JAHP_13_4_1055-1066

Introduction

Osteoporosis is a prevalent bone disorder associated with compromised skeletal biomechanics (Bansley et al., 2021). However, physiological imbalances in bone cells result in an imbalance between bone resorption and formation, leading to weakened bone microarchitecture, reduced strength, and an increased risk of cortical fractures (Chin et al., 2022). Osteoporosis is the leading cause of approximately one-third of fractures in developed countries, placing a substantial financial burden on healthcare systems (Chandran et al., 2023; Chin et al., 2022). Various animal models have been used to study osteoporosis, with the rat model being the most commonly chosen. This model can be induced through methods such as ovariectomy (OVX), orchidectomy (OCT), hypophysectomy, parathyroidectomy, immobilization, or dietary manipulation (Li et al., 2023; Saleh et al., 2020; Yousefzadeh et al., 2020). Of these, ovariectomy is considered the most reliable method for inducing osteoporosis that closely resembles the human condition (Li et al., 2023; Saleh et al., 2020; Yousefzadeh et al., 2020). Bone remodeling and modeling are progressive processes involving bone resorption and formation over an extended period (Chen et al., 2024; Mertiya et al., 2023). Calcium, the main constituent of hydroxyapatite minerals, is crucial for providing mechanical strength to the skeletal system (Osuchukwu et al., 2021). However, when calcium levels are imbalanced, the body’s bone reserves are mobilized through the stimulation of parathyroid hormone (PTH) release, which triggers bone resorption and remodeling (Langdahl et al., 2016). To counteract the bone loss in pre- and post-menopausal women, calcium supplements are often prescribed to prevent fractures and bone damage. However, recent studies indicate that calcium supplements have limited efficacy and can lead to cardiomyopathic effects (Park et al., 2022).

Additionally, commercially available calcite supplements, an inorganic polymorph of calcium carbonate, can have negative side effects in the body (Jin et al., 2024). This has led to the search for alternative biogenic sources of calcium carbonate polymorphs to replace calcite in osteoporosis management (Jeong et al., 2022; Park et al., 2022). Advances in technology have introduced cockle shell-derived calcium carbonate nanoparticles, a biocompatible, biodegradable, osteoconductive aragonite polymorph that is unstable in its nano-form and plays a key role as an enzyme activator in muscular contraction and bone tissue repair (Hammadi et al., 2017; Muhammad et al., 2019). Calcium carbonate exists in three polymorphic forms, with calcite being the most stable. Several studies have shown the efficacy of cockle shell-derived calcium carbonate in promoting osteogenic effects and forming scaffolds for bone repair (Danmaigoro et al., 2017; Muhammad et al., 2019). These findings have sparked interest in using aragonite as an alternative to calcite for osteoporosis prevention and bone strength restoration. To investigate the osteo-restorative potential of aragonite, this study aimed to use micron and nano-sized calcium carbonate derived from cockle shells to repair bone microstructural damage in type 1 osteoporotic rat models. Estrogen levels typically decrease after menopause, resulting in a 30% increase in bone resorption that outpaces bone formation (Noirrit-Esclassan et al., 2021). One effective way to enhance bone strength is through mineral deposition, which increases cortical thickness and helps resist pathological lesions. However, the role of cortical thickness in the pathophysiology of osteoporosis and the osteo-restorative effects of aragonite calcium carbonate nanoparticles (CAN) in osteoporosis remain poorly understood.

Materials and Methods

Synthesis and characterizations of aragonite cockle-shell derived calcium carbonate micro, nanoparticle and calcite calcium carbonate nanoparticles

The preparation and synthesis of macro, micro, and nanoparticles of calcium carbonate from cockleshells was carried out following a modified method from Danmaigoro et al. (2017). Initially, the washed cockle shells were oven-dried at 50 °C for 7 days using an oven (Merment UM 5000, Germany). After drying, the cockle shells were ground into particles using a pulverizing blending system (RT- 08 rpm 2500, Japan), and fine particles were separated by sieving through a steel sieve with a pore size of 75-90 μm (Endicott Ltd., England). The resulting macro- and micro-sized cockleshell-derived calcium carbonate (CS-CaCO3) was stored in a warm oven at 50 °C.

