Abstract

The current study aimed to explore the role of Cissus quadrangularis (L.) stem on osteogenic differentiation of human mesenchymal stem cells (hMSCs) and their application in bone regeneration. Orally administered natural products and its bioactive compounds often results in poor absorption, low solubility, and restricted bioavailability due to their vulnerability to intestinal hydrolytic and enzymatic degradation. To overcome these challenges, bioactive compounds extracted from C. quadrangularis were fabricated into solid lipid nanoparticles (CQ-SLNp) to enhance bioavailability. MTT assay confirmed that the CQ-SLNp found with safe and significantly increased hMSCs proliferation and IC₂₅ was identified. The impact of CQ-SLNp on stimulation of osteogenic differentiation in hMSCs at the lowest concentration was evaluated and compared to osteogenic standard medium alone after 4, 10 and 14 days, according to alkaline phosphatase (ALP) activity and alizarin red staining (ALZ) analysis. Bone morphogenic protein-receptor (BMPr) and Runt-related transcription factor 2 (RUNX2) associated mRNA levels has been analyzed. CQ-SLNp increased ALZ intensity and ALP activity. BMPr, RUNX2 and Wnt/β-catenin expressions found to be increased in osteogenesis induced hMSCs after 10 days compared to negative control. Although, the osteogenic differentiation was more effective in CQ-SLNp treatment when compared to CQ alone treated cells. We observed enhanced osteogenesis in CQ-SLNp alone treated hMSCs than standard differentiation medium. Taken together, the observed finding suggests that CQ-SLNp exhibited bioavailability and induced osteogenic differentiation of hMSCs, indicative of their potential applications for bone remodeling applications.

1. Introduction

Osteoarthritis (OA) is a common bone and joint disease, characterized by progressive loss of articular cartilage, osteophyte dysfunction, abnormal osteocyte differentiation and loss of joint function ¹. Osteocytes make up 95% of bone cells and play roles in responding to mechanical forces developing cracks due to mechanical stress and osteonecrosis. The death of osteocytes triggers bone restructuring by promoting the formation of bone tissue through the activation of cells that break down bone (osteoclasts) and cells that build bone tissue (osteoblasts) which release a protein called sclerostin that leads to bone resorption ². In addition, osteoarthritis (OA) recognized with the physiological conditions such as osteophyte formation, features of bone sclerosis, cyst in subchondral bone, articular cartilage, and synovium tissues ³. A formation of organic matric or cysts surrounded by less bone density bone uncoupled with calcification may suggest spatially differential regulation of osteoblasts and osteoclasts by Wnt/β-catenin signaling and the RUNX2 pathway ⁴. Sclerostin inhibited Wnt signaling, inhibit osteoblastic activity and terminate bone remodeling cycle ⁵.

Bone remodeling modulation might suppress osteophyte formation, improve progressive loss in cartilage and decreased bone marrow lesions (BMLs) in human OA ⁶. Osteogenic differentiation ability has been achieved successfully in human mesenchymal stem cells (hMSCs) isolated from several tissue sources, especially from bone marrow ⁷. MSCs differentiation into osteoblasts, osteoprogenitor cells which develop into osteo-precursors upon upregulation of Runx2 (Runt related transcription factor 2), bone marphogenic proteins (BMPs) and Wnt signaling. Proliferation and maturation of osteocytes begin with early-osteoblasts lineages and osteogenic precursors form osteoblasts ⁸.

