, according to FAO data ( 190 to ~ 200 eggs consumed per person per year in 2024), which implies a very sharp increase in consumption and production, generating approximately 8.58 million tons of eggshells considered waste, often disposed of in landfills without recovery [76] . The shell is mainly composed of approximately 94% calcium carbonate (CaCO 3 ), 1% calcium phosphate (Ca 3 (PO 4 ) 2 ), 1% magnesium carbonate (MgCO 3 ), and 4% organic substances [15] . The shell membrane, meanwhile, consists of approximately 69.2% protein and 2.7% lipids [15], [16] . Due to their nutritional richness, various initiatives have been undertaken to make use of eggshells. They have been used as fertilizer, catalysts for biodiesel production, animal feed additives and soil amendments [17], [18] . Eggshell waste is particularly rich in bioactive compounds, which is sparking growing interest in its reuse through the development of value-added products with a variety of commercial applications [19] . In addition, several studies have shown that eggshells can be used as a natural source of calcium for medical and dietary purposes, particularly for the production of calcium salts such as lactate, propionate, gluconate, citrate, and calcium acetate [20] . In adults, approximately 2.7g of eggshell powder provides the recommended daily intake of calcium [21] . In this context, the present study was conducted on albino Wistar rats to evaluate the protective effects of eggshell powder (ESP) and its derivatives, nano-calcium lactate (Ncal), prepared by the precipitation method, and calcium (CA) obtained by chemical transformation, as well as inorganic calcium carbonate (CaCO 3 ) on glucocorticoid-induced osteoporosis. Materials and methods 1. Sample collection The sample for this study consisted of chicken eggs from the market in Mostaganem, Algeria. These samples were randomly selected, and the eggs were then transferred to the laboratory for analysis. 2. Preparation of eggshell powder The eggshells were washed with distilled water, and then all internal membranes were removed. They were dried at room temperature, ground into a fine powder using a mortar, sieved, and stored in an airtight container [22] . 3. Analysis of proximate and energy composition The protein, moisture, total ash, crude fiber, crude fat, and carbohydrate contents of the eggshell powder were determined. 3.1. Moisture content The moisture content of the eggshell powder was determined using the method described by Osibona [23] . This procedure is based on removing water from the sample and measuring it by weight loss. A clean crucible was first weighed and dried in an oven (W 0 ); then 2.0 g of eggshell powder was weighed into the crucible (W 1 ) and dried at 105°C for 25 hours. After drying, the crucible was transferred to desiccators until it had cooled completely, then weighed again (W 2 ). The moisture content was calculated as follows: Moisture content= (W 1 -W 2 /W 1 -W 0 ) ×100 [24] 3.2. Protein content The protein composition of 2.0g of eggshell powder was determined using the Kjeldahl method, in the presence of a catalyst. The protein content was calculated by multiplying the amount of nitrogen by a factor of 6.25: Crude Protein (%) = N(%) × 6.25 [25], [26] 3.3. Ash content The ash content was determined by incinerating the sample at 650°C for 2 hours in a muffle furnace. A clean, dry crucible was weighed (W₀), then 2.0 g of eggshell powder was weighed into this crucible (W₁). After incineration, the crucible containing the residues were weighed again (W₂). The percentage of ash was then calculated based on the residual mass after calcination, using the following equation: Ash content (%) = (W 2 -W 0 /W 1 -W 0 ) × 100 [22], [27] 3.4. Fiber content Crude fiber was determined by weighing 2.0 g of eggshell powder W s, which was mixed with 20 ml of 1.25% H 2 SO 4 and boiled for 30 minutes. After cooling, the residue was recovered by filtration, treated with 20 ml of 1.25% NaOH, and boiled for 30 minutes. The mixture was filtered, and the residue was washed with hot distilled water, and then rinsed once with 10% HCL and subsequently with ethanol. The residue W 1 was left to dry in an oven overnight at 105°C, then cooled in a desiccator and weighed. The dried sample was incinerated at 550°C and the ashes W 2 were obtained after cooling. The raw fibers were determined according to the following equation: Crude fiber (%) = (W 1 -W 2 /W S ) × 100 [25], [28] 3.5. Fat content The crude fat content was determined using a Soxhlet apparatus. 2.0 g of eggshell powder W S was placed in an extraction cartridge. In a round-bottom flask W 0 , 80 ml of hexane were poured .the heating mantle was switched on after installing the cartridge at 60°C, and water was circulated through the condenser to ensure cooling. After 2 hours of extraction, the hexane was removed by drying and the flask was weighed W 1 . The crude fat content of the sample was calculated using the following equation [29], [24]. Crude fat (%) = ( W 1 − W 0 / W s ) × 100 3.6. Carbohydrate content The carbohydrate content was determined by subtracting 100% from the sum of the percentages of crude fat, crude protein, crude fiber, moisture, and ash in accordance with following formula: CHO (KJ/100g) = (crude fat % + crude protein % + crude fiber % + moisture % + ash % ) -100 [25] 4. Characterization of eggshell powder The chemical characterization of eggshell powder was revealed following XRD and FTIR analyses. 4.1. X-ray diffraction (XRD) The diffraction pattern of the powder samples was analyzed using a rotating sample holder in the Malvern Panalytical Empyrean XRD instrument, scanning the rang from 10° to 80° at a speed of 10°/min [9] . 4.2. Fourier transform infrared spectroscopy (FTIR) analysis For FTIR analysis, ATR acquisition was performed on powder samples using a diamond crystal on the Perkin Elmer Spectrum 100 instrument. The transmission spectrum of these samples was obtained in the Wavenumber range of 4000 − 380 cm − 1 [9] . 5. Eggshell derivatives and their characterization 5.1. Production of calcium acetate The chemical transformation begins by washing 35g of eggshells (halves and pieces) with 350 ml of distilled water on a magnetic stirrer ( Stuart,UK) at 3 × 30 minutes at 250 rpm at room temperature to remove impurities, particularly residual egg white proteins [31]. Subsequently, the washed shells were treated with 525 ml of 10% (w/v) acetic acid for 3 hours at room temperature to obtain calcium salts. The mixture was filtered to separate the eggshell membranes from the saline solutions using a plastic mesh sieve with porosity of 1 mm. The calcium salt solutions obtained were clarified by two successive steps of vacuum filtration using Whatman 114 and Whatman 1 filter paper, and the calcium acetate solution was evaporated at 100–130°C until 1/10 of initial volume was obtained. The crystals were obtained by adding 3 volumes of acetone to the saturated saline solution cooled to room temperature, followed by filtration. After drying at 60°C for 24 hours, the crushed salts were stored in plastic containers for further analysis. The yield of calcium acetate derived from eggshells was calculated using the mass yield and chemical yield, as shown in equations (1) and (2) [30]. Mass yield (g) = [m (calcium salt) / m (eggshells) ] × 100 (1) Chemical yield (g) = [m (calcium salt) / m (calcium salt ) (theoretical) ] (2) The analysis of calcium acetate derived from eggshells was performed using an FTIR-ATR spectrometer in the range of 650 to 4000 cm − 1 [30] . 5.2. Production of calcium nano-lactate Calcium nano-lactate was synthesized from calcium oxide derived chicken eggshells. Calcium oxide was synthesized using the incineration method, according to YusraM. and al. (2022) [32] , while calcium nano-lactate was synthesized using the precipitation method, according to Prayitno and al. (2016) [33]. A 6 mol/L lactic acid solution was mixed with a 1 mol/L CaO solution derived from eggshells in a ratio of 1:1.5 (v/v) at 50°C at a speed of 500 rpm/minute for 30 minutes. 50% ethanol was then added to the solution (v/v). After drying at 105°C for 72 hours, the calcium lactate was powdered and stored in a dry place [34] . The characterization of calcium nano-lactate derived from eggshells included Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD), in accordance with Dheyab and al. (2020) [34], [35] . 6. In vivo experimentation 6.1. Animals and adaptation conditions Fifty 2-months-old albino Wistar rats weighing between 100 and 120 g, supplied by the Pasteur Institute of Algeria, were used in this study. During the 7-day adaptation period, the rats were housed in polypropylene cages and kept under standard laboratory conditions (ambient temperature of 25 ± 2°C and a 12-hour light-dark cycle) with free access to food and water ad libitum. 6.2. Experimental protocol After the adaptation period, the rats were randomly divided into ten groups, with five rats per cage. Osteoporosis was induced in the animals in the different groups, with the exception of the negative control group, by intramuscular injections of methylprednisolone sodium succinate, a glucocorticoid (GC), administered at a dose of 10 mg/kg, three times a week for four weeks [36], [4], [8] . The rats were simultaneously subjected to different treatments during the four weeks of the study [13], [37] , as detailed below: Group 1: Negative control group (NC) administration of distilled water to healthy animals. Group 2: Positive control group (PC); induction of osteoporosis by glucocorticoids (GC) without treatment. Groups 3 and 4: Animals received eggshell powder (ESP) at doses of 20 and 40 mg/kg, respectively, diluted in 1 ml of distilled water, administered daily by oral gavage. Groups 5 and 6: Administration of calcium acetate (CA) derived from eggshells at doses of 20 and 40 mg/kg, respectively, diluted in 1 ml of distilled water, administered daily by oral gavage. Groups 7 and 8: Rats receive calcium nano-lactate derived from eggshells (NCal) at doses of 20 and 40 mg/kg, respectively, diluted in 1 ml of distilled water, administered daily by oral gavage. Groups 9 and 10: Administration of pharmaceutical calcium carbonate (CaCO 3 ) at doses of 20 and 40 mg/kg, respectively, diluted in 1 ml of distilled water, administered daily by oral gavage. 6.3. Controlled experimental parameters One week after treatment, the rats were fasted overnight and a blood sample was taken from the sinus. After dissection, both femurs were removed. 6.3.1. Analysis of serum biochemical parameters Alkaline phosphatase (ALP), calcium (Ca), and phosphorus (P) levels were measured in serum. ALP and phosphorus (P) levels were measured using the Fujifilm Dri-Chem 3500i analyzer. Calcium (Ca) levels were measured using the o-Cresolphthalein complexone method (ERBA Mannheim XL200). 6.3.2. Measurement of mineral content in the ash of left femur The left femur was weighed after being incinerated in a muffle furnace for 5 hours at 600°C. The resulting ashes were dissolved in 6 M hydrochlorid acid (HCl) [38]. Calcium, phosphorus, and magnesium content were measured using Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) [39] . 6.3.3. Histopathological examinations The right femurs were removed and then decalcified using a buffered formic acid solution for three consecutive days [40]. After fixing the femurs in 10% neutral buffered foemalin, they were then processed and embedded in paraffin [13] . Tissue sections 5 µm thick were obtained by cutting the paraffin blocks. The samples were then deparaffinized in xylene and rehydrated using a series of decreasing ethanol baths. Using the H&E solution, the treated tissue sections were then stained and observed under a microscope (Carl Zeiss, Axiovert 25, Oberkochen, Germany) [41]. 7. Statistical analysis Statistical analysis was performed using PAST software. Analysis of variance (ANOVA) followed by Tukey’s post hoc test was used to compare the different treatment groups with the negative control and positive control groups. A p-value < 0.05 was considered statistically significant. In the in vitro study, each parameter was evaluated in three independent replicates. For the in vivo study, the results represent the average of the values obtained from five rats per group. Ethical note All experimental procedures and protocols involving animals were carried out in accordance with Algerian legislation (Law No. 95–322/1995) on the protection of animals used for experimental and scientific purposes. They also comply with the guidelines of the Algerian Association for Laboratory Animal Science (AASEA, authorization No. 45/DGLPAG/DVA/SDA/14) concerning the protection of animals used for experimental and other scientific purposes. Results 1. Proximate and energy composition Proximate chemical analyses performed on the powder obtained from eggshells made it possible to evaluate its nutritional composition. The results, expressed as a percentage of fresh matter, reveal a moisture content of 1.16 ± 0.28%, a crude protein content of 2.06 ± 0.24%, an ash content of 92.66 ± 1.04%, and a crude fiber content of 0.66 ± 0.76%. The fat and carbohydrate content was also determined, with respective values of 3.12 ± 0.32% and 0.3 ± 0.08 (KJ/100g). 2. Characterization of eggshell powder 2.1. Results of X-ray diffraction analysis The X-ray diffractogram (XRD) of the eggshell powder is shown in Fig. 1 . The presence of several distinct peaks throughout the spectrum indicates that the material is crystalline in nature. Each peak corresponds to a specific crystallographic plane within the structure. In this particular case, the peak intensities correspond to those of calcite (CaCO 3 ), which is the most common crystalline form of eggshell powder. The major peak characteristic of CaCO 3 was observed at 29.6°, and some of the most intense peaks appear at 2θ values of approximately 23.2°, 26.7°, 36.2°, 39.6°, 43.3°, 47.7°, and 48.7°. Comparison of the positions of the measured peaks (2θ angles) and their intensities with a reference database confirms the identification of the crystalline phase. 2.2. Results of Fourier transform infrared spectrum results The Fourier transform infrared (FTIR) spectrum of eggshell powder was recorded in the range of 4000 − 380 cm⁻¹. The spectrum obtained shows several characteristic bands (Fig. 2 ). A board band observed between 3400 and 3200 cm⁻¹ was attributed to O–H stretching vibration. Similarly, weak bands around 2950 − 2850 cm⁻¹ correspond to aliphatic C–H stretching. The presence of a band in the 1700-1750cm⁻¹ region is associated with C = O vibration. In addition, the intense bands at 1450,875 and 713 cm⁻¹ are attributed respectively to antisymmetric vibrations and deformations characteristic of the carbonate group (CO 3 −2 ). Finally, the shoulder observed around 2515cm⁻¹ corresponds to the signal reported for calcium carbonate. 3. Eggshell derivatives and their characteristics 3.1. Calcium acetate production The chemical transformation process of 35 g of eggshells using acetic acid resulted in a mass yield of calcium acetate derived from eggshells of approximately 33.33 ± 2.91g/35 g of PES and a chemical yield of 23.68 ± 2.07%. The Fourier transform infrared (FTIR) spectrum of calcium acetate derived from eggshells was recorded in the range of 4000 to 650 cm⁻¹. The spectrum reveals several characteristic peaks corresponding to different functional groups and bonds present in the molecule (Fig. 3 ). The broad band observed in the 3117–3709 cm⁻¹ region is indicative of O–H stretching vibrations, typical of hydroxyl groups present in calcium acetate hydrate. The peaks in the 2850–3000 cm⁻¹ range correspond to C–H stretching vibrations, attributed to the alkyl groups linked to acetate. The intense peaks around 1714 cm ⁻¹ are characteristic of the stretching vibrations of the carbonyl group (C = O), corresponding to the acetyl group of calcium acetate. In addition, peaks in the range 1418–1443 cm⁻¹ indicate O–H bending vibrations, common to carboxylic acids and their salts. The peak at 1054 cm⁻¹ is associated with C–O stretching vibrations related to the acetate group. Furthermore, the peaks below 669 cm⁻¹ can be attributed to metal-oxygen bonds, in particular calcium-oxygen interactions. The notable peaks around 1027 cm⁻¹ probably correspond to C–O stretching or bending vibrations associated with the carboxylate group of calcium acetate, while those between 419–669 cm − 1 may be due to bending modes or out of plane deformations involving the acetate group and its interactions with calcium. This analysis provides an in depth understanding of the functional groups present in the calcium acetate sample based on the FTIR spectrum. 