For precipitation, the stored products were treated using a top-bottom approach on a hotplate stirring machine (6 Positions, Systematic Multi-Hotplate Stirrers, Korea). The resulting solution was filtered through filter paper with a pore size of 18.0 cm (Smith, Malaysia). The sediment was then dried in the hot oven at 50 °C for 5 days. Finally, the dried calcium carbonate particles were processed further by rolling them on a programmable roll miller (BML-6” Korea) to obtain nanosized particles. The synthesized aragonite cockle-derived calcium carbonate nanoparticles (CAN) were stored in sealed bottles and kept in a dry oven at 50 °C.

The characterization was performed following the guidelines established by the Nanotechnology Characterization Laboratory (NCL), as previously reported by Danmaigoro et al. (2017). The particle size, surface morphology, Zeta size, Charge, and dispersity of macron, micron, and nano calcite and cockle shell-derived material were determined using High Resolution Transmission Electron Microscope (JOEL JEM-2100F, Japan). The surface morphology was further evaluated using Field Emission Scanning Microscopy (FESEM) (JOEL 7600F, Germany). Additionally, the particle in aqueous solution surface charge and polydispersity index were assessed using a Zeta Sizer Nano ZS (Malvern, England).

Sample size of animal model

The animal sample size was estimated based on previous studies by Çamili et al. (2022) and using SigmaPlot software (SigmaPlot V.12.0, United States). The sample power was calculated with an alpha error of 0.05 and 90% power to detect significant differences across the six groups, with a minimum of 6 rats per treatment group. To account for potential dropouts due to death or other exclusion criteria during the nephrectomy procedure, 5 additional rats were included in each group.

Experimental animals and feeding conditions

This study was conducted under the guidelines and approval of the Institutional Animal Care and Use Committee (AUP-UDUS-2022-023), of Usmanu Danfodiyo University, Sokoto, at the university’s animal house facility. All experiments adhered to national and international animal care protocols and complied with the ARRIVE guidelines. Sixty (60) healthy male and female Wistar rats, approximately 8 weeks old and weighing 220 ± 5 g, were obtained from Usmanu Danfodiyo University, Sokoto Animal Breeding Unit. The rats were housed in a group of 4-5 per cage and kept in the laboratory environment with controlled temperature (24–27 °C), and humidity (50-60%). They were fed a standard rat chow diet and provided with tap water ad libitum. The rats were acclimatized for two weeks before the start of the experimental period.

Surgical Induction of osteoporosis in rats

The induction of the type I osteoporotic model was performed by exposing the rats to bilateral ovariectomy (OVX) and orchidectomy (OCT), following the standard procedure outlined by Ren et al. (2016). After anesthetizing the rats, an abdominal incision was made along the wall in female rats and the pre-scrotal incision in male rats. The ovaries were located, and the ovarian arteries were ligated with absorbable sutures. The ovaries were then carefully excised, and the sutures were used to close the muscles (with non-absorbable sutures) and the skin (with non-absorbable sutures). Simultaneously, OCT was conducted following the procedure described by Sophocleous and Idris (2014).

Animal experimentation

A randomized control trial (RCT) experimental design was employed, with rats selected and grouped based on sex. Eight (8) weeks post-surgery, 50% bone resorption was achieved and confirmed radiographically as evidence of osteoporosis. The rat models were then randomly assigned to one of the six groups. Group 1 - 4 consisted of OCT and OVX rats each treated with either Micron calcite calcium carbonate (CCM) (n=10), Nano calcite calcium carbonate (CCN) (n=10), Nano aragonite calcium carbonate (CAN) (n=10), or Micron aragonite calcium carbonate (CAM) (n=10) each receiving 1 mL (5 mg/mL). Group 5, the Sham group (negative control), underwent Laparotomy without any organ removal (n=10), while Group 6, the intact rate (Positive Control) (n=10) were treated with 1 mL of normal saline orally every day for eight weeks (Figure 1).

Confirmation of osteoporosis and bone defect assessment

The osteoporotic rat models were confirmed through craniocaudal X-ray imaging of the femur and tibia, using a Stationary Siemens X-ray machine (Ma, 500, India) set at 50kVp, 6.2 mAs, and 50 ms exposure. Radiographs were assessed for cortical thickness and bone remodeling both before and after the treatment. All radiological evaluations were conducted by independent observers who were blinded to the study groups.