Phytochemicals from plant owing their health benefits, such as carnosine, berberine, catechin increased the osteogenic differentiation in vitro and in vivo ⁹. Wu et al. ¹⁰ have identified the natural compounds such as ginsenoside, icartin and hydroxycholesterol have identified with osteogenic potentials with low toxicity and high utility. In addition, zingerone [from ginger] stimulate the bone transcription factor, Runx2 in hMSCs ¹¹ and naringin [active component of Rhizoma drynariae] stimulate osteogenic differentiation and anti-osteoporotic properties in the osteoblast cell line ¹². Most of the secondary metabolites from plants have been failed in further drug development, due to challenges such as poor absorption, low solubility, and limited bioavailability particularly when administered orally. These difficulties arise from susceptibility of natural products and their bioactive compounds to intestinal hydrolytic and enzymatic degradation. Currently the applications of solid lipid nanoparticles (SLNp) as carriers, enabling the bioavailability through effective absorption of active metabolites such as triptolide, metformin, silymarin, berberine, and pomegranate extract with pharmacological potential are most successful ^{13, 14, 15, 16}. In this context, Icariin loaded nanoplatform effectively enhance osteogenesis and bone regeneration when compared to free icariin ^{17, 18}. Khezri et al. ¹⁹ confirmed that the curcumin-containing nanoscaffolds enhancing the cellular and molecular mechanisms behind the osteogenic differentiation from mesenchymal stem cells (MSCs).

Cissus quadrangularis L. belongs to the grape family which is also known as veldt grape, adamant creeper, or devil's backbone. It has been used to relieve pain and mend bone fractures from the ancient time ²⁰. C. quadrangularis has been demonstrated to help decrease joint pain and arthritis symptoms. It improved exercise-induced joint discomfort with chronic joint pain in humans ²¹. Furthermore, C. quadrangularis was more successful in reducing swelling and decreasing inflammation than typical pharmaceuticals used to treat rheumatoid arthritis ²². Potu et al. ²³ confirmed that the C. quadrangularis exhibited higher proliferation, differentiation and calcification rates which confirms the osteoblasts (osteoblastogenesis) and extracellular matrix calcification in bone marrow mesenchymal stem cells undergo osteogenic stimulation. In the present study, based on the above literature we aimed to identify and entrap the phytochemicals of C. quadrangularis L. with phosphatidylcholine and hyaluronic acid to improve the intracellular uptake, bioavailability and approached as efficient drug deliver to stimulate osteocyte differentiation and maturation signaling pathway to prevent osteoarthritis.

2. Materials and Method

Chemicals

Hyaluronic acid, dimethyl sulfoxide (DMSO), phosphatidylcholine, MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2h-tetrazolium bromide], hydrogen peroxide (H₂O₂) were purchased from Sigma chemical company (St. Louis, MO, USA). Cell staining agents such as ethidium bromide, propidium iodide, acridine orange and mitochondrial stain, jc-1 (MMP assay) were also purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell growth media, phosphate buffered saline (PBS), fetal bovine serum (FBS) and molecular biology-related chemicals were obtained from Gibco chemicals (Grand Island, NY), USA.

Plant material

C. quadrangularis L. (veldt grape) and Salvia hispanica L. (chia seed) were procured from iHerbs and Spices in Riyadh, Saudi Arabia. The identification and authentication of the plant names and species were carried out by Taxonomist (Dr. V. Duraipandiyan, Professor, Department of Botany, College of Science, King Saud University, Riyadh). The plant specimen for C. quadrangularis L. (CQ) and KSU-SH-07 for Salvia hispanica L., (SH) were deposited and displayed in public herbarium, King Saud University, Riyadh-11451.

Extraction

C. quadrangularis L. and Salvia hispanica L. underwent shade drying, and grinding using a commercial blender, respectively. A quantity of 400 gm of CQ powder was immersed in 1200 ml of laboratory grade methanol for 72 hr, the content was kept in a mechanical shaker. Following the 72 hr period, the phytoconstituents bound with methanol were collected by filtration and reconstituted using a rotary evaporator. The concentrated CQ extract (CQE) underwent filtration, then subsequently stored at -20ºC until needed for further use. Following this, 200 gm of Salvia hispanica L. seed powder were extracted with ethyl acetate to extract phospholipids. The extraction progress of Salvia hispanica L. seeds have been carried out as same as the CQ seed progress.

