Plants being rich sources of natural entities have been used for their medicinal value since antiquity. The compounds isolated from medicinal plants have potential to serve as lead molecules for the development of safer and better alternatives to synthetic drugs against various diseases. Herbal plants have harvested significant attention from the scientific community due to its traditional use in various indigenous healing practices. This plant contains multiple bioactive molecules that encourage osteoblast growth, regulate inflammatory responses and support the buildup of the extracellular matrix (Ekor, 2014). Recent research by the scientist is actively investigating how such herbal remedies can be effectively applied in modern bone healing process (Bodeker et al., 2005).

Several plant species are good sources of drugs which have therapeutic properties or exert beneficial pharmacological effects on the body, thus it has been seen that around 80% of global population prefer to use herbal drugs against cancer, hepatic dysfunction, AIDS, diarrhoea, hyper- or hypo-glycaemia, graft rejection, autoimmune disorders, as antioxidants and for immune dysfunctions (Jouad et al., 2001). From the ancient time Plants have been a part of traditional medicines round the globe. They contain a variety of phytochemicals such as phenolic compounds (e.g. phenolic acids, flavonoids and tannins), alkaloids, terpenes  (Yu et al.,  2021) and even functional phytohormones (Kim et al.,  2020), which are known to exert, directly or indirectly, a multitude of healing effects and regeneration.

Bone, though a rigid tissue is an equally dynamic one that is continuously moulded, shaped and repaired. Once formed, bone undergoes a continuous renewing process called bone remodelling. This process of bone remodelling involves break down (resorption, by osteoclasts) and build-up (synthesis, by osteoblasts), occurring at a microscopic scale throughout the skeleton.

Extract of rhizome of turmeric (Curcuma longa Linn.) has been used as anti-inflammatory agents in Ayurvedic and Traditional Chinese Medicine (TCM). The turmeric extracts containing substances called curcuminoids prevent formation of osteoclasts (osteoclastogenesis) and destruction of periarticular bone. Curcumin is a yellow phenolic compound present naturally in various types of herbs, especially in turmeric. It possesses antirheumatic, hepatoprotective, antidiabetic, neuroprotective, nephroprotective, hypoglycaemic and cardioprotective activities. It also suppresses thrombosis and provides protection against myocardial infarction (Sou, 2012). Despite its reported benefits, multiple factors often limit the practical and clinical applications of curcumin. For instance, poor water solubility and physicochemical instability, low pharmacokinetics and bioavailability, poor bioactive absorption, rapid metabolization, low penetration and targeting efficacy, sensitivity to alkaline conditions, metal ions heat and light (Flora et al., 2013). Due to low oral absorption has been the limitation its uses in clinical medicine, therefore present study was conducted to formulate a nanoparticle, which enhances the oral absorption and increases the systemic bioavailability.

Nanotechnology is an evolving field that utilizes the physicochemical attributes of nanomaterials as a means to control their dimensions, surface area and shape in order to generate different nanoscale-sized materials. The nanoparticulate drug delivery systems appear to be a promising strategy for curcumin delivery for its enhanced system availability and could be exploited for its therapeutic nature in prevention and alleviation of the symptoms of osteoporosis.

Thus, keeping in view of the above facts regarding medicinal properties of curcumin and preparation of novel nanoparticle formulations, this study was designed to develop an efficient drug delivery system for aqueous extract of Curcuma longa.

Isolation, culture and expansion of primary rat foetal osteoblasts
 
Long and cranial bones from Wistar rats were collected from Department of Veterinary Pharmacology and Toxicology, College of Veterinary and Animal Sciences, G. B. Pant University of Agriculture and Technology, Pantnagar.

The isolation, culture, expansion and characterization of the osteoblasts were done by the method as standardized in our lab (Yang et al., 2015).
 
Giemsa staining for cell morphology
 
Giemsa stain, a polychromatic stain, was used to study the morphology of cells (Meirelles and Nardi, 2003).
 
Nano-curcumin extracts preparation
 
Nanocurcumin preparation was procured from Department of Molecular Biology and Genetic Engineering, G.B. Pant University of Agriculture and Technology, Pantnagar. The size of the nanoparticle was confirmed by zeta potential and sizes were <55 nm.
 