Preparation of bones for morphometry

The rats were euthanized at 10th weeks post–induction of osteoporosis using an overdose of Nembutal (200 mg/kg) (Euthatal, England). The femurs were carefully dissected, with all surrounding muscles removed. Various measurements were taken, including the weight, femoral length (measured parallel to the femoral diaphysis between the femoral head and distal extremities) using digital callipers, and proximal and distal epiphyseal width of the left femur. These measurements were recorded in millimeters (to the nearest 0.01 mm), and the weight was measured in grams (g). The specific femoral bone traits measured included: femoral length (FL) from the superior (articular) surface of the femoral head to the distal femoral condyles, proximal epiphyseal width (PEW) from the greater trochanter to the medial surface of the femoral head and distal epiphyseal width (DEW), which is the mediolateral distance across the distal femoral extremities. Additionally, the bone mineral density (BMD) of the left femur was calculated by comparing the mass of the bone in air with its weight when submerged in a specific volume of distilled water according to the formula adopted from Sheu and Diamond (2016).

D= Density, W1= Weight in air, W2= Weight in water, P= Density of distilled water at a given temperature (g/cm3).

Haematology, serum biochemical, and proteomic bone turnover evaluation

Whole blood and serum samples were collected from the animal model upon confirmation of osteoporosis and after the experiment. Blood was drawn into EDTA tubes for hematology analysis and plain tubes for serum biochemistry and proteomic bone turnover matrix analysis. Blood cell counts were determined using a Haemoanalyser (Auto-analyser Sysmex KX-21N). The serum samples from the plain tubes were centrifuged at 3000 rpm for 10 minutes, then stored at -20 oC for biochemical analysis. For proteomic bone turnover biomarkers, the samples were stored at -80 oC.

Biochemical parameters were evaluated using commercially available immunoassay kits. These included Blood Urea Nitrogen (BUN), Creatinine (Crea), total cholesterol (TC), and electrolytes such as Calcium (Ca), Phosphorus (P), sodium (Na+), Potassium (K+) and Chloride (Cl-) and ALP activity, using dimension Xpand plus integrated chemistry system (Dimension® EXL™ 200, Siemens, Germany). Bone resorption [Rat Bone Serum Carboxyl Terminal Collagen 1 51 Cross Link (CTX-1)] and formation [Rat Bone Serum Procollagen Type 1 N-Terminal Protein (P1NP)] markers were assessed using Elabscience® Rat C-telopeptide of Type I Collagen (CTX-I) and Rat Procollagen Type N-Terminal Peptide (PINP) ELISA Kits. The competitive inhibition enzyme immunoassay technique was employed, and protein concentrations were quantified using a spectrophotometer at a wavelength of 450 nm.

Histopathological evaluation of the femoral bone

Two weeks post-treatment, the models were euthanized and longitudinal sections of the femoral bone were preserved in 10% formalin (NBF-Neutral buffered formalin). The femoral bone samples were decalcified by incubation in a decalcifying agent (Nitric acid 5%, Sigma-Aldrich) with continued stirring for 7 days. Decalcification was confirmed by puncturing the samples with a needle to assess resistance.

The samples were then processed using the paraffin-embedded method. The blocks were sectioned at 5 µm and placed on coated slide glasses. The tissue sections were stained using the standard HandE (H and E Abcam, USA) (Sheu and Diamond, 2016). The stained slides were examined using a Leica microscope (DM4M, USA) and captured with a Moticam pro-282A 10.0MP.

Statistical analysis

All data were presented as Mean ± Standard deviation (SD). Two-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison post-hoc test was used to assess the difference between the experimental treatment groups and sexes, A significance level of 95% (P<0.05) was applied. Statistical analyses were conducted using GraphPad Prism 9 Software (GraphPad, USA).

Results

Physicochemical properties of the macro, micro nano-calcite, and cockle shell-derived calcium carbonate

Transmission electron microscopy (TEM) analysis of both CCM and CAM particles showed average diameter size of 202 ± 52 nm and 140 ± 39 nm respectively. In contrast, the CCN and CAN measure 83 ± 23 nm and 32 ± 08 nm respectively (Figure 2a, b, c, d). Field emission microscopy (FESEM) reveals that CAN were spherical with a rough, porous surface while CCN exhibited sharp edges with a hexagonal surface shape with a roughened texture on both micro and nanosize form (Figure 2e, f, g, h).

In aqueous colloids, the zeta size for CCM and CAM particles were 339.2 ± 43 nm and 242 ± 20 nm and 345 ± 34 and 123 ± 23 nm for (CCN and CAN) nano particles respectively (Figure 2i, j, k, l). The Zeta potential values were 13.1 ± 2.2 mV for CCN and -3.0 ± 0.2 mV for CAN (Figure 2m, n, o, p) below. The polydispersity index (PDI) for CCM and CCN were 0.45 and 0.22, respectively, while the PDI for CAM and CAN were 0.41 and 0.14 respectively (Figure 2q).