3.2. Production of calcium lactate derived from eggshells The production of nano-calcium lactate (NCal) from calcium oxide derived from eggshells was achieved using the precipitation method. A yield of 88g of NCal was obtained from a 6 mol/L lactic acid solution and a 1 mol/L CaO solution derived from eggshells. The Fourier transform infrared (FTIR) spectrum of calcium nano-lactate is shown in Fig. 4 . The FTIR spectrum provided for calcium nano-lactate shows distinct peaks corresponding to various functional groups present in the molecule (Fig. 4 ). The broad absorption band around 3155 cm − 1 indicates O–H stretching vibrations, which are typical of hydroxyl groups and suggest the presence of water molecules, either in the form of hydration water or intrinsic to the lactate structure. The peaks in the 2850–3000 cm − 1 region correspond to C–H stretching vibrations of alkyl groups present in lactate molecule. A notable peak at 1715 cm − 1 corresponds to C = O stretching vibrations of carboxylate group, which is a key characteristic of lactate anion. In addition, the peak at 1458 cm − 1 is associated with the asymmetric stretching vibrations of the carboxylate group, while the peak at 1421 cm- 1 corresponds to the symmetric stretching of the same group. The peaks around 1300–1400 cm − 1 can be attributed to C–H bending vibrations in lactate. The region between 1200 and 1000 cm − 1 shows peaks related to C–O stretching vibrations, indicating the presence of carboxylate and hydroxyl groups. The peaks observed below 600 cm − 1 , particularly those around 500 cm − 1 , can be associated with calcium-oxygen interactions, indicating metal coordination within be compound. The presence of peaks at 1100 − 1000 cm − 1 suggests C–O stretching, typical of alcohol groups. Other smaller peaks in the region below 1000 cm − 1 indicate various bending vibration that are consistent with the structure of calcium lactate. Overall, the FTIR spectrum confirms the presence of hydroxyl, carboxylate, and alkyl groups in calcium lactate, which is consistent with its known chemical structure. Characterization was also performed using XRD (Fig. 5 ). The presence of several marked peaks throughout the spectrum indicates the presence of a crystalline material, which corresponds to calcium lactate. Each peak corresponds to a specific crystallographic plane within the crystal structure. In this case, the peak intensities correspond to those of calcium lactate, indicating that the sample exhibits the most common crystalline from of this compound. 4. Results of in vivo experimentation 4.1. Serum biochemical parameter analysis The results of serum biochemical markers related to bone metabolism are shown in Table 1 . Serum calcium levels were within normal limits in healthy animals in group 1 (9.86 ± 0.08 mg/dl), unlike in rats receiving glucocorticoid doses. There was a highly significant decrease in serum calcium (8.68 ± 0.42 mg/dl; p < 0.05), confirming the induction of calcium metabolism imbalance. In groups treated with various forms of calcium supplements derived from eggshells, variable improvements in blood calcium levels were observed depending on the type of salt and the dose administered. Treatment with eggshell powder (ESP) resulted in a significant increase in serum calcium compared to the negative control group (p < 0.05) at a dose of 20 mg/kg (9.02 ± 0.13 mg/dl). However, a significant improvement compared to the positive control group was only observed at a dose of 40 mg/kg (9.54 ± 0.37 mg/dl). A similar profile was observed with galenic calcium carbonate (CaCO3), which induced a significant increase in serum calcium (p < 0.05), with a significant corrective effect compared to the positive control group only at the highest dose, reaching values close to normal. Furthermore, the groups treated with calcium nano-lactate (NCal) and calcium acetate (CA) showed more moderate increases. NCal at 40 mg/kg reached 8.98 ± 0.13 mg/dl, while CA recorded levels of 9.02 ± 0.40 mg/dl at 20 mg/kg and 8.72 ± 0.36 mg/dl at 40 mg/kg, with no significant difference compared to the positive control group (p < 0.05). As shown in Table 1 , the serum phosphorus level recorded in animals in the positive control group (8.2 ± 0.46 mg/dl) was lower than that in the negative control group (9.08 ± 0.08 mg/dl) (p < 0.05). However, a significant increase in serum P levels was observed in rats treated with ESP 40 mg/kg and CaCO 3 40 mg/kg, reaching (9.7 ± 0.75 and 9.56 ± 0.25 mg/dL), respectively, compared to the positive control. In contrast, in the group treated with CA 40 mg/kg, the serum P level (7.34 ± 0.74 mg/dl) was significantly lower than that of the negative control group (p < 0.05). As shown in Table 1 , compared to the negative control group, the serum alkaline phosphatase (ALP) activity of the positive control group increased to 195.4 ± 15.5 U/L. Administration of either 20 or 40 (mg/kg) of ESP or CaCO 3 to the rats revealed a significant decrease (p < 0.05) in serum ALP levels compared to the positive control group, reaching respectively (149.2 ± 15.2 U/L and 152.6 ± 8.26 U/L) for the ESP groups, and (155 ± 4.5 U/L and 167 ± 5.2 U/L) for the groups treated with CaCO 3 . In contrast, in the groups treated with nano-calcium lactate NCal and calcium acetate CA at different doses, the decrease in ALP levels was not significant compared to the positive control group (p < 0.05) Table 1 Effects of different treatments of eggshells and their derivatives on serum calcium, phosphorus, and alkaline phosphatase levels in rats with glucocorticoid-induced osteoporosis Groups Calcium (mg/dl) Phosphorus (mg/dl) Alkaline phosphatase (U/L) G1 Negative control 9.86 ± 0.08 9.08 ± 0,08 168.6± 2.3 G2 Positive control 8.68 ± 0.42 ** 8.2 ± 0.46 195,4 ± 15,5 G3 Powder eggshell(20mg/kg) 9.02 ± 0.13 ** 8.9 ± 0.1 149,2 ± 15,2 ## G4 Powder eggshell(40mg/kg) 9.54 ± 0.37 ## 9.7 ± 0.75 ## 152,6 ± 8,26 ## G5 Eggshell nano-calcium lactate (20mg/kg) 9.18± 0.26** 9.06 ± 0.11 182 ± 22.21 G6 eggshell nano–calcium lactate (40mg/kg) 8.98 ± 0.13 ** 8.78 ± 0.25 178 ± 26.2 G7 Eggshell-derived calcium acetate(20mg/kg) 9.02 ± 0.40 ** 8.72 ± 0.54 169.8 ± 1.7 G8 Eggshell-derived calcium acetate(40mg/kg) 8.72 ± 0.36 ** 7.34 ± 0.74 ** 192.4 ± 3.7 G9 Calcium carbonate(20mg/kg) 9.22 ± 0.30 ** 8.92 ± 0.16 155 ± 4,5 ## G10 Calcium carbonate(40mg/kg) 9.48 ± 0.21 ## 9.56 ± 0.25 ## 167 ± 5,2 ## p 0.0002 0.00005 0.0002 Data expressed as median (IQR) (n = 5). A one-way ANOVA was used for statistical analysis, followed by Tukey’s post-hoc test. ** p < 0.05 compared to negative control, ## p < 0.05 compared to positive control. 4.2. Mineral content in Femoral Bone Ash Analysis of bone mineral composition revealed highly significant differences between the experimental groups (p < 0.001). The positive control group (G2) showed a marked decrease in calcium, phosphorus, and magnesium content compared to the negative control group (G1), confirming the alteration in bone mineralization induced by glucocorticoids. Supplementation with eggshell powder (ESP) resulted in a significant dose-dependent restoration of bone minerals, with values close to those of the negative control at a dose of 40 mg/kg (G4), indicating high remineralizing efficacy. The groups treated with nano-calcium lactate derived from eggshells (G5-G6) also showed significant improvement, particularly for phosphorus and magnesium, suggesting better bioavailability linked to nanometric form. In contrast, calcium acetate (G7-G8) and calcium carbonate (G9-G10) induced a moderate increase in mineral parameters, less marked than that observed with ESP and NCal, particularly for phosphorus (Table 2 ). Table 2 Calcium, phosphorus, and magnesium levels in the left femoral bone ash of healthy rats and glucocorticoid-induced osteoporotic rats treated with ESP, NCal, CA, and CaCO 3 (n = 5). Groups Calcium (mg/L) Phosphorus (mg/L) Magnesium (mg/L) G1 Negative control 1881.534 ± 54.64 1190.32 ± 49.15 38.57 ± 1.10 G2 Positive control 1695.36 ± 54.23 a 949.14 ± 26.55 a 35.54 ± 0.70 a G3 Powder eggshell(20mg/kg) 1792.358 ± 7.14 b 988.28 ± 6.32 a 36.88 ± 0.06 a G4 Powder eggshell(40mg/kg) 1866.04 ± 47.33 b 1139.32 ± 23.27 b 37.67 ± 0.19 b G5 Eggshellnano-calcium lactate (20mg/kg) 1770.142 ± 38.89 a 1096.144 ± 52.58 b 36.8 ± 0.16 b G6 eggshell nano–calcium lactate (40mg/kg) 1833.84 ± 52.15 b 1126.58 ± 24.54 b 37.31 ± 0.37 b G7 Eggshell-derived calcium acetate(20mg/kg) 1786.934 ± 8.56 1009.056 ± 54.38 a 36.59 ± 0.55 a G8 Eggshell-derived calcium acetate(40mg/kg) 1800.046 ± 13.74 b 1061.326 ± 55.40 a 36.79 ± 0.13 a G9 Calcium carbonate(20mg/kg) 1832.72 ± 50.18 b 1030.70 ± 62.36 a 36.51 ± 0.33 a G10 Calcium carbonate(40mg/kg) 1845.84 ± 74.48 b 1062.78 ± 26.02 a 36.13 ± 0.83 a p 0.0002 0.00001 0.00002 Data are expressed as means ± standard deviation, with five rats per group. Differences between groups were analyzed using a one-way ANOVA test, followed by Tukey’s pairwise test. a,b: indicate a significant difference compared to the negative control group and the positive control group (osteoporotic rats), respectively. 4.3. Histopathological Analysis Figure 6 shows histological sections of the right femur of rats from the different experimental groups, stained with hematoxylin and eosin (H&E), revealed the bone architecture and the histopathological changes induced by the different treatments used. In rats from the negative control group, bone architecture was preserved and characterized by thick, well-organized, and intensely eosinophilic trabeculae, indicating good trabecular connectivity (Fig. 6 A). The trabeculae were lined with numerous resting osteoblasts, while a large number of viable osteocytes were observed within the lamellar bone matrix, indicating balanced bone remodeling. Conversely, rats in the positive control group, treated with glucocorticoids without calcium supplementation, showed marked histological changes reflecting significant bone resorption (Fig. 6 B and 6 C). The trabeculae appeared thin, disorganized, and weakly eosinophilic, associated with a decrease in the number of osteocytes and increased osteoclastic activity. Thinning of the cortical bone and enlargement of the medullary spaces were also observed, reflecting a loss of trabecular connectivity characteristic of osteoporosis. Bone sections from osteoporotic rats treated with eggshell powder (ESP 40mg/kg), calcium nano-lactate ( NCal 40 mg/kg), calcium acetate (CA 20 and 40 mg/kg), and calcium carbonate (CaCO 3 20 and 40 mg/kg), (Fig. 6 E, G, H, I, J and K, respectively) showed significant histological improvement. The bone trabeculae were thicker and better organized with a dense lamellar matrix, intensely stained with eosin, containing numerous evenly distributed osteocytes. In addition, the medullary spaces were reduced and no significant osteoclastic proliferation was observed, suggesting marked bone regeneration. In contrast, rats receiving lower doses, notably ESP at 20 mg/kg and NCal at 20 mg/kg (Fig. 6 D and F, respectively), showed histological features similar to those of the positive control group. The trabeculae remained thin, pale, and locally irregular, with a low number of osteocytes and peripheral osteoblasts. These results suggest limited improvement, indicating that these low doses do not have a significant effect on bone regeneration. Discussion 1. Characterization of eggshell powder and its derivatives: The results relating to proximate chemical composition of the eggshell powder obtained, particularly for moisture and crude protein, are similar to those reported for brown eggshells [43]. However, the lipid and fiber contents observed in our study are higher than those mentioned in the same study. Furthermore, the ash content obtained is comparable to that reported in a study on eggshell powder used to enrich bread [44] , but remains lower than that reported for white eggshell powder [43] . These differences in composition could be attributed to the origin of eggshells and the birds diet [43],[45]. The FTIR results for the eggshell powder studied confirm the presence of bands characteristic of calcium carbonate (CaCO 3 ), in agreement with the work reported in the literature. The characteristic bands observed at 713 and 874 cm − 1 , corresponding to the calcite phase of CaCO 3 , are similar to those reported in the reference study on calcite [46] . Calcite, the trigonal phase of calcium carbonate, is the main component of bird eggshells. At room temperature, it is the most stable polymorph of calcium carbonate [42] . Similarly, the results obtained by X-ray diffraction (XRD) confirm the crystallinity of this product and are similar to those reported for eggshell powder from different species [9] . The characteristic major peak for CaCO 3 was observed at 29.6° for all species studied. Eggshells can be used in the medical field as a source of calcium and as a food additive in the form of calcium lactate, citrate, gluconate, propionate, and acetate [20] . The mass and chemical yields of calcium acetate derived from eggshells are consistent with those reported in the work of Strelec and al. (2023) [30] and other studies [47], [50] . Furthermore, the yield of calcium nano-lactate (NCal) obtained is four times higher than that reported by Tam and al. (2025) [51] , who reported a yield of 20g of calcium lactate using a 16% lactic acid solution. Although the experimental protocols differ, this comparison highlights the decisive role of reagent concentration and the process used on the final product yield. The FTIR spectra obtained for acetate and calcium nano-lactate derived from eggshells reveal the presence of several characteristic peaks, attributed to different functional groups and chemical bonds constituting the molecules. These results are consistent with those reported by Chung and al. [52] and Tongkam and al. [53] for calcium acetate, as well as with the observations of McReynolds and al. [54] and Tesfamariam and al. [55] for calcium lactate. Furthermore, X-ray diffraction (XRD) analyses of NCal confirm the crystalline nature of this compound. Indeed, comparing the positions of the recorded peaks (2Ɵ- angles) and their intensities with reference data allows the material to be precisely identified [ 56], [57]. 