Quantification of amount of curcumin extracted in untreated curcumin and nanocurcumin preparation
 
The amount of curcumin extracted was estimated by measuring the absorbance of the final solution at 425nm i.e. the absorption maximum of curcumin and applying the following formula (Sadasivam et al., 2008).
 
Primer sets
 
Sequences of reported primers of OCN, OPN, ALP, MMP-9, Runx2, Collagen type I alpha 1, Collagen type III alpha 1, RANKL, M-CSF, CAT-K and the GAPDH (as a housekeeping) genes were used for quantification of different osteoblast and osteoclast by real time RT-PCR. Details of Real-time PCR primers for above genes showed in Table 1.


 
Isolation of total RNA
 
Total RNA was extracted from a using RNA isolation kit according to manufacturer’s instruction from each sample. The extracted RNA was further purified and treated with DNase I using RNeasy kit and was quantified by spectrophotometer considering the OD260/OD280 ratio, which was 1.8-2.0 for all the samples. The total RNA yields in each sample were diluted to 10 ng/µl for further analysis. Total RNA preparations were stored at -80°C till further use.
 
cDNA preparation
 
cDNA was prepared from DNase-free RNA of osteoblast cells for the characterization of osteoblasts. For cDNA synthesis, Revert Aid reverse transcriptase (RT) (200 units/µl) was used (Fermentas). The isolated RNA was subjected to RT-PCR.
 
Statistical analysis
 
All samples were amplified in triplicate and the mean values were calculated to calculate the relative expression of genes (Livak and Schmittgen, 2001). Two-way analysis of variance (two-way ANOVA) was used to compare the observations at 1% and 5% levels of significance.

The present study was conducted to study the effect of nanocurcumin on rat mesenchymal stem cell (rMSC)-derived osteoblast culture. An important aspect of the study was evaluation of osteoblast differentiation and mineralisation using gene specific primers, carried out to get an insight into the expression of different markers after challenging with nanocurcumin.
 
Isolation, culture and expansion of primary rat osteoblasts
    
The isolated bones were harvested aseptically for culture of primary osteoblasts. During primary culture, after 2-3 days, round or polygonal cells were observed migrating from bone pieces under phase contrast microscope (Fig 1). These bone pieces, as already stated, contain stem cell niches i.e. areas where stem cells reside. When cultured in suitable media, these stem cells grow and proliferate to form cells that vary in shape from fibroblast-like to cuboid i.e. with the passage of time, the cells started to change their morphology from round to various shapes. Initially this culture represented mixture of heterogeneous cell populations. After 5 days of culture, cells with protuberances were observed as adhering to the culture flask as can be seen in Fig 1 (B) Gu et al., (2012). With the passage of time, these cells begin to expand and attains confluency all along the surface of culture flasks. In this study, the first passage was done when approximately 80% confluency of cells was observed after 12-15 days of culture. After passage one, triangular cells were prevalent and packed closely in culture flask. Cell proliferation was uniform and throughout the culture flask. Similar results with respect to cell morphology have been reported by Yang et al., (2015).


 
In vitro evaluation of efficacy of nanocurcumin of primary rat osteoblast and qRT-PCR expression of osteoblast-and osteoclast-specific genes/markers
 
Study of the population dynamics of cell culture in control, curcumin and nanocurcumin treated groups
 
After seeding a known number of cells (3.75x10⁴ cells/well) in each well of a 24-well plate, the cells were challenged with untreated curcumin and nanocurcumin, while the third was kept as a control. At different intervals of time, the cells were detached to make a cell suspension. 10 µl of the cell suspension (Dilute cells in complete medium without serum) was taken in a PCR tube. The cells were counted by trypan blue cell counting method at different intervals of time (12h/0.5d, 24h/1d, 2d, 3d, 4d, 5d, 6d and 7d). The results of the cell count are given in Fig 2 graph was also plotted by taking time on x-axis and the number of cells counted on the y-axis to obtain a growth curve for each culture having control, curcumin treated and nano-curcumin treated cells respectively. As is evident from the data and the growth curve, the culture that was challenged with nano-curcumin had the highest number of viable cells, as compared to the control. The number of viable cells in the culture challenged with untreated curcumin was more than the control but less than the one treated with nano-curcumin. This indicated that the nano-curcumin preparation had a better effect at enhancing cell viability as compared to untreated curcumin. It can be inferred from these observations that nano-curcumin enhances cell survivability and is likely more osteo-protective as compared to pure curcumin.