Osteoporosis radiographic assessment

The OVX and OCT-induced osteoporotic models exhibited osteolytic cortical thinning with slight alterations in the trabecular pattern as evidence by radiolucency in the cortical diaphysis of femoral and tibial bones, 8-weeks post induction. These was in contrast to the control rat model, which display a radio-dense cortical diaphysis. Notably, significant restoration in bone remodeling was observed following treatment with CAN, where the cortical diaphyseal regions of the femur and tibia appeared radiopaque. This restoration was evident in both ovariectomised and orchidectomised osteoporotic rat models, as shown Figures 3 and 4.

Bone morphometry

Bone mineral density (BMD) of osteoporotic rats’ models treated with CCN, CCM, CAM, CAN, and control group showed significant difference with the treatment groups and across sexes (p<0.05). Specifically, rats treated with CCN and CAN exhibited increased femoral BMD compared with the controls group in the orchiectomized rats as shown in Figure 3a. with increased femoral BMD as compared to the control group as shown in Figure 5a. Additionally, the average of femoral lengths, proximal epiphyseal width, and distal epiphyseal width of the osteoporotic rat models treated with CCN, CCM, CAM, CAN, differed significantly (p<0.05) when compared to positive controls. These differences in the mean morphometric variables were observed across treatment groups and sexes, as demonstrated in Figure 5b, c, d.

Bone biomarkers

Bone formation (P1NP), bone resorption biomarkers (CTX-1) and alkaline phosphatase enzymes biomarkers: The average of bone formation biomarkers in the blood circulation of the osteoporotic rats model treated with CCN, CCM, CAM, CAN, and controls reveals various levels of significant difference between the treatment group (p<0.05) compared to the control groups, in addition to the interactive effect between treatment and sex group (p <0.05) as shown in the Figure 6. Particularly, the differences were notable between the groups administered with CCN and CAN, compared to the control groups. The mean bone resorption biomarkers of osteoporotic rats treated with CCN, CCM, CAM, CAN, controls, and SHAM show no significant difference between the treatment groups and with sex as well (p>0.05) as shown in Figure 6. The average non-specific bone resorption biomarker serum ALP of the osteoporotic rats treated with CCM, CAM and controls significantly differs between the treatment groups (p< 0.05) and the sex is shown in the table below. The rat models treated with CAN and CCN exhibited lower serum alkaline phosphatase (ALP) levels; the differences were not statistically significant (p> 0.05) (Table 1).

Hematology and serum protein level

The average PCV, RBC, and WBC of the different groups of osteoporotic rats treated with CCN, CCM, CAM, CAN, and controls showed no significant difference between the treatment groups and sex (p > 0.05), as shown in Table 2.

Table 1: Serum Biomarkers of osteoporotic rats induced osteoporotic rats’ model treated with Nano calcite calcium carbonate (CCN), Micron calcite calcium carbonate (CCM); Micro aragonite calcium carbonate (CAM); Nano aragonite calcium carbonate (CAN), and controls.

Parameters	Sex	+ve Control	CCN	CCM	CAM	CAN	SHAM
ALP (U/L)	M	320.2 ± 61a	210 ± 41b	192 ± 32 a,c	220 ± 24 a,d	205±29 e	214 ± 26f
	F	328 ± 71a	215 ± 38b	199± 39 a,c	229± 37 a,d	200 ± 24 a,b	234 19.3 a,b

Treatment group with the same superscript alphabet means no significant difference (p>0.05) while treatment group with different superscript alphabet means that they differ significantly (p<0.05), ALP: Alkaline Phosphatase, M: Male, F: Female, CCN: Nano calcite calcium carbonate; CCM: Micron calcite calcium carbonate; CAM: Micro aragonite calcium carbonate; CAN: Nano aragonite calcium carbonate; +ve Control: Positive group Model; SHAM: Negative control Model.

Table 2: Haematology and serum protein level of osteoporotic rats induce osteoporotic rats’ model treated with nano calcite calcium carbonate (CCN), micron calcite calcium carbonate (CCM); micro aragonite calcium carbonate (CAM); nano aragonite calcium carbonate (CAN), and controls.