Chemical analysis using GC-MS

CQ and SH extracts were assessed using gas chromatography (GC) coupled with a 5975 inert mass-spectrometer (MSD) [Agilent 7890A, Agilent Technologies, USA]. GC-MS column featured with 30m length, 0.25mm inner diameter, and 0.25µm film thickness, a Triple-Axis detector (MSD) and a liquid sampler. Pure LS or SH extracts (1 ml) subsequently injected, employing a split ratio of 30:1, respectively. The initial temperature was fixed at 280°C and the column temperature was maintained at 300°C throughout the analysis. Helium, operating at a flow rate of 1mL/min, served as the carrier gas. The electron ionization energy was set as 70 eV. Data collection occurred post full-scan mass spectra within the 40-550 AMU scan range. The LS or SH constituents were then quantified based on peak area percentage.

Formulation solid lipid nanoparticle containing C. quadrangularis L. seed

Chia seed phospholipids were employed as the lipid phase during the formulation of solid lipid nanoparticles (SLNp) according to solvent emulsification evaporation method. In brief, a lipid phase mixture comprising chia seed phospholipids (15 µL), phosphatidylethanolamine (10 µL), hyaluronic acid and 25mg of CQ extract (CQE) were dissolved in 20 ml acetone and melted on a hot plate equipped with a water bath, surpassing its melting point. Simultaneously, a surfactant solution, containing stabilizing agents like Tween 20 and lecithin, was prepared in an aqueous phase (30 ml). The molten chia seed phospholipid was emulsified with the surfactant solution to form a pre-emulsion. High-pressure ultrasonication was then applied to reduce particle size, resulting in the creation of solid lipid nanoparticles. The nanoemulsion was allowed to cool, leading to the solidification of lipid nanoparticles. The final SLNp formulation underwent characterization for crucial parameters, including particle size and polydispersity index, to ensure optimal performance and stability.

Characterization of prepared CQ-SLNp

Total drug content in CQ phytocomponents loaded CQ-SLNp was quantified from 1 mL of freshly prepared CQ-SLNp using a spectrophotometer at 420 nm. Presence of functional chemical interaction were analyzed using FT-IR (Agilent, USA). Using Zetasizer (NANO-Zs90) the particle size was determined and dynamic light scattering (DLS) technique used to calculate the z-average diameter. The morphology, shape, size, and morphology of CQ-SLNp were analyzed using high-resolution scanning electron microscopy (SEM, JEOL, Japan). Negative staining with 2% uranyl acetate (w/v) was employed against sample.

Cell lines and cell culture

Present study, the hMSCs cells were purchased from ATCC (American type culture collections, USA). Cells were cultured in a humidified atmosphere with 5% CO₂ at 37°C using DMEM (Dulbecco’s Modified Eagle’s medium) enriched with 10% FBS (fetal bovine serum), 2 mM _L-glutamate and 1% penicillin/ streptomycin (antibiotics) as growth medium. Upon reaching 80% confluence, growth media was removed and trypsinized using 0.25% trypsin/EDTA solution. Further, the cell pellets were collected following centrifugation at 3500 rpm for 5 min at room temperature. The corresponding cell numbers were seeded onto 96, 24, or 6 wells plate for MTT assay, staining and gene expression analysis.

MTT assay

Following appropriate culture conditions with the confirmation of 80% confluence, the cells were subjected to MTT assay. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was carried out in hMSCs (growing in osteogenic differentiation medium) to assess the effect of CQE (0, 5, 10, 20, 40, 80, 160 µg/mL) and CQ-SLNp (0, 2, 4, 8, 16, 32, and 64 µg/mL) on neither inhibition nor enhance the cell viability after day-1, day-5 and day 10. Viable cells tend to convert the soluble dye (yellow) into insoluble formazan crystals (purple), which were subsequently solubilized using DMSO. The absorbance was measured spectrophotometrically at 570 nm, providing a quantitative representation of cell viability. The number of viable cells has been calculated in percentage according to the mean value of the sample/control absorbance × 100.

Alkaline phosphatase Assay

In a 96-well plate, CQE (20 and 40 µg/mL) and CQ-SLNp (4 and 8 µg/mL) were treated to osteogenic differentiation induced hMSCs, respectively. 80 μL of condition medium (on 4^th and10^th days) was mixed with 20 μL of 0.75 M 2-amino-2-methyl-1-propanol buffer and 100 μL of 10 mM p-nitrophenylphosphate solution and incubated for until color development. Earlier, 0.2 M NaOH was added to arrest the conversion of p-nitrophenylphosphate to p-nitrophenol. Absorbance was measured in a spectrophotometer at 450 nm.