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Quantitative PCR (Real time PCR) to study the expression profiles of selected biomarkers (genes) in response to the treatments given
 
The cells were collected at different stages, cDNA was prepared as per the procedure and the RNA extracted from osteoblasts in control, curcumin and nano-curcumin treatment groups at 12-hours, 1-day, 3-days and 7-days post treatment. A random cDNA sample was diluted with buffer to obtain different dilutions (1:2, 1:4, 1:6, 1:8, 1:10) and subjected to qRT-PCR using GAPDH primers.
 
Expression profiling of osteoblast genes like RUNX-2, OCN, OPN, ALP, COL-I and COL-III gene in control and treatment groups by using quantitative RT-PCR
 
Runx2 is detected in pre-osteoblasts, undergoes upregulation when osteoblasts are in immature stage and is downregulated when osteoblasts are matured. It is the first factor that directs determination of the osteoblast lineage and suppression of chondrogenic lineage Komori (2009). It was observed that the relative transcript abundance (fold expression) of Runx2 in control at 12 h, 1 d, 3 d and 7 d stages respectively as compared to GAPDH expression (taken as 1) and depicted in Fig 3.

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The expression of Runx2 gene were increased till it reached the highest value in cells treated with curcumin at 12 h stage though it decreased later on. Same trend was observed in the cells kept as control i.e. Runx2 expression increased till 3 d but start decreasing later on. However, Runx2 expression in control was lower than the cells treated with curcumin. In case of cells treated with nanocurcumin, there was uneven expression; however, it increased at 7 d stage of culture. These observations indicate that as compared to control, curcumin further enhanced Runx2 expression and helped in osteoblastogenesis in early stages. However, nanocurcumin also induced osteoblastogenesis but that was delayed. Alsoufy et al., (2024) reported that expression of Runx2 gene a osteogenic biomarkers was increases due to the melatonin which increases the bone remodelling and reduces the orthodontic relapse in sheep.

The observations made with respect to Runx2 expression are in agreement with the findings reported previously Komori (2009). Curcumin increased Runx2 expression in rMSCs when cells were grown in osteogenic medium (Gu et al., 2012). 

Osteocalcin is produced by osteoblasts under regulation and control by Vitamin-D and Runx2. Its levels increase during bone formation, whether physiological or pathological (fracture) and the highest levels are seen during puberty (Chapurlat and Confavreux, 2016). It was observed that the relative transcript abundance (Fold expression) of OCN in control, nonocurcumin and curcumin 1d, 3d and 7d stages depicted in Fig 4. It was observed from the data that the highest expression levels were seen in the cells treated with nanocurcumin. The increase in OCN expression in these cells was slow but persisted for a longer duration as compared to cells treated with simple curcumin extract. This shows that there was more active bone formation in the former as compared to later.

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The fact that OCN expression is regulated by Runx2 was also evident when the expression levels of OCN were compared with Runx2. This finding is corroborated with the finding as reported by Lee et al., (1997).  Moreover, a finding of this study supported by comparison of Runx2 and OCN expression in control with curcumin-treated cultures is that there are some constituents in crude turmeric extracts that can negate or reduce the beneficial effects of curcumin. These findings have been earlier reported by Funk et al., (2009), while investigating the effect of curcumininoids on SCW-induced model of arthritis in female rats.
 
Expression profiling of osteopontin (OPN) gene in control and treatment groups by using quantitative RT-PCR
 
It is produced by osteoblasts, osteoclasts as well as osteocytes and not much is known about its exact role as most of the evidence is only circumstantial (Chapurlat and Confavreux, 2016). The cDNA samples at 12-hours, 1-day, 3-days and 7-days post treatment were taken from each- control, curcumin and nanocurcumin treated cultures. It was observed that the relative transcript abundance (Fold expression) of OPN in Fig 5.

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As is evident from the expression levels, the highest OPN expression was seen in the cells treated with nanocurcumin, even though the increase in expression was slow. Similarly, the cells treated with curcumin recorded higher levels of OPN expression as compared to control, but lower levels as compared to nanocurcumin. Although OPN is produced by all the three types of bone cells, we can conclude that the different levels of expression were due to the corresponding number of osteoblasts in each group.