Parameters	Sex	+ve Control	CCN	CCM	CAM	CAN	SHAM
PCV (%)	M	50.0±9.0	47.0±7.0	58.5±12.0	54.9 ±9.69	49.2±12.1	53.1± 10.20
	F	48.0± 8.0	47.4±9.0	52.5±10.0	50.9 ±3.79	56.2 ±11.1	53.1±11.20
RBC (x 1012 /L)	M	6.40±0.61	6.23±0.34	6.66 ± 0.4	6.68 ± 0.45	6.10 ± 0.61	7.15 0.67
	F	6.60±0.31	6.33±0.3	6.16±0.33	6.68 ±0.45	6.23±0.61	7.15 0.67
WBC (x 109 /L)	M	12.01±3.43	10.93±2.35	14.88±3.7	12.88±3.76	13.01±3.43	11.78±2.93
	F	11.03± 2.43	12.53±2.55	13.88±3.2	15.28±3.26	13.21±3.13	10.48±2.13
Creatinine (mg/dL)	M	2.08 ± 0.32	2.08 ± 0.32	1.80 ± 0.35	1.62 ± 0.33	1.26±0.21	1.52 ± 0.08
	F	2.30 ± 0.16	2.30 ± 0.16	2.02 ± 0.19	0.74 ± 0.05	0.82±0.08	1.50 ± 0.07
Total Protein (g/dL)	M	6.5.60±1.14	7.2.40±2.85	7.4.6±2.04	6.5.8±2.35	6.8.6±1.14	6.9.00±1.83
	F	6.20±1.48	7.4.00±4.95	7.5.8±14.5	6.5.8±1.28	6.5.2±1.48	6.30±2.05
Total Bilirubin (mg/dL)	M	0.58±0.08	0.64±0.11	0.7±0.16	0.8 ± 0.21	0.68±0.08	0.72±0.08
	F	0.70±21	0.72±0.08	0.71 0.16	0.82 ± 0.21	0.69±0.08	0.82±0.07
Albumin (g/dL)	M	3.90±1.24	3.80±0..94	3.90±1.04	3.701.08	3.90±1.24	3.70±0.94
	F	3.80±1.14	4.00±1.91	4.14±1.22	3.80±1.11	3.76±1.82	3.80±1.14
Urea (mg/dL)	M	39.0±2.24	38.48±3.67	36.58±4.79	38.06±3.76	39.1±3.09	37.66±2.24
	F	39.0±4.24	37.28±4.20	38.58±3.79	38.16±3.76	37.10 ±3.09	40.66±1.09

PCV: Packed cell Volume; RBC: Red blood Cells; WBC: White blood Cells; M: Male, F: Female, CCN: Nano calcite calcium carbonate; CCM: Micron calcite calcium carbonate; CAM: Micro aragonite calcium carbonate; CAN: Nano aragonite calcium carbonate; +ve Control: Positive group Model; SHAM: Negative control Model.

Table 3: Electrolyte level in osteoporotic rats induced osteoporotic rats’ model treated with nano calcite calcium carbonate (CCN), micron calcite calcium carbonate (CCM); micro aragonite calcium carbonate (CAM); nano aragonite calcium carbonate (CAN), and controls.

		+ve Control	CCN	CCM	CAM	CAN	SHAM
Calcium (mg/dL)	M	8.59 ± 2.22a	11.03± 0.26ab	9.06 ± 0.21	10.24±0.21	11.54±0.21ac	11.06±0.41
	F	8.40± 1.16	10.18± 1.29	9.10± 1.16	10.08±0.19	12.14±0.39	11.7±0.36
Potassium (mmol/L)	M	4.12±0.26	5.04± 0.21	6.88±0.37	6.10±0.46	6.92 ± 0.5	5.88±0.37
	F	4.32±0.25	5.00± 0.22	5.98±0.15	6.88±0.50	6.04± 0.52	5.98±0.15
Sodium (mmol/L)	M	148.80±4.30	142.00± 5.22	139.80±8.59	141.00±1.00	142.00±9.41	138.80±2.59
	F	145.60±6.82	140.10± 4.00	138.20±9.64	139.60±0.89	145.00±8.00	146.20±1.64
Bicarbonate (mmol/L)	M	24.00±4.47	22.40± 2.07	22.00±1.58	24.40±2.70	20.00±2.92	23.00±4.58
	F	23.80±5.80	21.40± 2.07	24.40±3.14	25.40±2.14	22.80±1.30	24.40±3.14

Treatment group with the same superscript alphabet means no significant difference (p>0.05) while treatment group with different superscript alphabet means that they differ significantly (p<0.05), M: Male, F: Female CCN: Nano calcite calcium carbonate; CCM: Micron calcite calcium carbonate; CAM: Micro aragonite calcium carbonate; CAN: Nano aragonite calcium carbonate; +ve Control: Positive group Model; SHAM: Negative control Model.