Mineralization

After the cytotoxicity assay, 20 and 40 µg/mL dose of CQE and 4 and 8 µg/mL dose of CQ-SLNp were selected as effective and safe dose to study the mineralization during osteogenic differentiation. The cells were then analyzed for calcium production at 10 and 14 days by staining with 10% Alizarin red solution (the dye specifically binds to calcium). Followed by fixation, the staining cells were washed three times with PBS and the fixed stains were eluted using 1mL of 10% cetylpyridium chloride (Sigma) were added to each well and incubated for 20 min. To quantify the stain accumulation, 100μL of this eluted stain was utilized to read at 550nm using a microplate reader ²⁴. A standard curve has been prepared using Alizarin Red stain and cetylpyridium chloride. The calcium deposition was expressed as molar equivalent of calcium. One mole of Alizarin Red binds to two moles of calcium in an Alizarin Red S-calcium complex.

JC-1 Staining assay

Mitochondrial membrane potential was measured in bone cell stem cells that were undergoing differentiation on the 4th and 10th days of the process. The cells exposed to CQE and CQ SLNp were treated with JC 1 dye for 20 minutes at 37°C, in an environment. After staining the cells were rinsed to get rid of dye. Examined under a fluorescence microscope. Changes in color, from green (indicating membrane potential) to red (indicating high mitochondrial membrane potential) showed the status of mitochondria and images were taken for record keeping purposes. 500 stained cells were randomly selected for analysis and the mitochondrial membrane potential was manually calculated with images captured during the process.

Gene expression analysis

Total RNA and cDNA were synthesized using Fastlane® cell cDNA kit from control, CQE (20 and 40 µg/mL) and CQ-SLNp (4 and 8 µg/mL) treated cells (7500 Fast Real-Time PCR System, Applied Biosystems, USA). The genes corresponding to the osteogenic differentiation, such as RUNX2 (Runt-related transcription factor 2), BMP-2 (bone morphogenetic protein-2), BMP-4, COL1A1 (collagen 1A1), VDR-1 (vitamin D receptor), ALPL (alkaline phosphatase) were quantified on 4^th day. In addition, the gene associated with osteoblast signaling and maturation, such as FRZ-2 (Frizzled protein 2), Wnt 5a, β-catenin related mRNA were quantified on 14^th day against the reference gene, β-actin by the method of Yuan et al ²⁵. All the primers were purchased from Qiagen, and sequence were verified in primer-BLAST, National Library of Medicine, online software and OriGene Global, Rockville, MD 20850, USA. The qPCR was performed using 25µL reaction volume and 40 cycles were allowed for amplification using Applied Biosystems 7500 Fast Real-Time PCR System (USA). Two negative controls were included for each gene by removing template cDNA. The relative expression levels were calculated by the formula: ΔΔCt (comparative threshold) = amplification values of CQE or CQ-SLNp treated (ΔCt) - ΔCt (positive control). The amplification of each target was normalized to its corresponding mRNA levels of β-actin using the 2^ΔΔCt method using Applied Biosystems software.

Quantification of protein using ELISA

On 10^th day, the amount of osteocyte differentiation and maturation related proteins such as, SPARC (osteonectin), SPP1 osteopontin (OPN or bone /sialoprotein I (BSP-1 or BNSP), SPP1 (secreted phosphoprotein 1), BGLAP (osteocalcin or bone gamma-carboxyglutamic acid-containing protein in control, CQE (µg/mL) and CQ-SLNp (8 µg/mL) treated cells using high-sensitivity ELISA-kits (Quantikine, R&D Systems, Minneapolis, MN, USA). The assay kits detect both soluble and receptor-bound proteins and thus measures the total concentration of proteins. The values were expressed as pg/mg protein for all the analyzed proteins.