2. Effects of eggshell powder and its derivatives on glucocorticoid-induced osteoporotic rats 2. Effects of eggshell powder and its derivatives on glucocorticoid-induced osteoporotic rats Calcium is one of the essential nutrients involved in many metabolic functions in the human body [58] . A sufficient intake of this mineral is essential for growth, maintaining bone health, and preventing the risk of osteoporosis and associated fractures [59] . The medical management of osteoporosis continues to face the constant challenge of preventing associated fractures [60], [13] . The convergence between the nutritional and practical applications of eggshells and those of bone tissue justifies their promotion as a potential source of dietary calcium [61] . In this context, the present study used an appropriate animal model of glucocorticoid-induced osteoporotic bone loss to compare the efficacy of eggshell powder (ESP), considered a source of inorganic calcium, with that of calcium acetate (CA) and nano-calcium lactate (NCal) derived from eggshells, representing sources of organic calcium, as well as synthetic calcium carbonate, in preventing the onset of osteoporosis. The administration of different sources of calcium during glucocorticoid treatment induced significant variations in bone biochemical parameters, particularly serum calcium and phosphorus levels and alkaline phosphatase activity, depending on the experimental group. The marked decrease in serum calcium observed in the positive control group confirms the alteration in calcium-phosphorus metabolism induced by glucocorticoids, characterized by inhibition of intestinal calcium absorption and increased renal excretion. Conversely, 30 days of supplementation with eggshell powder (ESP) or synthetic calcium carbonate significantly restored serum calcium, with a more pronounced dose-dependent effect at 40 mg/kg. These results are consistent with those of Nagamma et al. [62] , who reported a significant increase in serum calcium in rats supplemented with calcium from eggshells compared to untreated ovariectomized animals. This improvement could be attributed to the mineral richness of eggshells and the presence of trace compounds that may promote calcium bioavailability. However, Kareem et al. (2025) showed that nanoscale eggshell calcium carbonate (NPES) induced a greater increase in serum calcium than that observed with micronized ESP or inorganic calcium carbonate (CaCO 3 ) [16] . This superiority of NPES could be explained by its high specific surface area, which facilitates solubilization, gastrointestinal absorption, and cellular uptake of calcium [63] . In contrast, the groups treated with calcium acetate (CA) and nanometric calcium lactate (NCal) showed no significant difference compared to the positive control group. However, treatment with NCal appears to slightly improve serum calcium levels compared to CA. these results are consistent with the observations reported by Ueda et al. (2013) , according to which calcium acetate and lactate are rapidly dissociated and eliminated in the digestive tract, thus limiting their intestinal residence time and, consequently, their bioavailability [64] . With regard to serum phosphorus, the groups supplemented with ESP and CaCO 3 at a dose of 40 mg/kg showed a significant increase compared to the positive control group, in agreement with the results of Kareem et al. (2025) [16] . Given that the bone mineral content, particularly calcium (Ca) and phosphorus (P), is a fundamental indicator of bone quality, these results suggest an improvement in the bone mineralization process under the effect of these treatments [65] . In the NCal group, a moderate and insignificant increase in serum phosphorus was observed, while calcium acetate led to a marked decrease in this parameter. These results confirm the well-established role of acetate as a calcium phosphate chelator, reducing its intestinal absorption [66] . This property gives calcium acetate particular therapeutic interest, allowing both calcium intake and a reduction in phosphate load thus contributing to the prevention of hypocalcemia and secondary hyperparathyroidism, which is often involved in bone remodeling disorders [67] . These observations suggest that the beneficial effects of calcium acetate may go beyond simply correcting mineral metabolism, notably by improving metabolic acidosis and modulating inflammatory processes [67] . Analysis of alkaline phosphatase (ALP) activity, a key marker of bone remodeling, showed a significant increase in the positive control group, reflecting an intensification of glucocorticoid-induced resorption, as reported by Latif et al. (2021) [68] . Conversely, all groups supplemented with different sources of calcium showed a significant reduction in ALP, consistent with the findings of Kareem et al. (2025) for NPES and CaCO 3 [16] . These data indicate that the calcium supplements tested contribute to limiting excessive bone remodeling associated with estrogen deficiency, thereby promoting more balanced bone metabolism and improved bone structural integrity [69]. Comparative analysis of the mineral composition of femoral ash reveals marked differences in efficacy between eggshell powder (ESP), nano-calcium lactate (NCal), and calcium acetate (CA) in restoring bone mineralization impaired by glucocorticoids. The GIOP group had the lowest calcium, phosphorus, and magnesium levels, confirming the onset f severe osteoporosis linked to inhibition of bone formation and accelerated mineral loss [16] . Among the supplements tested, ESP stood out for providing the most complete and consistent restoration of femoral mineral content, with a significant and concomitant increase in calcium, phosphorus, and magnesium. This superiority suggests that the effect of ESP is not based solely on elemental calcium intake, but also on the presence of a naturally balanced mineral matrix rich in bioactive trace elements such as magnesium, zinc, and strontium. These elements are recognized for their synergistic role in osteoblast differentiation, osteoid maturation, and hydroxyapatite crystal stabilization [70] . Thus, ESP appears to be a multifunctional biomaterial that promotes physiological and sustainable bone remineralization. NCal showed moderate efficacy, characterized mainly by a significant improvement in bone phosphorus and magnesium levels, while calcium restoration remained partial, particularly at the lowest doses. This response could be explained by nanometric size of NCal, which increases the specific surface area and improves intestinal calcium absorption. However, the absence of a complex mineral matrix comparable to that of ESP could limit its overall remineralizing potential. Nevertheless, at a dose of 40 mg/kg, NCal showed a more marked mineral improvement, suggesting a dose-response relationship and efficacy dependent on the concentration administered, in agreement with the observations of Kareem et al. [16]. Conversely, CA showed the most limited effectiveness in restoring bone mineral content. Although it provides a soluble source of calcium, its impact on femoral calcium and phosphorus levels remained modest, particularly at low doses. This reduced efficacy could be attributed to its rapid dissociation and elimination in the digestive tract, as well as its well-documented role as a phosphate chelator, limiting intestinal phosphorus absorption and, consequently, adequate bone hydroxyapatite formation [66]. Thus, CA appears to act more as a regulator of systemic calcium-phosphorus metabolism than as a direct bone remineralizing agent. From a comparative perspective, the remineralizing efficacy of the supplements tested can be ranked as follows: ESP < NCal < CA, with ESP clearly superior at a dose of 40 mg/kg. This ranking highlights the importance not only of the physicochemical from of calcium, but also of its mineral matrix and functional bioavailability in the prevention and correction of glucocorticoid-induced bone loss. These results suggest that ESP represents a natural and particularly promising alternative to conventional calcium salts, combining remineralizing efficacy, mineral synergy, and the potential to improve bone remodeling. NCal could be an interesting option at adequate doses, while CA seems more appropriate as an adjunctive treatment aimed at calcium-phosphorus regulation rather than structural bone restoration. Histologically, treatment with eggshell powder (ESP) and its derivatives offered better protection against bone loss in osteoporotic rats. Among the supplements evaluated, ESP and CaCO 3 showed the most marked histological effects, characterized by preservation of bone micro-architecture, improved trabecular continuity, and reduced inter-trabecular spaces. These results are consistent with those reported by Kareem et al. [16] , who observed a significant improvement in bone mineral density (BMD) in animals treated with ESP and CaCO 3 . The superior protective effect of eggshell powder could be attributed to its complex and naturally balanced mineral composition, rich in calcium (Ca), magnesium (Mg), and phosphorus (P), which are essential elements for maintaining bone integrity. Calcium is the main mineral constituent of bone tissue, in the form of calcium phosphate crystallized as hydroxyapatite, ensuring bone rigidity and strength. Magnesium, on the order hand, plays a key role in mineral and bone homeostasis by modulating the secretion of hormones involved in skeletal metabolism, regulating bone cell activity, and participating in the formation, growth, and stabilization of hydroxyapatite crystals [39] . This mineral synergy gives ESP a functional advantage over isolated calcium salts. In contrast, the positive control group showed marked trabecular disorganization, characterized by homogeneous thinning of the bone trabeculae and widening of the inter-trabecular spaces. These morphological changes are typical of glucocorticoid-induced osteoporosis and are consistent with the observations reported by Zhang et al. (2020) in ovariectomized rats [71] . Glucocorticoids exert direct deleterious effects on bone cells by inhibiting osteoblast differentiation and proliferation, while promoting their apoptosis via the activation of pro-apoptotic pathways (Bim, Bak, p53) and the inhibition of the Wnt/β-catenin pathway, which is essential for osteogenesis [72]. At the same time, they induce osteocyte apoptosis, compromising mechano-sensitivity and bone micro-architecture stability, while prolonging osteoclast survival through an increase in the RANKL/OPG ration, thereby intensifying bone resorption. Systemically, glucocorticoids reduce intestinal calcium absorption, increase renal excretion, and disrupt hormone regulation (vitamine D 3 , PTH, testosterone), aggravating bone demineralization and promoting skeletal fragility [72]. In osteoporotic rats treated with calcium supplements derived from eggshells, namely calcium acetate and nano lactate, histological bone sections showed active remineralization and improved tissue cohesion. The increase in the number of osteocytes and osteoblasts indicates stimulation of bone remodeling and reactivation of bone formation [73] . The bioavailability of calcium has a decisive influence on bone mineralization processes and osteogenic cell activity. The most easily absorbed forms of calcium promote osteoblastic differentiation and contribute to increased bone mineral density [ 74]. Calcium ions play a fundamental role in regulating the cellular processes involved in bone formation and regeneration. Mainly present in the form of calcium phosphates in the bone matrix, they actively participate in the calcification and maturation of bone tissue. In addition, calcium is involved in bone regeneration through various cell signaling mechanisms, stimulating mature bone cells by producing nitric oxide, inducing the differentiation of precursor cells into osteoblasts, and activating the ERK1/2 and PI3K/Akt pathways, which are involved in bone synthesis and osteoblast survival, respectively. Finally, calcium ions modulate the functional cycle of osteoblasts by Influencing both their formation and resorptive activity [75] . All of these mechanisms support the correlation observed between calcium bioavailability and improvement in bone histological and mineral parameters. These results confirm the biochemical and mineral data, suggesting a direct correlation between calcium bioavailability and bone regeneration. Conversely, low doses (20 mg/kg) of ESP and NCal induced only a partial improvement in bone micro-architecture, with trabeculae remaining thin, discontinuous, and poorly organized. These results highlight a marked dose-response relationship, indicating that even in the presence of a highly bioavailable source of calcium, insufficient calcium intake remains unable to compensate for the mineral loss induced by glucocorticoid treatment, particularly in pathological contexts characterized by accelerated bone remodeling or bone aging [74] . Conclusion In conclusion, this study highlights the potential of eggshells and their derivatives as effective natural sources of calcium in the prevention of glucocoticoid-induced osteoporosis. Eggshell powder, particularly at high doses, proved to be the most effective in improving biochemical markers, mineralization, and bone micro-architecture. Nano-lactate and calcium acetate showed dose-dependent efficacy, but less than that of eggshell powder. These results position the use of eggshells as a sustainable and promising alternative to conventional calcium supplements in the management of secondary osteoporosis. Declarations Data Availability Data are provided within the manuscript Acknowledgment The authors are grateful to the Ministry of Higher Education and Scientific Research and to University Abdelhamid Ibn Badis, Algeria, for supporting this research work within the framework of the Research Project (PRFU), Code: D00L01UN270120220002, entitled “Biovalorization and evaluation of the nutraceutical and therapeutic power of natural bioactive molecules.” Funding This research was supported by the Ministry of Higher Education and Scientific Research, Algeria Competing Interests The authors declare no competing interests. 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Une stratégie métabolomique révèle le mécanisme ostéogénique des peptides de collagène osseux de yak (Bosgrunniens) sur l'ostéoporose induite par ovariectomie chez le rat. Food &Function , 11 (2), 1498-1512. Henneicke, H., Gasparini, S. J., Brennan-Speranza, T. C., Zhou, H., & Seibel, M. J. (2014). Glucocorticoids and bone: local effects and systemic implications. Trends in Endocrinology & Metabolism , 25 (4), 197-211. Prideaux, M., Findlay, DM, et Atkins, GJ (2016). Ostéocytes : les cellules maîtresses du remodelage osseux. Opinion actuelle en pharmacologie , 28 , 24-30. Voulgaridou, G., Papadopoulou, S. K., Detopoulou, P., Tsoumana, D., Giaginis, C., Kondyli, F. S., ... &Pritsa, A. (2023). Vitamin D and calcium in osteoporosis, and the role of bone turnover markers: A narrative review of recent data from RCTs. Diseases, 11(1), 29. Jeong, J., Kim, J. H., Shim, J. H., Hwang, N. S., &Heo, C. Y. (2019). Bioactive calcium phosphate materials and applications in bone regeneration. Biomaterials research, 23(1), 4. Nada, EA, Kaddorah, MEA, El Jamal, M., Hamad, A., & Mansour, FR (2025). Les déchets de coquilles d'œufs comme ressource durable pour la préparation de nanoparticules ; synthèse, caractérisation et applications. Environmental Nanotechnology, Monitoring & Management , 24 , 101092. Supplementary Files graphicalabstract.