Additionally, the mean difference in serum protein levels such as creatinine, total protein, total bilirubin, albumin, and urea were not statistically significant between the treatment groups nor in sex, as shown in Table 2.

Serum electrolyte level

The mean serum calcium deposits were slightly elevated in the models treated with CAN, CAM, and CCN compared to the other treated rat models. The average serum electrolyte (calcium) level of osteoporotic rats treated with CCN and CAN, compared to the control reveals a significant difference between the treatment groups and sex (p<0.05), with no statistically significant (p> 0.05), variation in the level of potassium, sodium, bicarbonate among the treatment group and sex as shown in Table 3.

Histopathological assessment

The histological features demonstrated were similar to those observed on the radiographic findings. The representative photomicrograph of the histological section depicted in Figure 7 of the control rat model reveals that the trabecular bone matrix with an osteopenic appearance (Figure 7a). The study reveals a high intertrabecular spacing in the OCT and OVX control models compared to the SHAM rat model. The trabecular bone structure and intertrabecular cavity in the rat model treated with CAN and CCN were identical to the SHAM rats model, with distinct cortical thickening in the model treated with CAN, as when compared to the control group, and proportionate to the model treated with CCM and CAM that present a discrete space in the trabecular cavity and intertrabecular septum due to cellular and mineral deposits (Figure 7b, d). The SHAM rat model reveals a similar less dense trabecular network with the rat model treated with CAM, CCM, and CCN, compared to a dense trabecular network and intertrabecular cavity of the rat model treated with CAN (Figure 7b, c, d). A slight cortical thickening was observed in the rat model treated with CAN and CCN compared to the CAM, CCM, and control OCT and OVX models. The micrographs of the CAN and CCN reveal their spongy trabeculae as compared to the eroded trabeculae on the rat model treated with CCM, CAM, and controls (Figure 7a, b, c, d, e). The control and model administered with CCN and CCM show resorption of bone with dispersed trabeculae, fragmented bone parenchyma, and a smaller number of osteocytes and osteogenic activities, indicating osteopenia (Figure 7b, c, d, e). However, there is improvement in the morphological architecture of the bone matrix of rat models treated with CAN, CAM, and CCN, with more osteocyte lacunae and thickening of the cortical region of the femur (Figure 7b, c, e).

Discussion

Osteoporosis is characterized by an imbalance in bone metabolism, leading to decreased bone mass and thinning of the trabecular bone. This results in increased bone resorption, a deterioration to cortical bone microstructure, and heightened bone fragility (Wang et al., 2022). it is a systemic condition that predominantly affects women after menopause due to impaired ovarian function and a decline in hormone production (Thapa et al., 2022). Animal models, such as bilateral ovariectomy and orchidectomy in rats are commonly used in research because they replicate the histological changes observed in osteoporosis (Yousefzadeh et al., 2020). These models are particularly valuable for studying the effects and mechanisms of drug-induced osteorestorative treatments, as the changes in bone microstructure resemble human processes, making them a relevant model for postmenopausal osteoporosis. In comparison to ovariectomized rats, orchidectomised rats show a more modest bone mass loss, which aligns with the slower rate of bone loss seen in males, as they generally respond less to sex hormone deficiency than females, even within the same age group. Radiography is a fundamental tool for diagnosing osteoporosis, highlighting cortical thinning in affected bones, and bone mineral density (BMD) is used to assess bone strength. However, a deeper understanding of bone density through histomorphology and immunohistochemistry is crucial for more comprehensive insight into the disease (Dudakovic et al., 2022; Payne et al., 2011). Micro-computed tomography (µCT) offers high-resolution images of bone and trabecular structures, but its high cost limits its widespread use in many studies (Payne et al., 2011). Currently, no single technique can fully assess bone quality; instead, a combination of methods, including bone mineral density (BMD) and micromorphological evaluations, is employed (Payne et al., 2011), As a result, this efficacy study utilised a combination of radiography, BMD, and micromorphology techniques and a more comprehensive analysis.