As already stated that OPN expression is a circumstantial evidence, it can be concluded that nanocurcumin is better at inducing osteoblast and osteocyte differentiation as compared to untreated curcumin. These findings are in agreement with the findings reported earlier by Mohammadi et al., (2015) who reported overexpression of OPN in leukemic stem cells when treated with curcumin.

BAP is an isoform of ALP produced by osteoblasts in bones. It is believed that it probably helps in calcification of the bone matrix. Being a secretory product of the osteoblasts, its concentration increases during active bone formation. Its levels are high in fracture healing, osteomalacia and Paget’s disease; however, the concentration remains normal during osteoporosis. The cDNA samples at 12-hours, 1-day, 3-days and 7-days post treatment were taken from each-control, curcumin and nanocurcumin treated cultures and the relative transcript abundance (fold expression) of ALP depicted in Fig 6.

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However, the results were found to be significant (p<0.05) for all treatments. Increase in ALP expression in response to treatment with different curcumin formulations has been already reported by Gu et al., (2012) and our results are in agreement with the study.

Different types of collagen are found in different tissues and organs. Collagen-I is widely distributed but it forms the bulk of organic part of bone. Col-III is another variant of collagen found in the bone. As depicted in Fig  7 and 8, the cDNA samples at 12-hours, 1-day, 3-days and 7-days post treatment were taken from each control, curcumin and nanocurcumin treated cultures. It was observed that the relative transcript abundance (fold expression) of Col-I and Col-III in control, nanocurcumin and curcumin were at 12h, 1d, 3d and 7d stages depicted in Fig 7 and 8.

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It is evident from the figure that in control, Col-I and Col-III expression levels increased rapidly till they reached the highest on 1-day post treatment and started to decline till 7th day post treatment. In case of cells treated with curcumin, Col-I expression increased consistently till day-7, while Col-III expression increased till day 3 and then had a rapid decrease. The level of Col-I expression increased slowly till day-3 and rapidly afterwards till day-7 in the cells treated with nanocurcumin preparation. However, Col-III showed a pulsating trend in these cells. The differences in ratios of Col-I and Col-III have been reported earlier (Sokolov et al., 1992). It was observed that nanocurcumin preparation brought about a delayed but the highest increase in the expression of collagen. These observations indicated that the nanocurcumin formulation was better than untreated curcumin at inducing collagen expression and subsequent deposition in the bone matrix.
 
Expression profiling of RANKL, CAT-K, MMP-9 and M-CSF gene in control and treatment groups by using quantitative RT-PCR
 
RANKL induces osteoclast differentiation through RANKL/RANK/OPG pathway. When RANKL binds to RANK, it initiates a cascade of events which culminates in increased expression of osteoclast specific markers, thus bringing about bone resorption (Boyce et al., 2007). It was observed that the relative transcript abundance (fold expression) of RANKL in control, nanocurcumin and curcumin treated were at 12 h, 1 d, 3 d and 7 d stages depicted in Fig 9.

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As in figure, there was decreased expression of RANKL in all the three treatment groups. However, the decrease in RANKL expression was more in the cells treated with nanocurcumin, as compared to the cells treated with curcumin alone. This observation indicates that nanocurcumin is more effective in preventing osteoclast differentiation and consequent bone resorption as compared to aqueous extracts of untreated curcumin.

The expression patterns obtained from the quantitative Real Time PCR were significant (p<0.01) for all treatments. Similar results were reported Li et al., (2015). It was also reported a decrease in RANK/RANKL/OPG signalling on supplementation with curcumin in glucocorticoid-induced osteoporosis model (Li et al., 2015).

CATK is found in lysosomes, activated at low pH by autocatalytic cleavage and released into the resorption lacunae during breakdown of bone tissue e.g. postmenopausal osteoporosis (Chapurlat, 2014). It was observed that the relative transcript abundance (fold expression) of CATK in control, nanocurcumin and curcumin were at 12h, 1d, 3d and 7d stages depicted in Fig 10.  As is evident from the figures 10, at most of the stages of study from 12 hours to 7 days, CATK levels were the least in the cells treated with nanocurcumin. CATK expression level was increased in cells cultured with untreated curcumin extracts at the 7th day of study, although low levels of CATK were observed with nanocurcumin preparation till the end.