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9012106","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":609014751,"identity":"0b77575d-2c59-47cc-963a-f3f8f7c02ed1","order_by":0,"name":"souad cheraitia","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIiWNgGAWjYBACA2YIzcPA3sAAZgNpYrXwHIBoAdIEtMBZEglEajFnZz784WPbNhn+mW8MPxdU2DDwSBPQY9nMliY5s+02j8TtHGPpGWfSGHj4Egg47DCPGTMvUAvD7RwDad62wwz2PIT8cpjH+DNIi/zNM8a/QVp4iNACMvw2j8ENHjNporSA/TLj3G0ewzNpZdY8Z9J4CGox5z98+MOHstv2cscPb77NU2EjR1ALEuAAxxEJGoAp5QEpqkfBKBgFo2AEAQCfSznsVcOgdAAAAABJRU5ErkJggg==","orcid":"","institution":"Université Abdelhamid Ibn Badis de Mostaganem: Universite Abdelhamid Ibn Badis de Mostaganem","correspondingAuthor":true,"prefix":"","firstName":"souad","middleName":"","lastName":"cheraitia","suffix":""},{"id":609014752,"identity":"d93e9d78-c1fa-4cd6-adb3-4dcbaf24d5d8","order_by":1,"name":"Soumia Keddari","email":"","orcid":"","institution":"University of Abdelhamid Ibn Badis of Mostaganem: Universite Abdelhamid Ibn Badis de 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Houari Boumediene","correspondingAuthor":false,"prefix":"","firstName":"Yasmina","middleName":"Mokhtaria","lastName":"Boufadi","suffix":""},{"id":609014756,"identity":"3af5aa0a-5b80-4a91-b15a-783de37d81bf","order_by":5,"name":"Djahira Hamed","email":"","orcid":"","institution":"Centre Universitaire Aïn Témouchent: Universite de Ain Temouchent Belhadj Bouchaib","correspondingAuthor":false,"prefix":"","firstName":"Djahira","middleName":"","lastName":"Hamed","suffix":""},{"id":609014757,"identity":"9691470b-f87a-4532-94b8-055a76df3dc0","order_by":6,"name":"Abla Bouhend","email":"","orcid":"","institution":"Université Abdelhamid Ibn Badis de Mostaganem: Universite Abdelhamid Ibn Badis de Mostaganem","correspondingAuthor":false,"prefix":"","firstName":"Abla","middleName":"","lastName":"Bouhend","suffix":""}],"badges":[],"createdAt":"2026-03-02 15:59:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9012106/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9012106/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105307053,"identity":"2348f2dc-effe-473f-b510-244abc5f1b78","added_by":"auto","created_at":"2026-03-24 14:44:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":40589,"visible":true,"origin":"","legend":"\u003cp\u003eXRD spectrum of eggshell powder\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9012106/v1/d3a7ae5219734be71670c2d6.png"},{"id":105307047,"identity":"0225282a-572d-4254-9c22-8ade2622026e","added_by":"auto","created_at":"2026-03-24 14:44:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":101225,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectrum of eggshell powder\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9012106/v1/58bfd33dfa92789d14fbdf37.png"},{"id":105307046,"identity":"e99b3573-ce52-453d-8caa-5e906d24e8bb","added_by":"auto","created_at":"2026-03-24 14:44:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":138160,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectrum of calcium acetate derived from eggshells\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9012106/v1/fa7c14e9642eeb67e5dd4a3c.png"},{"id":105564992,"identity":"a9796142-6ef9-4443-844f-c133ce69b967","added_by":"auto","created_at":"2026-03-27 12:51:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":88897,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectrum of calcium lactate derived from eggshells\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9012106/v1/05a40abef5927037e5bfe6be.png"},{"id":105307048,"identity":"24b903d5-4bbb-4019-b774-e9ec8294236a","added_by":"auto","created_at":"2026-03-24 14:44:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":58821,"visible":true,"origin":"","legend":"\u003cp\u003eXRD spectrum of calcium lactate derived from eggshells\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9012106/v1/7060d1884808a3762a9066c6.png"},{"id":105307049,"identity":"56b497ba-ff17-474b-a810-778b28437d3e","added_by":"auto","created_at":"2026-03-24 14:44:32","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3573552,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of administering eggshell powder (ESP), nano-calcium lactate (NCal), calcium acetate (CA), and calcium carbonate (CaCO3), at different doses, on the histopathology of femur in rats with glucocorticoid-induced osteoporosis (Magnification ×100)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Photomicrograph of a normal rat femur (negative control) showing thick, strongly eosinophilic bone trabeculae (short black arrows), containing uniform osteocytes (OC) and marked proliferation of osteoblasts at the bone periphery (OB). \u003cstrong\u003e(B)\u003c/strong\u003e And \u003cstrong\u003e(C)\u003c/strong\u003e Femurs of osteoporotic rats (treated with GLu only) showing thin, weakly eosinophilic bone trabeculae (short black arrows), with a reduced number of osteocytes (OC) and few osteoblasts (OB), as well as thinning of the cortex\u003cstrong\u003e. (D)\u003c/strong\u003e And \u003cstrong\u003e(E)\u003c/strong\u003eOsteoporotic rats treated with PES at 20 mg/kg and 40 mg/kg, respectively: \u003cstrong\u003e(D)\u003c/strong\u003eThin, poorly connected bone trabeculae (short black arrows), with a reduced number of osteocytes (OC) and few osteoblasts at the periphery (OB). \u003cstrong\u003e(E)\u003c/strong\u003eThicker and partially interconnected bone trabeculae, containing uniform osteocytes (OC) and a proliferation of osteoblasts at the edge (OB). \u003cstrong\u003e(F), (G), (H),\u003c/strong\u003e and \u003cstrong\u003e(I)\u003c/strong\u003e Osteoporotic rats treated with NCal 20 mg/kg, NCal 40 mg/kg, CA 20 mg/kg and CA 40 mg/kg, respectively: \u003cstrong\u003e(F) \u003c/strong\u003eThin and poorly connected trabeculae (short black arrows), with a low number of osteocytes (OC) and osteoblasts (OB). (\u003cstrong\u003eG), (H)\u003c/strong\u003e and \u003cstrong\u003e(I)\u003c/strong\u003e Focal, eosinophilic and well-connected lamellar trabeculae (short black arrows), with numerous osteocytes (OC) and osteoblasts (OB) at the bone border. \u003cstrong\u003e(J)\u003c/strong\u003e And \u003cstrong\u003e(K)\u003c/strong\u003eOsteoporotic rats treated with CaCO₃ 20 mg/kg and CaCO₃ 40 mg/kg, respectively: Focal, eosinophilic, and well-interconnected lamellar beams (short black arrows), with a large number of osteocytes (OC) and osteoblasts (OB) at the bone border.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9012106/v1/d9131bfb6b2b3e45e9b1bac9.png"},{"id":109296537,"identity":"e3340efe-c1fa-4802-9c79-7f152fc6b0f0","added_by":"auto","created_at":"2026-05-15 08:48:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4828141,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9012106/v1/ac0b16b2-1798-4765-b565-da05ae2300f3.pdf"},{"id":105307050,"identity":"451ccfb2-3d9c-4c8f-b547-657954e0cca3","added_by":"auto","created_at":"2026-03-24 14:44:32","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":239544,"visible":true,"origin":"","legend":"","description":"","filename":"graphicalabstract.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9012106/v1/df30fde53bab93361111ab4c.pdf"}],"financialInterests":"","formattedTitle":"Protective Effects of Eggshell Powder and Eggshell-Derived Calcium Compounds on Glucocorticoid-Induced Osteoporosis in Albinos Wistar Rats","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOsteoporosis (OP) is a major public health problem worldwide, mainly due to aging populations. It is a generalized metabolic disease of bone tissue \u003cb\u003e[1], [2]\u003c/b\u003e. It is defined by a decrease in bone strength, resulting in increased vulnerability to fractures. The mechanical strength of bone depends on both the quantity and quality of bone tissue \u003cb\u003e[3]\u003c/b\u003e. Osteoporosis is a common condition that affects both men and women, with a significantly higher incidence in women. Recognized as a major public health issue, it causes approximately 1.3\u0026nbsp;million bone fractures per year, representing an estimated economic cost of nearly \u003cspan\u003e$\u003c/span\u003e10\u0026nbsp;billion in the United States \u003cb\u003e[4].\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOsteoporosis is divided into two main forms: primary and secondary.\u003c/p\u003e \u003cp\u003eType I primary osteoporosis is linked to the decline in estrogen in women after menopause, while type II osteoporosis, know as senile osteoporosis, is a consequence of physiological aging. Secondary osteoporosis is closely linked to various underlying causes such as vitamin D deficiency, insufficient calcium intake, or iatrogenic factors \u003cb\u003e[5]\u003c/b\u003e. Furthermore, glucocorticoid-induced osteoporosis is the most common form of secondary osteoporosis, accounting for nearly 20% of all reported cases of osteoporosis \u003cb\u003e[6], [7]\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eGlucocorticoids are widely prescribed for the treatment of various inflammatory and autoimmune conditions. However, glucocorticoid-induced osteoporosis (GIO) is one of the most common and severe adverse complications associated with their administration \u003cb\u003e[8]\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eIn healthy adults, bone tissue undergoes continuous remodeling as a result of a closely coordinated physiological process. During this mechanism, osteoclasts ensure the resorption of old bone tissue, while osteoblasts are involved in the formation and mineralization of newly formed bone tissue, thus contributing to the maintenance and structural development of the skeleton \u003cb\u003e[9, 10]\u003c/b\u003e. The use of glucocorticoids leads to inhibition of osteoblast activity, stimulation of bone resorption by osteoclasts, reduced intestinal calcium absorption, and secondary hyperparathyroidism. The interaction of these mechanisms results in a rapid decrease in bone mineral density (BMD) \u003cb\u003e[11]\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eSeveral therapeutic options are available for the management of secondary osteoporosis. Previous studies have shown that certain nutritional compound, particularly eggshells, have beneficial effects by promoting bone mineralization and stimulating osteoblast activity in patients with osteoporosis \u003cb\u003e[12]\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eCalcium extracted from eggshells is currently widely used as a low-cost calcium supplement. Recent studies have shown that eggshells are highly effective source of calcium for stimulating bone mineralization in ovariectomized rats \u003cb\u003e[13]\u003c/b\u003e. Therefore, the disposal of eggshells without recovery represents an avoidable loss of valuable natural resources \u003cb\u003e[14].\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGlobal egg production has increased dramatically, from 0.75 trillion to 1.63 trillion eggs between 1994 and 2021, according to FAO data ( 190 to ~\u0026thinsp;200 eggs consumed per person per year in 2024), which implies a very sharp increase in consumption and production, generating approximately 8.58\u0026nbsp;million tons of eggshells considered waste, often disposed of in landfills without recovery \u003cb\u003e[76]\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eThe shell is mainly composed of approximately 94% calcium carbonate (CaCO\u003csub\u003e3\u003c/sub\u003e), 1% calcium phosphate (Ca\u003csub\u003e3\u003c/sub\u003e (PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e), 1% magnesium carbonate (MgCO\u003csub\u003e3\u003c/sub\u003e), and 4% organic substances \u003cb\u003e[15]\u003c/b\u003e. The shell membrane, meanwhile, consists of approximately 69.2% protein and 2.7% lipids \u003cb\u003e[15], [16]\u003c/b\u003e. Due to their nutritional richness, various initiatives have been undertaken to make use of eggshells. They have been used as fertilizer, catalysts for biodiesel production, animal feed additives and soil amendments \u003cb\u003e[17], [18]\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eEggshell waste is particularly rich in bioactive compounds, which is sparking growing interest in its reuse through the development of value-added products with a variety of commercial applications \u003cb\u003e[19]\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eIn addition, several studies have shown that eggshells can be used as a natural source of calcium for medical and dietary purposes, particularly for the production of calcium salts such as lactate, propionate, gluconate, citrate, and calcium acetate \u003cb\u003e[20]\u003c/b\u003e. In adults, approximately 2.7g of eggshell powder provides the recommended daily intake of calcium \u003cb\u003e[21]\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eIn this context, the present study was conducted on albino Wistar rats to evaluate the protective effects of eggshell powder (ESP) and its derivatives, nano-calcium lactate (Ncal), prepared by the precipitation method, and calcium (CA) obtained by chemical transformation, as well as inorganic calcium carbonate (CaCO\u003csub\u003e3\u003c/sub\u003e) on glucocorticoid-induced osteoporosis.\u003c/p\u003e"},{"header":"Materials and methods","content":"\n\u003ch3\u003e1. Sample collection\u003c/h3\u003e\n\u003cp\u003eThe sample for this study consisted of chicken eggs from the market in Mostaganem, Algeria. These samples were randomly selected, and the eggs were then transferred to the laboratory for analysis.\u003c/p\u003e\n\u003ch3\u003e2. Preparation of eggshell powder\u003c/h3\u003e\n\u003cp\u003eThe eggshells were washed with distilled water, and then all internal membranes were removed. They were dried at room temperature, ground into a fine powder using a mortar, sieved, and stored in an airtight container \u003cb\u003e[22]\u003c/b\u003e.\u003c/p\u003e\n\u003ch3\u003e3. Analysis of proximate and energy composition\u003c/h3\u003e\n\u003cp\u003eThe protein, moisture, total ash, crude fiber, crude fat, and carbohydrate contents of the eggshell powder were determined.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Moisture content\u003c/h2\u003e \u003cp\u003eThe moisture content of the eggshell powder was determined using the method described by Osibona \u003cb\u003e[23]\u003c/b\u003e. This procedure is based on removing water from the sample and measuring it by weight loss. A clean crucible was first weighed and dried in an oven (W\u003csub\u003e0\u003c/sub\u003e); then 2.0 g of eggshell powder was weighed into the crucible (W\u003csub\u003e1\u003c/sub\u003e) and dried at 105\u0026deg;C for 25 hours. After drying, the crucible was transferred to desiccators until it had cooled completely, then weighed again (W\u003csub\u003e2\u003c/sub\u003e). The moisture content was calculated as follows:\u003c/p\u003e \u003cp\u003eMoisture content= (W\u003csub\u003e1\u003c/sub\u003e-W\u003csub\u003e2\u003c/sub\u003e/W\u003csub\u003e1\u003c/sub\u003e-W\u003csub\u003e0\u003c/sub\u003e) \u0026times;100 \u003cb\u003e[24]\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Protein content\u003c/h2\u003e \u003cp\u003eThe protein composition of 2.0g of eggshell powder was determined using the Kjeldahl method, in the presence of a catalyst. The protein content was calculated by multiplying the amount of nitrogen by a factor of 6.25:\u003c/p\u003e \u003cp\u003eCrude Protein (%)\u0026thinsp;=\u0026thinsp;N(%) \u0026times; 6.25 \u003cb\u003e[25], [26]\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Ash content\u003c/h2\u003e \u003cp\u003eThe ash content was determined by incinerating the sample at 650\u0026deg;C for 2 hours in a muffle furnace. A clean, dry crucible was weighed (W₀), then 2.0 g of eggshell powder was weighed into this crucible (W₁). After incineration, the crucible containing the residues were weighed again (W₂). The percentage of ash was then calculated based on the residual mass after calcination, using the following equation:\u003c/p\u003e \u003cp\u003eAsh content (%) = (W\u003csub\u003e2\u003c/sub\u003e-W\u003csub\u003e0\u003c/sub\u003e/W\u003csub\u003e1\u003c/sub\u003e-W\u003csub\u003e0\u003c/sub\u003e) \u0026times; 100 \u003cb\u003e[22], [27]\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Fiber content\u003c/h2\u003e \u003cp\u003eCrude fiber was determined by weighing 2.0 g of eggshell powder W\u003csub\u003es,\u003c/sub\u003e which was mixed with 20 ml of 1.25% H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e and boiled for 30 minutes. After cooling, the residue was recovered by filtration, treated with 20 ml of 1.25% NaOH, and boiled for 30 minutes. The mixture was filtered, and the residue was washed with hot distilled water, and then rinsed once with 10% HCL and subsequently with ethanol. The residue W\u003csub\u003e1\u003c/sub\u003e was left to dry in an oven overnight at 105\u0026deg;C, then cooled in a desiccator and weighed.\u003c/p\u003e \u003cp\u003eThe dried sample was incinerated at 550\u0026deg;C and the ashes W\u003csub\u003e2\u003c/sub\u003e were obtained after cooling.