Statistical analysis

The experimental data was analyzed by one-way analysis of variance (ANOVA). Then post-hoc analysis with Tukey's range test was carried out to compare and analyze the data within and between the groups using SPSS/28.5 software package (IBM, New York, USA). For all the comparisons, differences were considered statistically significant at p ≤ 0.05 and p ≤ 0.001 ²⁶.

3. Results

C. quadrangularis L. extract phytochemical profiling

The methanol extract of C. quadrangularis L. has been extensively analyzed using Gas Chromatography Mass Spectrometry (GC-MS) have been presented in Figure 1. The analysis revealed a range of compounds with biological activities, including antimicrobial, antioxidant, anticancer and anti-inflammatory effects. Some notable compounds identified in the analysis include acid methyl ester, benzyl nitrile, 2,3,5,6-tetrafluoroanisole and 3 isoquinolinamine. The presence of these compounds may contribute to the extract's biological potential. The presence of phytochemicals in C. quadrangularis L. highlights its value and supports its traditional use in different therapeutic applications. Detailed information about the constituents of this plant has been presented in Table 2.

Characterization of prepared CQ-SLNp

Physiochemical characterization of the solid lipid nanoparticles (SLNp) loaded with C. quadrangularis L. (CQ) extract using various analytical techniques. In SEM, we carefully examined the CQ SLNp and observed particle size between 76 to 125 nm (Figures 2A and 2B). These images provided insights into the surface structure and morphology of the nanoparticles allowing us to better understand their properties. Particle size analysis revealed that the nanoparticles exhibited a distribution with a size of 65nm (Figures 2C and 2D). This uniformity in size distribution is crucial for ensuring their effectiveness and suitability, for applications indicating a well-controlled synthesis process.

Fourier Transform Infrared Spectroscopy (FT-IR) analysis played a role in distinguishing the characteristics of the CQ extract and the CQ loaded SLNp (as shown in Figures 2E and 2F) revealing significant differences in their spectra. CQ Extract: The spectrum displayed peaks at 3321.46, 2931.93 and 2246.27cm^-1 along with a wide absorption band ranging from 3500 to 2000cm^-1. The peaks such as 1630.50, 1020.93 and 1415.03 cm^-1 were observed only in CQE. CQ loaded SLNp; This was characterized by peaks at 3284.14 cm^-1 featuring a pattern beyond 3500 cm^-1 and specific peaks at 2195.90, 2186.29, 2158.96 and 2226.37cm^-1. Furthermore, new peaks were detected at wavelengths as; 2124.30, 2073.17, 2054.19, 2034.26, 2020.61cm^-1 and 1998.14, 1973.48 and 1943.21cm^-1. The comparative analysis emphasized variations in % transmittance between the spectra of the CQ extract and the CQ loaded SLNp indicating changes in composition after encapsulation process took place. The presence of peaks in the CQ loaded SLNp spectrum implies modifications or interactions during the solid lipid phase encapsulation process that might influence the effectiveness and functionality of these nanoparticles. The FT IR analysis has provided evidence that the encapsulation process was successful. This is evident, from the chemical profiles and the presence of peaks in the spectrum of CQ-SLNp compared to the CQ extract. These differences highlight the changes in groups and potential modifications in chemical composition that occur during encapsulation.

Cell Viability Assessment

The undifferentiated hMSCs were cultured in 10% FBS containing growth medium for 48 hr, after confluence the characterization osteogenic differentiation was carried out. The undifferentiated hMSCs were cultured in basic growth medium showed negative to ALP activity and Ca²⁺ deposition negative in Alizarin red staining. While the hMSCs cultured in osteogenic differentiation media, the Alizarin red staining found with high red accumulation confirmed the Ca²⁺ accumulation and osteogenic differentiation. In addition, we found increased ALP activity after day 10.

To ensure that either CQE or CQ-SLNp did not compromise cell health cell viability was assessed. The hMSCs cultured in osteogenic differentiation medium were treated with increasing concentration of CQE (0, 5, 10, 20, 40, 80, 160 µg/mL) and CQ-SLNp (0, 2, 4, 8, 16, 32, and 64 µg/mL). The cell growth stimulation was extensive between day 1 to day 10 when compared to the untreated hMSCs on day 1 (Figure 3-i) or day 10 (Figure 3-ii). The cell proliferation was increased on day 5, after day 5 no further increase of the cell number was identified.