The results suggest that the administration of CCN and CAN has played a role in the osteo-restorative and regenerative processes, as radiographs clearly demonstrate both the health status of the bones and the impact of therapeutic interventions. Peripheral quantitative assessment of bone structural morphology provides valuable insights into osteoporosis in the rat model (Wang et al., 2022). Bone mineral density (BMD) is a direct method for assessing bone structure and defects, offering detailed information about the geometry of the bone extremities. This contrasts with other techniques that require expensive machinery for bone structural analysis. In this study, both orchidectomy and ovariectomy-induced osteoporosis led to a decrease in bone mass in the femur, which is evidenced by reduced cortical thickness in the femoral shaft as shown in the radiographs. These changes align with the understanding that bone modeling and remodeling affect bone size, shape, internal structure, mass density, and mechanical strength. The observed decrease in bone mass due to orchidectomy and ovariectomy has been well-documented in several studies (Ryu et al., 2015; Wang et al., 2022).

Bone mineral density (BMD) serves as a surrogate measure for bone strength and is a key contributor to bone quality (Wang et al., 2022). In cases of bone loss, the trabeculae become more pronounced, making BMD a crucial indicator for assessing bone biomechanical strength. In this study, a more significant increase in bone mineral density was observed in the model groups treated with CAN, CAM, and CCN, demonstrating improved biomechanical properties compared to the control group rats. While BMD is commonly used to assess bone diseases, it has limitations in clinical settings, particularly due to the slow response of bone tissue to changes in cortical and trabecular structure (Schini et al., 2023). Biomarkers of bone resorption and formation are more sensitive and specific, offering valuable insights into bone remodeling dynamics (Schini et al., 2023). These biomarkers provide information on increased bone turnover metabolites, which is essential for understanding both aging and pathological bone conditions. The results of bone turnover biomarkers in this study align with the observed microstructural changes in the bone. Furthermore, the findings regarding bone resorption and formation biomarkers are consistent with research by Schini et al. (2023) and Shetty et al. (2016), particularly among women undergoing daily teriparatide therapy. These biomarkers help monitor ongoing bone remodeling and assess the pharmacodynamics of osteoporosis treatments, which are essential for optimizing calcium supplementation and tracking patient compliance. CTX-1, a well-established biochemical marker for bone resorption, continues to hold clinical relevance. In many studies, oral calcium supplementation has been shown to mitigate increased bone turnover, making CTX-1 a reliable predictor of bone resorption in postmenopausal women (Ivaska et al., 2022; Schini et al., 2023; Shetty et al., 2016). The reduced levels of CTX-1 in rats treated with CAN and CCN, compared to the control and CCM-treated rats, indicate a decrease in the degradation of type I collagen in the bone. This suggests a reduction in bone resorption, which, if unchecked, leads to increased bone fragility in osteoporosis.

Moreover, the NIP-1 biomarker levels observed in all rat models in this study support its potential as an indicator of bone health status in osteoporosis, particularly in relation to the inflammatory processes involved in bone remodeling. These findings reinforce the emerging relevance of NIP-1 as a marker for neuroinflammation and bone status, particularly in evaluating the osteorestorative effects of the various supplements used in this study (Ashcherkin et al., 2023).

The observed changes in electrolytes and minerals highlight the dynamic processes of bone mineralization and development, particularly during bone formation and resorption in the periosteal and cortical regions (Ashcherkin et al., 2023). Calcium salt deposition during bone development is influenced by nutritional status, as well as immunological and biochemical factors (Ashcherkin et al., 2023). Alkaline phosphatase (ALP) plays a key role in bone metabolism, osteoblast differentiation, and osteogenic activity. Despite the administration of CAM and CCM, the rat models still showed a lower modulating effect on bone metabolism, offering insights into the progression of bone metabolism in older individuals. ALP is a component of many cell membranes, with osteocytes exhibiting higher levels of the bone-specific isoenzyme (Chen et al., 2021; Cheng and Zhong, 2023). ALP levels increase in response to osteoblastic activity and bone remodeling (Su et al., 2023), with multiple studies reporting elevated ALP in rat models of induced osteoporosis (LeBoff et al., 2022; Ryu et al., 2015; Shetty et al., 2016; Su et al., 2023).