\u003c/p\u003e \u003cp\u003eThe raw fibers were determined according to the following equation:\u003c/p\u003e \u003cp\u003eCrude fiber (%) = (W\u003csub\u003e1\u003c/sub\u003e-W\u003csub\u003e2\u003c/sub\u003e/W\u003csub\u003eS\u003c/sub\u003e) \u0026times; 100 \u003cb\u003e[25], [28]\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Fat content\u003c/h2\u003e \u003cp\u003eThe crude fat content was determined using a Soxhlet apparatus. 2.0 g of eggshell powder W\u003csub\u003eS\u003c/sub\u003e was placed in an extraction cartridge. In a round-bottom flask W\u003csub\u003e0\u003c/sub\u003e, 80 ml of hexane were poured .the heating mantle was switched on after installing the cartridge at 60\u0026deg;C, and water was circulated through the condenser to ensure cooling. After 2 hours of extraction, the hexane was removed by drying and the flask was weighed W\u003csub\u003e1\u003c/sub\u003e. The crude fat content of the sample was calculated using the following equation \u003cb\u003e[29], [24].\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCrude fat \u003cb\u003e(%) = (\u003c/b\u003eW\u003csub\u003e1 \u0026minus;\u003c/sub\u003e W\u003csub\u003e0 /\u003c/sub\u003eW\u003csub\u003es\u003c/sub\u003e) \u0026times; 100\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Carbohydrate content\u003c/h2\u003e \u003cp\u003e The carbohydrate content was determined by subtracting 100% from the sum of the percentages of crude fat, crude protein, crude fiber, moisture, and ash in accordance with following formula:\u003c/p\u003e \u003cp\u003eCHO (KJ/100g) = (crude fat % + crude protein % + crude fiber % + moisture % + ash % ) -100 \u003cb\u003e[25]\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e4. Characterization of eggshell powder\u003c/h3\u003e\n\u003cp\u003eThe chemical characterization of eggshell powder was revealed following XRD and FTIR analyses.\u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.1. X-ray diffraction (XRD)\u003c/h2\u003e \u003cp\u003eThe diffraction pattern of the powder samples was analyzed using a rotating sample holder in the Malvern Panalytical Empyrean XRD instrument, scanning the rang from 10\u0026deg; to 80\u0026deg; at a speed of 10\u0026deg;/min \u003cb\u003e[9]\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.2. Fourier transform infrared spectroscopy (FTIR) analysis\u003c/h2\u003e \u003cp\u003eFor FTIR analysis, ATR acquisition was performed on powder samples using a diamond crystal on the Perkin Elmer Spectrum 100 instrument. The transmission spectrum of these samples was obtained in the Wavenumber range of 4000\u0026thinsp;\u0026minus;\u0026thinsp;380 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e \u003cb\u003e[9]\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e5. Eggshell derivatives and their characterization\u003c/h3\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e5.1. Production of calcium acetate\u003c/h2\u003e \u003cp\u003eThe chemical transformation begins by washing 35g of eggshells (halves and pieces) with 350 ml of distilled water on a magnetic stirrer ( Stuart,UK) at 3 \u0026times; 30 minutes at 250 rpm at room temperature to remove impurities, particularly residual egg white proteins\u003cb\u003e[31].\u003c/b\u003e Subsequently, the washed shells were treated with 525 ml of 10% (w/v) acetic acid for 3 hours at room temperature to obtain calcium salts. The mixture was filtered to separate the eggshell membranes from the saline solutions using a plastic mesh sieve with porosity of 1 mm. The calcium salt solutions obtained were clarified by two successive steps of vacuum filtration using Whatman 114 and Whatman 1 filter paper, and the calcium acetate solution was evaporated at 100\u0026ndash;130\u0026deg;C until 1/10 of initial volume was obtained. The crystals were obtained by adding 3 volumes of acetone to the saturated saline solution cooled to room temperature, followed by filtration. After drying at 60\u0026deg;C for 24 hours, the crushed salts were stored in plastic containers for further analysis.\u003c/p\u003e \u003cp\u003eThe yield of calcium acetate derived from eggshells was calculated using the mass yield and chemical yield, as shown in equations (1) and (2) \u003cb\u003e[30].\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMass yield (g) \u003cb\u003e=\u003c/b\u003e [m \u003csub\u003e(calcium salt)\u003c/sub\u003e\u003cb\u003e/\u003c/b\u003e m \u003csub\u003e(eggshells)\u003c/sub\u003e ] \u0026times; 100 (1)\u003c/p\u003e \u003cp\u003eChemical yield (g) \u003cb\u003e=\u003c/b\u003e [m \u003csub\u003e(calcium salt)\u003c/sub\u003e\u003cb\u003e/\u003c/b\u003e m \u003csub\u003e(calcium salt )\u003c/sub\u003e (theoretical) ] (2)\u003c/p\u003e \u003cp\u003eThe analysis of calcium acetate derived from eggshells was performed using an FTIR-ATR spectrometer in the range of 650 to 4000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e \u003cb\u003e[30]\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e5.2. Production of calcium nano-lactate\u003c/h2\u003e \u003cp\u003eCalcium nano-lactate was synthesized from calcium oxide derived chicken eggshells. Calcium oxide was synthesized using the incineration method, according to \u003cb\u003eYusraM. and al. (2022) [32]\u003c/b\u003e, while calcium nano-lactate was synthesized using the precipitation method, according to \u003cb\u003ePrayitno and al. (2016) [33].\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA 6 mol/L lactic acid solution was mixed with a 1 mol/L CaO solution derived from eggshells in a ratio of 1:1.5 (v/v) at 50\u0026deg;C at a speed of 500 rpm/minute for 30 minutes. 50% ethanol was then added to the solution (v/v). After drying at 105\u0026deg;C for 72 hours, the calcium lactate was powdered and stored in a dry place \u003cb\u003e[34]\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eThe characterization of calcium nano-lactate derived from eggshells included Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD), in accordance with \u003cb\u003eDheyab and al. (2020) [34], [35]\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e6. In vivo experimentation\u003c/h3\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e6.1. Animals and adaptation conditions\u003c/h2\u003e \u003cp\u003eFifty 2-months-old albino Wistar rats weighing between 100 and 120 g, supplied by the Pasteur Institute of Algeria, were used in this study. During the 7-day adaptation period, the rats were housed in polypropylene cages and kept under standard laboratory conditions (ambient temperature of 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and a 12-hour light-dark cycle) with free access to food and water ad libitum.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e6.2. Experimental protocol\u003c/h2\u003e \u003cp\u003eAfter the adaptation period, the rats were randomly divided into ten groups, with five rats per cage. Osteoporosis was induced in the animals in the different groups, with the exception of the negative control group, by intramuscular injections of methylprednisolone sodium succinate, a glucocorticoid (GC), administered at a dose of 10 mg/kg, three times a week for four weeks \u003cb\u003e[36], [4], [8]\u003c/b\u003e. The rats were simultaneously subjected to different treatments during the four weeks of the study \u003cb\u003e[13], [37]\u003c/b\u003e, as detailed below:\u003c/p\u003e \u003cp\u003eGroup 1: Negative control group (NC) administration of distilled water to healthy animals.\u003c/p\u003e \u003cp\u003eGroup 2: Positive control group (PC); induction of osteoporosis by glucocorticoids (GC) without treatment.\u003c/p\u003e \u003cp\u003eGroups 3 and 4: Animals received eggshell powder (ESP) at doses of 20 and 40 mg/kg, respectively, diluted in 1 ml of distilled water, administered daily by oral gavage.\u003c/p\u003e \u003cp\u003eGroups 5 and 6: Administration of calcium acetate (CA) derived from eggshells at doses of 20 and 40 mg/kg, respectively, diluted in 1 ml of distilled water, administered daily by oral gavage.\u003c/p\u003e \u003cp\u003eGroups 7 and 8: Rats receive calcium nano-lactate derived from eggshells (NCal) at doses of 20 and 40 mg/kg, respectively, diluted in 1 ml of distilled water, administered daily by oral gavage.\u003c/p\u003e \u003cp\u003eGroups 9 and 10: Administration of pharmaceutical calcium carbonate (CaCO\u003csub\u003e3\u003c/sub\u003e) at doses of 20 and 40 mg/kg, respectively, diluted in 1 ml of distilled water, administered daily by oral gavage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e6.3. Controlled experimental parameters\u003c/h2\u003e \u003cp\u003eOne week after treatment, the rats were fasted overnight and a blood sample was taken from the sinus. After dissection, both femurs were removed.\u003c/p\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003e6.3.1. Analysis of serum biochemical parameters\u003c/h2\u003e \u003cp\u003eAlkaline phosphatase (ALP), calcium (Ca), and phosphorus (P) levels were measured in serum. ALP and phosphorus (P) levels were measured using the Fujifilm Dri-Chem 3500i analyzer. Calcium (Ca) levels were measured using the o-Cresolphthalein complexone method (ERBA Mannheim XL200).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003e6.3.2. Measurement of mineral content in the ash of left femur\u003c/h2\u003e \u003cp\u003eThe left femur was weighed after being incinerated in a muffle furnace for 5 hours at 600\u0026deg;C. The resulting ashes were dissolved in 6 M hydrochlorid acid (HCl) \u003cb\u003e[38].\u003c/b\u003e Calcium, phosphorus, and magnesium content were measured using Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) \u003cb\u003e[39]\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e \u003ch2\u003e6.3.3. Histopathological examinations\u003c/h2\u003e \u003cp\u003eThe right femurs were removed and then decalcified using a buffered formic acid solution for three consecutive days \u003cb\u003e[40].\u003c/b\u003e After fixing the femurs in 10% neutral buffered foemalin, they were then processed and embedded in paraffin \u003cb\u003e[13]\u003c/b\u003e. Tissue sections \u003cspan refid=\"Sec15\" class=\"InternalRef\"\u003e5\u003c/span\u003e \u0026micro;m thick were obtained by cutting the paraffin blocks. The samples were then deparaffinized in xylene and rehydrated using a series of decreasing ethanol baths. Using the H\u0026amp;E solution, the treated tissue sections were then stained and observed under a microscope (Carl Zeiss, Axiovert 25, Oberkochen, Germany) \u003cb\u003e[41].\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003e7. Statistical analysis\u003c/h3\u003e\n\u003cp\u003eStatistical analysis was performed using PAST software. Analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s post hoc test was used to compare the different treatment groups with the negative control and positive control groups. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003cp\u003eIn the in vitro study, each parameter was evaluated in three independent replicates. For the in vivo study, the results represent the average of the values obtained from five rats per group.\u003c/p\u003e\n\u003ch3\u003eEthical note\u003c/h3\u003e\n\u003cp\u003e All experimental procedures and protocols involving animals were carried out in accordance with Algerian legislation (Law No. 95\u0026ndash;322/1995) on the protection of animals used for experimental and scientific purposes. They also comply with the guidelines of the Algerian Association for Laboratory Animal Science (AASEA, authorization No. 45/DGLPAG/DVA/SDA/14) concerning the protection of animals used for experimental and other scientific purposes.\u003c/p\u003e"},{"header":"Results","content":"\u003ch3\u003e1. Proximate and energy composition\u003c/h3\u003e\n\u003cp\u003eProximate chemical analyses performed on the powder obtained from eggshells made it possible to evaluate its nutritional composition. The results, expressed as a percentage of fresh matter, reveal a moisture content of 1.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28%, a crude protein content of 2.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24%, an ash content of 92.66\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04%, and a crude fiber content of 0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76%. The fat and carbohydrate content was also determined, with respective values of 3.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32% and 0.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 (KJ/100g).\u003c/p\u003e\n\u003ch3\u003e2. Characterization of eggshell powder\u003c/h3\u003e\n\u003cdiv id=\"Sec30\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1. Results of X-ray diffraction analysis\u003c/h2\u003e\n \u003cp\u003eThe X-ray diffractogram (XRD) of the eggshell powder is shown in Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The presence of several distinct peaks throughout the spectrum indicates that the material is crystalline in nature. Each peak corresponds to a specific crystallographic plane within the structure. In this particular case, the peak intensities correspond to those of calcite (CaCO\u003csub\u003e3\u003c/sub\u003e), which is the most common crystalline form of eggshell powder. The major peak characteristic of CaCO\u003csub\u003e3\u003c/sub\u003e was observed at 29.6\u0026deg;, and some of the most intense peaks appear at 2\u0026theta; values of approximately 23.2\u0026deg;, 26.7\u0026deg;, 36.2\u0026deg;, 39.6\u0026deg;, 43.3\u0026deg;, 47.7\u0026deg;, and 48.7\u0026deg;. Comparison of the positions of the measured peaks (2\u0026theta; angles) and their intensities with a reference database confirms the identification of the crystalline phase.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2. Results of Fourier transform infrared spectrum results\u003c/h2\u003e\n \u003cp\u003eThe Fourier transform infrared (FTIR) spectrum of eggshell powder was recorded in the range of 4000\u0026thinsp;\u0026minus;\u0026thinsp;380 cm⁻\u0026sup1;. The spectrum obtained shows several characteristic bands (Fig. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). A board band observed between 3400 and 3200 cm⁻\u0026sup1; was attributed to O\u0026ndash;H stretching vibration. Similarly, weak bands around 2950\u0026thinsp;\u0026minus;\u0026thinsp;2850 cm⁻\u0026sup1; correspond to aliphatic C\u0026ndash;H stretching. The presence of a band in the 1700-1750cm⁻\u0026sup1; region is associated with C\u0026thinsp;=\u0026thinsp;O vibration. In addition, the intense bands at 1450,875 and 713 cm⁻\u0026sup1; are attributed respectively to antisymmetric vibrations and deformations characteristic of the carbonate group (CO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;2\u003c/sup\u003e). Finally, the shoulder observed around 2515cm⁻\u0026sup1; corresponds to the signal reported for calcium carbonate.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003e3. Eggshell derivatives and their characteristics\u003c/h3\u003e\n\u003cdiv id=\"Sec33\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1. Calcium acetate production\u003c/h2\u003e\n \u003cp\u003eThe chemical transformation process of 35 g of eggshells using acetic acid resulted in a mass yield of calcium acetate derived from eggshells of approximately 33.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.91g/35 g of PES and a chemical yield of 23.68\u0026thinsp;\u0026plusmn;\u0026thinsp;2.07%.\u003c/p\u003e\n \u003cp\u003eThe Fourier transform infrared (FTIR) spectrum of calcium acetate derived from eggshells was recorded in the range of 4000 to 650 cm⁻\u0026sup1;. The spectrum reveals several characteristic peaks corresponding to different functional groups and bonds present in the molecule (Fig. \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The broad band observed in the 3117\u0026ndash;3709 cm⁻\u0026sup1; region is indicative of O\u0026ndash;H stretching vibrations, typical of hydroxyl groups present in calcium acetate hydrate. The peaks in the 2850\u0026ndash;3000 cm⁻\u0026sup1; range correspond to C\u0026ndash;H stretching vibrations, attributed to the alkyl groups linked to acetate. The intense peaks around 1714 cm ⁻\u0026sup1; are characteristic of the stretching vibrations of the carbonyl group (C\u0026thinsp;=\u0026thinsp;O), corresponding to the acetyl group of calcium acetate.