Alizarin Red Staining Test and Alkaline Phosphatase

Analysis using Alizarin Red staining revealed that both the CQ and CQ-SLNp treatments effectively promoted calcium deposition in the matrix indicating maturation. The CQ treatment resulted in an increase in mineralization at 40 µg/mL when compared to 20 µg/mL or the control group. However, the CQ-SLNp group exhibited mineral deposition with staining intensity three times greater than that of the control group. The lower dose (4/ml) of CQ-SLNp was found with 2 times greater mineral deposition, it was significantly (p≤0.001) higher than the higher dose of CQE (Figure 4).

The ALP test showed that both the CQ and CQ-SLNp treatments significantly increased ALP activity both in 4^th and 10^th day when compared to the control group. However, the CQ-SLNp group exhibited the increase in ALP activity indicating an effect on osteogenic differentiation. Specifically, cells treated with 40 µg/mL of CQ showed significantly higher level of ALP activity in 10^th day when compared to 4^th day (Figure 5-i). CQ-SLNp at 8 µg/mL treatment showed significantly increased level of ALP even at 4^th day; further on 10^th day 2-fold increase was observed when compared to the untreated control (Figure 5-ii).

Mitochondrial Potential Assay

JC-1 staining was done to check the health of the mitochondria. Cells treated with CQ-SLNp showed JC-1 formation indicating that the mitochondrial membrane potential was maintained, a sign of healthy and metabolically active cells. Additionally, the DAPI nuclear staining confirmed the cell viability showing that treatment, with CQ-SLNp led to an improvement, in integrity and density (Figure 6).

Quantitative polymerase chain reaction (qPCR) analysis

The analysis using polymerase chain reaction (qPCR) aimed to understand the gene expression profiles associated with stress, inflammation, nerve cell proliferation and osteogenic differentiation. The experimental results were compared the control group, with groups treated with CQ and CQ-SLNp. Osteogenesis and osteocyte maturation related genes expressions levels were compared between CQE (40 µg/mL) and CQ-SLNp (8 µg/mL) treated cells on 4^th day (osteogenic inducer) and 10^th day (maturation).

In the early 4^th day, most of the hMSCs were differentiated into osteocyte have been confirmed by the increased osteogenic inducer mRNA expression such as in the Figure 7 (i, ii) as, RUNX-1, BMP-2, BMP-4 up-to 3-fold when compared to CQE alone treated differentiation induced hMSCs. Meanwhile on the 10^th day, the up-regulated expression of osteogenic inducer genes was inclining to normal level in both CQE or CQ-SLNp treated cells.

The expression levels of osteogenesis stimulator markers, such as in the Figure 7 (iii, iv) as COLA1, ALPL and VDR-1 expression have been significantly (p≤0.05) increased on 4^th day, but during 10^th day the expression levels were increased upto 3-fold when compared to CQE treated cells.

Osteogenesis maturation linked signaling pathway associated traits such in the Figure 7 (v, vi) as, FRZ-2, Wnt-5a and β catenin were significantly (p≤0.001) increased to 3-fold in the CQ-SLNp group after 10 days when compared to early 4^th day expression. In CQE treated cells, the expressions of FRZ-2, Wnt-5a and β catenin were found to be non-significant in 4^th day; but on 10^th day the expression level found to be significant (p≤0.01).

Protein expression

The results for osteocyte differentiation signaling and maturation associated protein levels showed that both CQE (40 µg/mL) and CQ-SLNp (8 µg/mL) treated cells effectively increased SPARC (osteonectin), SPP1 osteopontin (OPN or bone /sialoprotein I (BSP-1 or BNSP), Wnt-5a, BGLAP (osteocalcin or bone gamma-carboxyglutamic acid-containing protein on 10^th day when compared to 4^th day (Figure 8-i). In addition, we observed a significantly (p≤0.001) decreased levels of oxidative stress (such as LPO, NOX-2) and pro inflammatory genes (such as IL1β, TNF-α) during the 10th day. Overall, the cells treated with CQ-SLNp exhibited a significantly higher reduction in this stress and inflammatory markers. This suggests that the nanoparticle formulation has osteogenesis, anti-inflammatory and anti-oxidative effects (Figure 8-ii).