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It was also reported that curcumin supplementation decreased CAT-K levels in GIOP mice model (Li et al., 2015). The results of this study indicated that nanocurcumin was more effective at preventing osteoclastogenesis as compared to untreated curcumin.

MMP9 brings about the degradation of the extracellular matrix. Increased levels of MMP9 are seen in circulation in osteoporotic patients, which are indicative of activated osteoclasts (Li et al., 2015). The cDNA samples at 12-hours, 1-day, 3-days and 7-days post treatment were taken from each control, curcumin and nanocurcumin treated cultures and the relative transcript abundance (fold expression) of MMP9 as depicted in Fig 11. In this study, MMP9 expression was surprisingly high at 3-day post treatment in cells treated with curcumin and 7-day post treatment in cells treated with nanocurcumin. The levels of MMP9 decreased after the 3^rd day post treatment in curcumin treated cells. On statistical analysis of ΔCT values obtained from the quantitative Real Time PCR, the results were found to be non-significant (p>0.05) for all treatments when time and treatment were considered independently. The interaction of the two factors was found to be significant (p<0.01). It has been reported earlier that curcumin inhibits MMP9 expression, so the results of our study are in agreement with time Buhrmann et al., (2011).

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Macrophage colony stimulating factor is a cytokine released by osteoblasts in response to parathyroid hormone. Once released, it acts on nearby osteoclasts causing them to differentiate and become active. As a result, it increases bone resorption (Takeshita et al., 2000). The cDNA samples at 12-hours, 1-day, 3-days and 7-days post treatment were taken from each control, curcumin and nanocurcumin treated cultures and were depicted in Fig 12. From the Fig 12, it is clear that M-CSF expression was more in cells treated with curcumin than the cells treated with nanocurcumin formulation and control. In the cells treated with curcumin extracts, there was an unexpected rise in the expression of M-CSF levels at day-3 post treatment, which is almost opposite of that has been reported earlier (Chapurlat, 2014). In spite of that it can be concluded from the observations that nanocurcumin is better at preventing osteoclastogenesis as compared to untreated curcumin as it decreased MCSF expression. Jain et al., (2023) reported that hydroxyapatite-collagen composite can be used as along with autolologus bone marrow concentrate as alternative therapy to bone graft in clinical cases to enhance the fracture healing. Awasthi et al., 2024 reported in their study that Anti-osteoporotic Activity of Punica granatum Seed, Bambusa arundinaceae  Leaves and  Trichosanthes diocio Fruit Ethanolic Extract In vivo and in vitro study.

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The expression study of different markers specific to osteoblasts revealed that nanocurcumin has a significant effect on their up regulation as compared to control and curcumin. The relative expression levels of osteoblast-specific markers like Runx2, OCN, OPN, ALP, Col-I and Col-III were significantly increased in nanocurcumin as compared to control and curcumin, which means that nanocurcumin has a better effect on bones and can be used as a potential anti-osteoporotic drug. The expression study of different markers specific to osteoclasts revealed that nanocurcumin had a significant effect on their downregulation as compared to control and curcumin. The relative expression levels of osteoclast-specific markers like RANKL, CAT-K, MMP9 and MCSF were significantly reduced in nanocurcumin treated cells as compared to control and curcumin. This also strengthened the hypothesis that nanocurcumin can be used as an anti-osteoporotic drug.

It was observed that the aqueous solubility of curcumin in the nanocurcumin was increased 6 times as compared to untreated turmeric. First, the calcium ions react with the hydroxyl group of curcumin and increase its solubility. The second advantage is that calcium oxide (lime) provides calcium supplementation which is necessary for bone health.

Hence the present study suggests that nanocurcumin had a potential role as a drug candidate in prevention and treatment of osteoporosis as compared to curcumin given alone. Nanocurcumin enhanced the formation and differentiation of osteoblasts and inhibited the formation and differentiation of osteoclasts i.e. it favoured bone formation over bone destruction.

Author are highly obliged to the Director Research and Dean, College Basic Sciences and Humanity, G.B. Pant University of Agriculture and Technology for providing all the basic facility and financial support to cried out this research work.

All the authors declare that there is no conflict of interest for publication of this article and also give their consent for publication.