\u003c/p\u003e\n \u003cp\u003eIn addition, peaks in the range 1418\u0026ndash;1443 cm⁻\u0026sup1; indicate O\u0026ndash;H bending vibrations, common to carboxylic acids and their salts. The peak at 1054 cm⁻\u0026sup1; is associated with C\u0026ndash;O stretching vibrations related to the acetate group. Furthermore, the peaks below 669 cm⁻\u0026sup1; can be attributed to metal-oxygen bonds, in particular calcium-oxygen interactions. The notable peaks around 1027 cm⁻\u0026sup1; probably correspond to C\u0026ndash;O stretching or bending vibrations associated with the carboxylate group of calcium acetate, while those between 419\u0026ndash;669 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e may be due to bending modes or out of plane deformations involving the acetate group and its interactions with calcium. This analysis provides an in depth understanding of the functional groups present in the calcium acetate sample based on the FTIR spectrum.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec34\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2. Production of calcium lactate derived from eggshells\u003c/h2\u003e\n \u003cp\u003eThe production of nano-calcium lactate (NCal) from calcium oxide derived from eggshells was achieved using the precipitation method. A yield of 88g of NCal was obtained from a 6 mol/L lactic acid solution and a 1 mol/L CaO solution derived from eggshells.\u003c/p\u003e\n \u003cp\u003eThe Fourier transform infrared (FTIR) spectrum of calcium nano-lactate is shown in Fig. \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The FTIR spectrum provided for calcium nano-lactate shows distinct peaks corresponding to various functional groups present in the molecule (Fig. \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The broad absorption band around 3155 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e indicates O\u0026ndash;H stretching vibrations, which are typical of hydroxyl groups and suggest the presence of water molecules, either in the form of hydration water or intrinsic to the lactate structure. The peaks in the 2850\u0026ndash;3000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e region correspond to C\u0026ndash;H stretching vibrations of alkyl groups present in lactate molecule. A notable peak at 1715 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e corresponds to C\u0026thinsp;=\u0026thinsp;O stretching vibrations of carboxylate group, which is a key characteristic of lactate anion. In addition, the peak at 1458 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is associated with the asymmetric stretching vibrations of the carboxylate group, while the peak at 1421 cm-\u003csup\u003e1\u003c/sup\u003e corresponds to the symmetric stretching of the same group. The peaks around 1300\u0026ndash;1400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e can be attributed to C\u0026ndash;H bending vibrations in lactate. The region between 1200 and 1000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e shows peaks related to C\u0026ndash;O stretching vibrations, indicating the presence of carboxylate and hydroxyl groups. The peaks observed below 600 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, particularly those around 500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, can be associated with calcium-oxygen interactions, indicating metal coordination within be compound. The presence of peaks at 1100\u0026thinsp;\u0026minus;\u0026thinsp;1000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e suggests C\u0026ndash;O stretching, typical of alcohol groups. Other smaller peaks in the region below 1000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e indicate various bending vibration that are consistent with the structure of calcium lactate. Overall, the FTIR spectrum confirms the presence of hydroxyl, carboxylate, and alkyl groups in calcium lactate, which is consistent with its known chemical structure.\u003c/p\u003e\n \u003cp\u003eCharacterization was also performed using XRD (Fig. \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The presence of several marked peaks throughout the spectrum indicates the presence of a crystalline material, which corresponds to calcium lactate. Each peak corresponds to a specific crystallographic plane within the crystal structure. In this case, the peak intensities correspond to those of calcium lactate, indicating that the sample exhibits the most common crystalline from of this compound.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003e4. Results of in vivo experimentation\u003c/h3\u003e\n\u003cdiv id=\"Sec36\" class=\"Section2\"\u003e\n \u003ch2\u003e4.1. Serum biochemical parameter analysis\u003c/h2\u003e\n \u003cp\u003eThe results of serum biochemical markers related to bone metabolism are shown in Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Serum calcium levels were within normal limits in healthy animals in group 1 (9.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 mg/dl), unlike in rats receiving glucocorticoid doses. There was a highly significant decrease in serum calcium (8.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42 mg/dl; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), confirming the induction of calcium metabolism imbalance.\u003c/p\u003e\n \u003cp\u003eIn groups treated with various forms of calcium supplements derived from eggshells, variable improvements in blood calcium levels were observed depending on the type of salt and the dose administered.\u003c/p\u003e\n \u003cp\u003eTreatment with eggshell powder (ESP) resulted in a significant increase in serum calcium compared to the negative control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) at a dose of 20 mg/kg (9.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 mg/dl). However, a significant improvement compared to the positive control group was only observed at a dose of 40 mg/kg (9.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37 mg/dl). A similar profile was observed with galenic calcium carbonate (CaCO3), which induced a significant increase in serum calcium (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with a significant corrective effect compared to the positive control group only at the highest dose, reaching values close to normal.\u003c/p\u003e\n \u003cp\u003eFurthermore, the groups treated with calcium nano-lactate (NCal) and calcium acetate (CA) showed more moderate increases. NCal at 40 mg/kg reached 8.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 mg/dl, while CA recorded levels of 9.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40 mg/dl at 20 mg/kg and 8.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36 mg/dl at 40 mg/kg, with no significant difference compared to the positive control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n \u003cp\u003eAs shown in Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the serum phosphorus level recorded in animals in the positive control group (8.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46 mg/dl) was lower than that in the negative control group (9.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 mg/dl) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, a significant increase in serum P levels was observed in rats treated with ESP 40 mg/kg and CaCO\u003csub\u003e3\u003c/sub\u003e 40 mg/kg, reaching (9.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75 and 9.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25 mg/dL), respectively, compared to the positive control. In contrast, in the group treated with CA 40 mg/kg, the serum P level (7.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74 mg/dl) was significantly lower than that of the negative control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n \u003cp\u003eAs shown in Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, compared to the negative control group, the serum alkaline phosphatase (ALP) activity of the positive control group increased to 195.4\u0026thinsp;\u0026plusmn;\u0026thinsp;15.5 U/L.\u003c/p\u003e\n \u003cp\u003eAdministration of either 20 or 40 (mg/kg) of ESP or CaCO\u003csub\u003e3\u003c/sub\u003e to the rats revealed a significant decrease (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in serum ALP levels compared to the positive control group, reaching respectively (149.2\u0026thinsp;\u0026plusmn;\u0026thinsp;15.2 U/L and 152.6\u0026thinsp;\u0026plusmn;\u0026thinsp;8.26 U/L) for the ESP groups, and (155\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5 U/L and 167\u0026thinsp;\u0026plusmn;\u0026thinsp;5.2 U/L) for the groups treated with CaCO\u003csub\u003e3\u003c/sub\u003e.\u003c/p\u003e\n \u003cp\u003eIn contrast, in the groups treated with nano-calcium lactate NCal and calcium acetate CA at different doses, the decrease in ALP levels was not significant compared to the positive control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05)\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffects of different treatments of eggshells and their derivatives on serum calcium, phosphorus, and alkaline phosphatase levels in rats with glucocorticoid-induced osteoporosis\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eGroups\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eCalcium (mg/dl)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003ePhosphorus (mg/dl)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003eAlkaline phosphatase (U/L)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eG1\u003c/p\u003e\n \u003cp\u003eNegative control\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e9.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e9.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0,08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e168.6\u0026plusmn; 2.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eG2\u003c/p\u003e\n \u003cp\u003ePositive control\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e8.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e8.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e195,4\u0026thinsp;\u0026plusmn;\u0026thinsp;15,5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eG3\u003c/p\u003e\n \u003cp\u003ePowder eggshell(20mg/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e9.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e8.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e149,2\u0026thinsp;\u0026plusmn;\u0026thinsp;15,2\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eG4\u003c/p\u003e\n \u003cp\u003ePowder eggshell(40mg/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e9.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e9.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e152,6\u0026thinsp;\u0026plusmn;\u0026thinsp;8,26\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eG5\u003c/p\u003e\n \u003cp\u003eEggshell nano-calcium lactate (20mg/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e9.18\u0026plusmn; 0.26**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e9.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e182\u0026thinsp;\u0026plusmn;\u0026thinsp;22.21\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eG6\u003c/p\u003e\n \u003cp\u003eeggshell nano\u0026ndash;calcium lactate (40mg/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e8.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e8.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e178\u0026thinsp;\u0026plusmn;\u0026thinsp;26.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eG7\u003c/p\u003e\n \u003cp\u003eEggshell-derived calcium acetate(20mg/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e9.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e8.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e169.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eG8\u003c/p\u003e\n \u003cp\u003eEggshell-derived calcium acetate(40mg/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e8.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e7.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e192.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eG9\u003c/p\u003e\n \u003cp\u003eCalcium carbonate(20mg/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e9.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e8.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e155\u0026thinsp;\u0026plusmn;\u0026thinsp;4,5\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eG10\u003c/p\u003e\n \u003cp\u003eCalcium carbonate(40mg/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e9.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e9.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e167\u0026thinsp;\u0026plusmn;\u0026thinsp;5,2\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003ep\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e0.0002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e0.00005\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e0.0002\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eData expressed as median (IQR) (n\u0026thinsp;=\u0026thinsp;5). A one-way ANOVA was used for statistical analysis, followed by Tukey\u0026rsquo;s post-hoc test. \u003csup\u003e**\u003c/sup\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05 compared to negative control, \u003csup\u003e##\u003c/sup\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05 compared to positive control.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec37\" class=\"Section2\"\u003e\n \u003ch2\u003e4.2. Mineral content in Femoral Bone Ash\u003c/h2\u003e\n \u003cp\u003eAnalysis of bone mineral composition revealed highly significant differences between the experimental groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The positive control group (G2) showed a marked decrease in calcium, phosphorus, and magnesium content compared to the negative control group (G1), confirming the alteration in bone mineralization induced by glucocorticoids.\u003c/p\u003e\n \u003cp\u003eSupplementation with eggshell powder (ESP) resulted in a significant dose-dependent restoration of bone minerals, with values close to those of the negative control at a dose of 40 mg/kg (G4), indicating high remineralizing efficacy. The groups treated with nano-calcium lactate derived from eggshells (G5-G6) also showed significant improvement, particularly for phosphorus and magnesium, suggesting better bioavailability linked to nanometric form.\u003c/p\u003e\n \u003cp\u003eIn contrast, calcium acetate (G7-G8) and calcium carbonate (G9-G10) induced a moderate increase in mineral parameters, less marked than that observed with ESP and NCal, particularly for phosphorus (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eCalcium, phosphorus, and magnesium levels in the left femoral bone ash of healthy rats and glucocorticoid-induced osteoporotic rats treated with ESP, NCal, CA, and CaCO\u003csub\u003e3\u003c/sub\u003e (n\u0026thinsp;=\u0026thinsp;5).\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eGroups\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eCalcium (mg/L)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003ePhosphorus (mg/L)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003eMagnesium (mg/L)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eG1\u003c/p\u003e\n \u003cp\u003eNegative control\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e1881.534 \u0026plusmn; 54.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e1190.32 \u0026plusmn; 49.