The reduction in serum ALP levels in the rat models treated with CAN and CCN, compared to those treated with CCM, CAM, or the control, indicates a decline in osteoblastic activity and bone turnover, consistent with findings in earlier research (Chen et al., 2021; Cheng and Zhong, 2023; Shu et al., 2022). This decline also correlates with the observed decrease in bone mineral density (BMD) in the CCM, CAM, and control groups. Given that alkaline phosphatase is an essential cofactor in bone mineralization, its synthesis, and a by-product of bone remodeling, it serves as a critical marker for bone formation and is widely used to evaluate the effectiveness of osteoporosis therapies.

Bone morphology in the rat models treated with CAN, CAM, and CCN showed increased bone thickness, greater trabecular spacing, and enhanced distribution of osteocytes, along with alterations in the medullary cavity, likely due to their higher mineral content. These structural changes serve as critical indicators for evaluating therapeutic interventions. The increased periosteal thickening observed in the femoral bone of the control osteoporosis model suggests active bone resorption within the femoral cortical region, pointing to an ongoing pathological process. In contrast, the cortical thickness in the femoral bones of rats treated with CCN and CAN reflects a key feature related to bone mechanical strength. The cortical thinning seen in the CCM and control groups, however, highlights an imbalance between bone resorption and formation typical of osteoporosis. The observed reduction in bone resorption activity in the CAN and CCN-treated rats provides strong evidence for the bone-restorative effects of these treatments, particularly as it demonstrates reduced resorption at the endocortical and intramedullary regions of the femoral bones.

The dense arrangement of osteocytes at the metaphyseal periosteum in the rat model treated with CAN, when compared to the other treatment groups, aligns with findings reported by Xu et al. (2023). This is particularly evident with the increased number of osteoclasts in the osteoporotic model, which is associated with periosteal bone turnover activity. However, the reduction in intramedullary bone resorption activity in the rat models treated with CAN and CAM, as compared to those treated with CCN and CCM, supports the notion that endosteal and intramedullary resorption is more active in osteoporotic bones. This is also consistent with the slower response of cortical bone compared to trabecular bone, as previously reported in global studies (Wang et al., 2022). The changes observed in the cortical wall of the femur cannot be attributed to the absence of a Haversian system, which would result in very low bone remodeling. Instead, the more significant changes were observed in the cancellous bone region, which is consistent with the abundant cancellous bone seen in the models treated with CAN, CAM, and CCN.

Conclusion

This study highlights the ability of CAN to promote osteoblastic bone formation while inhibiting osteoclastic activity in osteoporotic models. its administration effectively mitigates bone loss and facilitates osteorestoration of type 1 osteoporosis in rat models. These findings support the use of CAN as an effective osteorestorative treatment for type 1 osteoporosis.

Acknowledgments

The authors wish to acknowledge the assistance rendered by Mustapha Jimoh for his technical support in histology tissue processing and Prof Zuki Abubakar @ Zakaria and Dr. Archibold Garikayi Bakare for their proofreading of the entire manuscript.

Novelty Statement

This study uniquely compares the osteo-restorative effects of calcite and aragonite calcium carbonate in micro- and nano-scale forms using a Type 1 osteoporosis model. It introduces a new perspective on how polymorphic structure and particle size influence bone regeneration, providing insight into the development of cost-effective and biocompatible calcium-based therapies for osteoporosis

Author’s Contribution

AD, HA, ZAJ, and FOOA: Conceptualized the study, developed the research methodology, and supervised the entire project. They contributed to data interpretation and manuscript writing.

SJ: Conducted data collection, executed the experiments, and interpreted the results, ultimately drafting the article.

MAM: Assisted with data curation, contributed to statistical analyses, and participated in revising the manuscript for critical intellectual content.

AD, HA, ZAJ, FOOA, and MSI: Revised and endorsed the study trial while providing a critical review of the draft.

AAA: Assisted with project administration, offered technical expertise, and took part in the final revision of the article.

All authors endorsed the final manuscript for onward publication.

Ethical Approval

All the procedures involving animals were performed following ethical guidelines approved by Usmanu Danfodiyo University, Institutional Animal Care and Use Committee (UDUS-IACUC) (Approval No: AUP-UDUS-2022-023).

Data availability

The data supporting the findings of this study are available upon request from the corresponding author.

Funding declaration

This study was funded by the Tertiary Education Trust Fund (TetFund) through the National Research Grant 2021. Serial No. 50). The funding body has no role in the design, execution, and analysis of the study.

Generative AI and AI-assisted technology statement

No AI tools were used in this article.

Conflict of interest

The authors have declared no conflict of interest.

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