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e38.57 \u0026plusmn; 1.10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eG2\u003c/p\u003e\n \u003cp\u003ePositive control\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e1695.36 \u0026plusmn; 54.23\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e949.14 \u0026plusmn; 26.55\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e35.54 \u0026plusmn; 0.70 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eG3\u003c/p\u003e\n \u003cp\u003ePowder eggshell(20mg/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e1792.358 \u0026plusmn; 7.14\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e988.28 \u0026plusmn; 6.32\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e36.88 \u0026plusmn; 0.06 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eG4\u003c/p\u003e\n \u003cp\u003ePowder eggshell(40mg/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e1866.04 \u0026plusmn; 47.33\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e1139.32 \u0026plusmn; 23.27\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e37.67 \u0026plusmn; 0.19\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eG5\u003c/p\u003e\n \u003cp\u003eEggshellnano-calcium lactate (20mg/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e1770.142 \u0026plusmn; 38.89\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e1096.144 \u0026plusmn; 52.58\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e36.8 \u0026plusmn; 0.16 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eG6\u003c/p\u003e\n \u003cp\u003eeggshell nano\u0026ndash;calcium lactate (40mg/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e1833.84 \u0026plusmn; 52.15\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e1126.58 \u0026plusmn; 24.54\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e37.31 \u0026plusmn; 0.37 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eG7\u003c/p\u003e\n \u003cp\u003eEggshell-derived calcium acetate(20mg/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e1786.934 \u0026plusmn; 8.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e1009.056 \u0026plusmn; 54.38\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e36.59 \u0026plusmn; 0.55 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eG8\u003c/p\u003e\n \u003cp\u003eEggshell-derived calcium acetate(40mg/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e1800.046 \u0026plusmn; 13.74\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e1061.326 \u0026plusmn; 55.40\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e36.79 \u0026plusmn; 0.13\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eG9\u003c/p\u003e\n \u003cp\u003eCalcium carbonate(20mg/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e1832.72 \u0026plusmn; 50.18\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e1030.70 \u0026plusmn; 62.36\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e36.51 \u0026plusmn; 0.33\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eG10\u003c/p\u003e\n \u003cp\u003eCalcium carbonate(40mg/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e1845.84 \u0026plusmn; 74.48\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e1062.78 \u0026plusmn; 26.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e36.13 \u0026plusmn; 0.83\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003ep\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e0.0002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e0.00001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e0.00002\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eData are expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, with five rats per group. Differences between groups were analyzed using a one-way ANOVA test, followed by Tukey\u0026rsquo;s pairwise test.\u003c/p\u003e\n \u003cp\u003ea,b: indicate a significant difference compared to the negative control group and the positive control group (osteoporotic rats), respectively.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec38\" class=\"Section2\"\u003e\n \u003ch2\u003e4.3. Histopathological Analysis\u003c/h2\u003e\n \u003cp\u003eFigure \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e shows histological sections of the right femur of rats from the different experimental groups, stained with hematoxylin and eosin (H\u0026amp;E), revealed the bone architecture and the histopathological changes induced by the different treatments used.\u003c/p\u003e\n \u003cp\u003eIn rats from the negative control group, bone architecture was preserved and characterized by thick, well-organized, and intensely eosinophilic trabeculae, indicating good trabecular connectivity (Fig. \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The trabeculae were lined with numerous resting osteoblasts, while a large number of viable osteocytes were observed within the lamellar bone matrix, indicating balanced bone remodeling.\u003c/p\u003e\n \u003cp\u003eConversely, rats in the positive control group, treated with glucocorticoids without calcium supplementation, showed marked histological changes reflecting significant bone resorption (Fig. \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). The trabeculae appeared thin, disorganized, and weakly eosinophilic, associated with a decrease in the number of osteocytes and increased osteoclastic activity. Thinning of the cortical bone and enlargement of the medullary spaces were also observed, reflecting a loss of trabecular connectivity characteristic of osteoporosis.\u003c/p\u003e\n \u003cp\u003eBone sections from osteoporotic rats treated with eggshell powder (ESP 40mg/kg), calcium nano-lactate ( NCal 40 mg/kg), calcium acetate (CA 20 and 40 mg/kg), and calcium carbonate (CaCO\u003csub\u003e3\u003c/sub\u003e 20 and 40 mg/kg), (Fig. \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, G, H, I, J and K, respectively) showed significant histological improvement. The bone trabeculae were thicker and better organized with a dense lamellar matrix, intensely stained with eosin, containing numerous evenly distributed osteocytes. In addition, the medullary spaces were reduced and no significant osteoclastic proliferation was observed, suggesting marked bone regeneration.\u003c/p\u003e\n \u003cp\u003eIn contrast, rats receiving lower doses, notably ESP at 20 mg/kg and NCal at 20 mg/kg (Fig. \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD and F, respectively), showed histological features similar to those of the positive control group. The trabeculae remained thin, pale, and locally irregular, with a low number of osteocytes and peripheral osteoblasts. These results suggest limited improvement, indicating that these low doses do not have a significant effect on bone regeneration.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\n\u003ch3\u003e1. Characterization of eggshell powder and its derivatives:\u003c/h3\u003e\n\u003cp\u003eThe results relating to proximate chemical composition of the eggshell powder obtained, particularly for moisture and crude protein, are similar to those reported for brown eggshells \u003cb\u003e[43].\u003c/b\u003e However, the lipid and fiber contents observed in our study are higher than those mentioned in the same study. Furthermore, the ash content obtained is comparable to that reported in a study on eggshell powder used to enrich bread \u003cb\u003e[44]\u003c/b\u003e, but remains lower than that reported for white eggshell powder \u003cb\u003e[43]\u003c/b\u003e. These differences in composition could be attributed to the origin of eggshells and the birds diet \u003cb\u003e[43],[45].\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe FTIR results for the eggshell powder studied confirm the presence of bands characteristic of calcium carbonate (CaCO\u003csub\u003e3\u003c/sub\u003e), in agreement with the work reported in the literature. The characteristic bands observed at 713 and 874 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, corresponding to the calcite phase of CaCO\u003csub\u003e3\u003c/sub\u003e, are similar to those reported in the reference study on calcite \u003cb\u003e[46]\u003c/b\u003e. Calcite, the trigonal phase of calcium carbonate, is the main component of bird eggshells. At room temperature, it is the most stable polymorph of calcium carbonate \u003cb\u003e[42]\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eSimilarly, the results obtained by X-ray diffraction (XRD) confirm the crystallinity of this product and are similar to those reported for eggshell powder from different species \u003cb\u003e[9]\u003c/b\u003e. The characteristic major peak for CaCO\u003csub\u003e3\u003c/sub\u003e was observed at 29.6\u0026deg; for all species studied.\u003c/p\u003e \u003cp\u003eEggshells can be used in the medical field as a source of calcium and as a food additive in the form of calcium lactate, citrate, gluconate, propionate, and acetate \u003cb\u003e[20]\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eThe mass and chemical yields of calcium acetate derived from eggshells are consistent with those reported in the work of \u003cb\u003eStrelec and al. (2023) [30]\u003c/b\u003e and other studies \u003cb\u003e[47], [50]\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eFurthermore, the yield of calcium nano-lactate (NCal) obtained is four times higher than that reported by \u003cb\u003eTam and al. (2025) [51]\u003c/b\u003e, who reported a yield of 20g of calcium lactate using a 16% lactic acid solution. Although the experimental protocols differ, this comparison highlights the decisive role of reagent concentration and the process used on the final product yield.\u003c/p\u003e \u003cp\u003eThe FTIR spectra obtained for acetate and calcium nano-lactate derived from eggshells reveal the presence of several characteristic peaks, attributed to different functional groups and chemical bonds constituting the molecules. These results are consistent with those reported by \u003cb\u003eChung and al. [52]\u003c/b\u003e and \u003cb\u003eTongkam and al. [53]\u003c/b\u003e for calcium acetate, as well as with the observations of \u003cb\u003eMcReynolds and al. [54]\u003c/b\u003e and \u003cb\u003eTesfamariam and al. [55]\u003c/b\u003e for calcium lactate. Furthermore, X-ray diffraction (XRD) analyses of NCal confirm the crystalline nature of this compound. Indeed, comparing the positions of the recorded peaks (2Ɵ- angles) and their intensities with reference data allows the material to be precisely identified [\u003cb\u003e56], [57].\u003c/b\u003e\u003c/p\u003e\n\u003ch3\u003e2. Effects of eggshell powder and its derivatives on glucocorticoid-induced osteoporotic rats\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003e2. Effects of eggshell powder and its derivatives on glucocorticoid-induced osteoporotic rats\u003c/div\u003e \u003cp\u003eCalcium is one of the essential nutrients involved in many metabolic functions in the human body \u003cb\u003e[58]\u003c/b\u003e. A sufficient intake of this mineral is essential for growth, maintaining bone health, and preventing the risk of osteoporosis and associated fractures \u003cb\u003e[59]\u003c/b\u003e. The medical management of osteoporosis continues to face the constant challenge of preventing associated fractures \u003cb\u003e[60], [13]\u003c/b\u003e. The convergence between the nutritional and practical applications of eggshells and those of bone tissue justifies their promotion as a potential source of dietary calcium \u003cb\u003e[61]\u003c/b\u003e. In this context, the present study used an appropriate animal model of glucocorticoid-induced osteoporotic bone loss to compare the efficacy of eggshell powder (ESP), considered a source of inorganic calcium, with that of calcium acetate (CA) and nano-calcium lactate (NCal) derived from eggshells, representing sources of organic calcium, as well as synthetic calcium carbonate, in preventing the onset of osteoporosis.\u003c/p\u003e \u003cp\u003eThe administration of different sources of calcium during glucocorticoid treatment induced significant variations in bone biochemical parameters, particularly serum calcium and phosphorus levels and alkaline phosphatase activity, depending on the experimental group. The marked decrease in serum calcium observed in the positive control group confirms the alteration in calcium-phosphorus metabolism induced by glucocorticoids, characterized by inhibition of intestinal calcium absorption and increased renal excretion. Conversely, 30 days of supplementation with eggshell powder (ESP) or synthetic calcium carbonate significantly restored serum calcium, with a more pronounced dose-dependent effect at 40 mg/kg. These results are consistent with those of \u003cb\u003eNagamma et al. [62]\u003c/b\u003e, who reported a significant increase in serum calcium in rats supplemented with calcium from eggshells compared to untreated ovariectomized animals. This improvement could be attributed to the mineral richness of eggshells and the presence of trace compounds that may promote calcium bioavailability.\u003c/p\u003e \u003cp\u003eHowever, \u003cb\u003eKareem et al. (2025)\u003c/b\u003e showed that nanoscale eggshell calcium carbonate (NPES) induced a greater increase in serum calcium than that observed with micronized ESP or inorganic calcium carbonate (CaCO\u003csub\u003e3\u003c/sub\u003e) \u003cb\u003e[16]\u003c/b\u003e. This superiority of NPES could be explained by its high specific surface area, which facilitates solubilization, gastrointestinal absorption, and cellular uptake of calcium \u003cb\u003e[63]\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eIn contrast, the groups treated with calcium acetate (CA) and nanometric calcium lactate (NCal) showed no significant difference compared to the positive control group. However, treatment with NCal appears to slightly improve serum calcium levels compared to CA. these results are consistent with the observations reported by \u003cb\u003eUeda et al. (2013)\u003c/b\u003e, according to which calcium acetate and lactate are rapidly dissociated and eliminated in the digestive tract, thus limiting their intestinal residence time and, consequently, their bioavailability \u003cb\u003e[64]\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eWith regard to serum phosphorus, the groups supplemented with ESP and CaCO\u003csub\u003e3\u003c/sub\u003e at a dose of 40 mg/kg showed a significant increase compared to the positive control group, in agreement with the results of \u003cb\u003eKareem et al. (2025) [16]\u003c/b\u003e. Given that the bone mineral content, particularly calcium (Ca) and phosphorus (P), is a fundamental indicator of bone quality, these results suggest an improvement in the bone mineralization process under the effect of these treatments \u003cb\u003e[65]\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eIn the NCal group, a moderate and insignificant increase in serum phosphorus was observed, while calcium acetate led to a marked decrease in this parameter. These results confirm the well-established role of acetate as a calcium phosphate chelator, reducing its intestinal absorption \u003cb\u003e[66]\u003c/b\u003e. This property gives calcium acetate particular therapeutic interest, allowing both calcium intake and a reduction in phosphate load thus contributing to the prevention of hypocalcemia and secondary hyperparathyroidism, which is often involved in bone remodeling disorders \u003cb\u003e[67]\u003c/b\u003e. These observations suggest that the beneficial effects of calcium acetate may go beyond simply correcting mineral metabolism, notably by improving metabolic acidosis and modulating inflammatory processes \u003cb\u003e[67]\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eAnalysis of alkaline phosphatase (ALP) activity, a key marker of bone remodeling, showed a significant increase in the positive control group, reflecting an intensification of glucocorticoid-induced resorption, as reported by \u003cb\u003eLatif et al. (2021) [68]\u003c/b\u003e. Conversely, all groups supplemented with different sources of calcium showed a significant reduction in ALP, consistent with the findings of \u003cb\u003eKareem et al. (2025)\u003c/b\u003e for NPES and CaCO\u003csub\u003e3\u003c/sub\u003e \u003cb\u003e[16]\u003c/b\u003e. These data indicate that the calcium supplements tested contribute to limiting excessive bone remodeling associated with estrogen deficiency, thereby promoting more balanced bone metabolism and improved bone structural integrity \u003cb\u003e[69].\u003c/b\u003e\u003c/p\u003e \u003cp\u003eComparative analysis of the mineral composition of femoral ash reveals marked differences in efficacy between eggshell powder (ESP), nano-calcium lactate (NCal), and calcium acetate (CA) in restoring bone mineralization impaired by glucocorticoids. The GIOP group had the lowest calcium, phosphorus, and magnesium levels, confirming the onset f severe osteoporosis linked to inhibition of bone formation and accelerated mineral loss \u003cb\u003e[16]\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eAmong the supplements tested, ESP stood out for providing the most complete and consistent restoration of femoral mineral content, with a significant and concomitant increase in calcium, phosphorus, and magnesium. This superiority suggests that the effect of ESP is not based solely on elemental calcium intake, but also on the presence of a naturally balanced mineral matrix rich in bioactive trace elements such as magnesium, zinc, and strontium. These elements are recognized for their synergistic role in osteoblast differentiation, osteoid maturation, and hydroxyapatite crystal stabilization \u003cb\u003e[70]\u003c/b\u003e. Thus, ESP appears to be a multifunctional biomaterial that promotes physiological and sustainable bone remineralization.\u003c/p\u003e \u003cp\u003eNCal showed moderate efficacy, characterized mainly by a significant improvement in bone phosphorus and magnesium levels, while calcium restoration remained partial, particularly at the lowest doses. This response could be explained by nanometric size of NCal, which increases the specific surface area and improves intestinal calcium absorption. However, the absence of a complex mineral matrix comparable to that of ESP could limit its overall remineralizing potential. Nevertheless, at a dose of 40 mg/kg, NCal showed a more marked mineral improvement, suggesting a dose-response relationship and efficacy dependent on the concentration administered, in agreement with the observations of \u003cb\u003eKareem et al. [16].\u003c/b\u003e\u003c/p\u003e \u003cp\u003eConversely, CA showed the most limited effectiveness in restoring bone mineral content. Although it provides a soluble source of calcium, its impact on femoral calcium and phosphorus levels remained modest, particularly at low doses. This reduced efficacy could be attributed to its rapid dissociation and elimination in the digestive tract, as well as its well-documented role as a phosphate chelator, limiting intestinal phosphorus absorption and, consequently, adequate bone hydroxyapatite formation \u003cb\u003e[66].\u003c/b\u003e Thus, CA appears to act more as a regulator of systemic calcium-phosphorus metabolism than as a direct bone remineralizing agent.\u003c/p\u003e \u003cp\u003eFrom a comparative perspective, the remineralizing efficacy of the supplements tested can be ranked as follows: ESP\u0026thinsp;\u0026lt;\u0026thinsp;NCal\u0026thinsp;\u0026lt;\u0026thinsp;CA, with ESP clearly superior at a dose of 40 mg/kg. This ranking highlights the importance not only of the physicochemical from of calcium, but also of its mineral matrix and functional bioavailability in the prevention and correction of glucocorticoid-induced bone loss.\u003c/p\u003e \u003cp\u003eThese results suggest that ESP represents a natural and particularly promising alternative to conventional calcium salts, combining remineralizing efficacy, mineral synergy, and the potential to improve bone remodeling. NCal could be an interesting option at adequate doses, while CA seems more appropriate as an adjunctive treatment aimed at calcium-phosphorus regulation rather than structural bone restoration.\u003c/p\u003e \u003cp\u003eHistologically, treatment with eggshell powder (ESP) and its derivatives offered better protection against bone loss in osteoporotic rats. Among the supplements evaluated, ESP and CaCO\u003csub\u003e3\u003c/sub\u003e showed the most marked histological effects, characterized by preservation of bone micro-architecture, improved trabecular continuity, and reduced inter-trabecular spaces. These results are consistent with those reported by \u003cb\u003eKareem et al. [16]\u003c/b\u003e, who observed a significant improvement in bone mineral density (BMD) in animals treated with ESP and CaCO\u003csub\u003e3\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eThe superior protective effect of eggshell powder could be attributed to its complex and naturally balanced mineral composition, rich in calcium (Ca), magnesium (Mg), and phosphorus (P), which are essential elements for maintaining bone integrity. Calcium is the main mineral constituent of bone tissue, in the form of calcium phosphate crystallized as hydroxyapatite, ensuring bone rigidity and strength. Magnesium, on the order hand, plays a key role in mineral and bone homeostasis by modulating the secretion of hormones involved in skeletal metabolism, regulating bone cell activity, and participating in the formation, growth, and stabilization of hydroxyapatite crystals \u003cb\u003e[39]\u003c/b\u003e. This mineral synergy gives ESP a functional advantage over isolated calcium salts.\u003c/p\u003e \u003cp\u003eIn contrast, the positive control group showed marked trabecular disorganization, characterized by homogeneous thinning of the bone trabeculae and widening of the inter-trabecular spaces. These morphological changes are typical of glucocorticoid-induced osteoporosis and are consistent with the observations reported by \u003cb\u003eZhang et al. (2020)\u003c/b\u003e in ovariectomized rats \u003cb\u003e[71]\u003c/b\u003e. Glucocorticoids exert direct deleterious effects on bone cells by inhibiting osteoblast differentiation and proliferation, while promoting their apoptosis via the activation of pro-apoptotic pathways (Bim, Bak, p53) and the inhibition of the Wnt/β-catenin pathway, which is essential for osteogenesis \u003cb\u003e[72].\u003c/b\u003e At the same time, they induce osteocyte apoptosis, compromising mechano-sensitivity and bone micro-architecture stability, while prolonging osteoclast survival through an increase in the RANKL/OPG ration, thereby intensifying bone resorption. Systemically, glucocorticoids reduce intestinal calcium absorption, increase renal excretion, and disrupt hormone regulation (vitamine D\u003csub\u003e3\u003c/sub\u003e, PTH, testosterone), aggravating bone demineralization and promoting skeletal fragility \u003cb\u003e[72].\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn osteoporotic rats treated with calcium supplements derived from eggshells, namely calcium acetate and nano lactate, histological bone sections showed active remineralization and improved tissue cohesion. The increase in the number of osteocytes and osteoblasts indicates stimulation of bone remodeling and reactivation of bone formation \u003cb\u003e[73]\u003c/b\u003e. The bioavailability of calcium has a decisive influence on bone mineralization processes and osteogenic cell activity. The most easily absorbed forms of calcium promote osteoblastic differentiation and contribute to increased bone mineral density [\u003cb\u003e74].\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCalcium ions play a fundamental role in regulating the cellular processes involved in bone formation and regeneration. Mainly present in the form of calcium phosphates in the bone matrix, they actively participate in the calcification and maturation of bone tissue. In addition, calcium is involved in bone regeneration through various cell signaling mechanisms, stimulating mature bone cells by producing nitric oxide, inducing the differentiation of precursor cells into osteoblasts, and activating the ERK1/2 and PI3K/Akt pathways, which are involved in bone synthesis and osteoblast survival, respectively. Finally, calcium ions modulate the functional cycle of osteoblasts by Influencing both their formation and resorptive activity \u003cb\u003e[75]\u003c/b\u003e. All of these mechanisms support the correlation observed between calcium bioavailability and improvement in bone histological and mineral parameters. These results confirm the biochemical and mineral data, suggesting a direct correlation between calcium bioavailability and bone regeneration.\u003c/p\u003e \u003cp\u003eConversely, low doses (20 mg/kg) of ESP and NCal induced only a partial improvement in bone micro-architecture, with trabeculae remaining thin, discontinuous, and poorly organized. These results highlight a marked dose-response relationship, indicating that even in the presence of a highly bioavailable source of calcium, insufficient calcium intake remains unable to compensate for the mineral loss induced by glucocorticoid treatment, particularly in pathological contexts characterized by accelerated bone remodeling or bone aging \u003cb\u003e[74]\u003c/b\u003e.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, this study highlights the potential of eggshells and their derivatives as effective natural sources of calcium in the prevention of glucocoticoid-induced osteoporosis. Eggshell powder, particularly at high doses, proved to be the most effective in improving biochemical markers, mineralization, and bone micro-architecture. Nano-lactate and calcium acetate showed dose-dependent efficacy, but less than that of eggshell powder. These results position the use of eggshells as a sustainable and promising alternative to conventional calcium supplements in the management of secondary osteoporosis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are provided within the manuscript\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003cbr\u003eThe authors are grateful to the Ministry of Higher Education and Scientific Research and to University Abdelhamid Ibn Badis, Algeria, for supporting this research work within the framework of the Research Project (PRFU), Code: D00L01UN270120220002, entitled “Biovalorization and evaluation of the nutraceutical and therapeutic power of natural bioactive molecules.”\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;This research was supported by the Ministry of Higher Education and Scientific Research, Algeria\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical note:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were complied with the Algerian legislation (Law number 95-322/1995) inherent to protect animals designed for experiments or other scientific purposes, also the guidelines of the Algerian Association of Experimental Animal Sciences (AASEA approved under the agreement number 45/DGLPAG/DVA/SDA/14).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.C. -\u0026nbsp;\u003cem\u003eConceptualization, Writing - original draft, Visualization\u003c/em\u003e; S.K.-\u0026nbsp;\u003cem\u003eSupervision, Resources, Writing - review \u0026amp; editing\u003c/em\u003e; A.L.- \u003cem\u003eMethodology, Software, Writing - review \u0026amp; editing;\u0026nbsp;\u003c/em\u003e; M.M.-\u0026nbsp;\u003cem\u003eValidation, Writing - review \u0026amp; editing\u003c/em\u003e; Y.M.B - Conceptualization, Supervision ; D.H - \u003cem\u003eMethodology, Writing - original draft, Writing - review \u0026amp; editing\u003c/em\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;; A.B.- \u003cem\u003eInvestigation, Writing - review \u0026amp; editing, Formal analysis\u003c/em\u003e. All authors reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eJuliet, E., \u0026amp;Compston, M. (2019). R. \u0026amp; McClung, WDL Ost\u0026eacute;oporose. Lancet , 393 , 364-376.\u003c/li\u003e\n \u003cli\u003eSong, Y., Zhang, F., Guo, J., Fan, Y., Zeng, H., Sun, M., ...\u0026amp;Ge, G. (2025). High-efficient discovering the potent anti-Notum agents from herbal medicines for combating glucocorticoid-induced osteoporosis. ActaPharmaceuticaSinica B.\u003c/li\u003e\n \u003cli\u003eBaraka, N. A. F., Ahmed, N. F., \u0026amp; Hussein, S. I. (2022). The effect of Rutin hydrate on Glucocorticoids induced osteoporosis in mandibular alveolar bone in Albino rats (Radiological, histological and histochemical study). The Saudi Dental Journal, 34(6), 464-472.\u003c/li\u003e\n \u003cli\u003eJia, B., Fei, C., Hao, D., Qiao, F., \u0026amp; Hu, H. (2022). 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Les d\u0026eacute;chets de coquilles d\u0026apos;\u0026oelig;ufs comme ressource durable pour la pr\u0026eacute;paration de nanoparticules ; synth\u0026egrave;se, caract\u0026eacute;risation et applications. \u003cem\u003eEnvironmental Nanotechnology, Monitoring \u0026amp; Management\u003c/em\u003e , \u003cem\u003e24\u003c/em\u003e , 101092.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"eggshell, glucocorticoids, osteoporosis, calcium acetate, calcium nano-lactate","lastPublishedDoi":"10.21203/rs.3.rs-9012106/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9012106/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGlucocorticoid-induced osteoporosis (GIO) is a common complication of prolonged steroid treatment, leading to impaired mineral metabolism and bone micro-architecture. This study evaluates the protective effects of eggshell powder (EPS) and its derivatives, particularly calcium acetate (CA) and nano-calcium lactate (NCal), in a model of methylprednisolone-induced osteoporosis in Wistar rats.\u003c/p\u003e\n\u003cp\u003eThe eggshells, collected locally, were characterized chemically and morphologically, as were their CA and NCal derivatives, which were obtained respectively by chemical transformation of the powder with acetic acid and by precipitation from calcium oxide and lactic acid, then characterized by FTIR an XRD, revealing the presence of characteristic functional groups \u0026nbsp;(O-H,C=O,C-O,CO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-2 \u003c/sup\u003e) and typical crystalline structure.\u003c/p\u003e\n\u003cp\u003eThe in vivo study was conducted on ten groups of female rats, including a negative control, a positive control, and eight groups treated with ESP, CA, NCal and CaCO\u003csub\u003e3 \u003c/sub\u003eat doses of 20 and 40 mg/kg. Biochemical parameters (calcium, phosphorus, and alkaline phosphatase ), femoral ash content, and histological analysis of the femur were evaluated.\u003c/p\u003e\n\u003cp\u003eThe results indicated a significant improvement in mineral metabolism in the treated groups, characterized by an increase in serum calcium and phosphorus concentrations, a reduction in alkaline phosphatase activity, and femoral remineralization confirmed by elevated calcium, phosphorus, and magnesium levels in femoral ash.\u003c/p\u003e\n\u003cp\u003eHistological analysis revealed partial to complete restoration of trabecular bone structure with thick, well-organized trabeculae rich in osteocytes, contrasting with the bone degradation observed in untreated osteoporotic rats.\u003c/p\u003e\n\u003cp\u003eIn conclusion, eggshell powder and its derivatives, particularly calcium acetate and calcium nano-lactate, show great potential as a natural source of calcium for the prevention and treatment of glucocorticoid-induced osteoporosis.\u003c/p\u003e","manuscriptTitle":"Protective Effects of Eggshell Powder and Eggshell-Derived Calcium Compounds on Glucocorticoid-Induced Osteoporosis in Albinos Wistar Rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-24 14:44:27","doi":"10.21203/rs.3.rs-9012106/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"525f58c3-dfd9-41e6-8a3e-d26aa0cddeaa","owner":[],"postedDate":"March 24th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Reject","date":"2026-05-14T11:40:54+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-14T15:42:59+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-24 14:44:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9012106","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9012106","identity":"rs-9012106","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}