4. Discussion

Our investigation findings indicate that the use of C. quadrangularis stem loaded lipid nanoparticles (CQ-SLNp) can potentially enhance the process of osteocyte differentiation from human mesenchymal stem cells (hMSCs). This enhancement is evident through an increase in alkaline phosphatase activity and mineral accumulation confirmed by Alizarin Red S staining, well as the heightened expression of key markers associated with osteogenesis, such as bone morphogenic protein receptor (BMPr) and Runt related transcription factor 2 (RUNX2). These results suggest that CQS SLNs possess a capacity to induce bone formation potentially by activating signaling pathways related to osteogenesis.

The role of C. quadrangularis promoting the regeneration of bone has been well documented despite the bioavailability and solubility challenges it presents. In this context, Zahan et al ³⁹ who demonstrated that C. quadrangularis has the potential to accelerate the healing of bone in rat models. This study has enabled us to progress to deliver compounds from C. quadrangularis with lipid nanoparticles (SLNs). In addition, nanoencapsulated drug delivery for improved drug availability and osteocyte formation [40, 41] ^{40, 41}.

Our research extensively explores the mechanisms for osteogenic differentiation. We specifically focus on the enhancement of BMPr and RUNX-2 gene expression. In this context Chen et al ⁴² and Lee et al ⁴³ have confirmed the importance of RUNX2, which is considerd as a major differentiation regulator for osteogenesis. RUNX2 emphasize its role in promoting differentiation and facilitating the bone formation process ^{42, 43}. Additionally, the function of RUNX2 including its involvement in various signaling pathways and regulation of gene transcription, during osteoblast development have been confirmed ^{44, 45}. By integrating these perspectives our findings provide an understanding of the processes involved in bone formation highlighting the significant contributions made by BMPr and RUNX2 within this field. We provide insights by employing SLN technology to address the bioavailability challenges of C. quadrangularis thereby enhancing its capabilities for applications in bone tissue engineering ⁴⁶. In addition, studies conducted by Parvathi ⁴⁷ and Tamburaci ⁴⁸ has explored the phytochemicals of CQ and their role in promoting differentiation and bone regeneration with a focus, on the advantages it offers for the functions of mesenchymal stem cells.

Our study aligns, with research that emphasizes the role of the Wnt/β catenin signaling pathway in osteogenesis bone remodeling and osteoblast differentiation ⁴⁹. We also delve into the use of lipid nanoparticles loaded with C. quadrangularis stem extracts (CQ-SLNp) for promoting bone growth when compared to free CQ extract. This exploration provides insights into harnessing nanoparticle modified extracts to facilitate bone regeneration. The Wnt/β catenin pathway is vital for bone development and it plays a role in maintaining bone balance. Impaired Wnt/β catenin pathways are associated with conditions like osteoporosis and osteogenesis imperfecta (OI) ⁵⁰ and the maintenance of bone mass ⁵¹. Activation of this pathway promotes bone formation while its inhibition can lead to a reduction in bone density ⁵². However, it's worth noting that targeting this pathway for purposes needs consideration due to potential risks such as tumorigenesis ⁵³. Furthermore, Stohs and Ray ⁵⁴ validate the efficacy of C. quadrangularis in promoting bone formation and facilitating the healing of fractures. Many studies emphasize the plants potential to enhance bone growth especially when utilized alongside SLN technology to enhance bioavailability ^{55, 56}. Our findings highlight the benefits of utilizing nanoparticle technology to enhance the effectiveness of herbal extracts in promoting optimal bone health. This represents an advancement in integrating medicine with contemporary therapeutic strategies.

The osteogenic differentiation observed significantly higher in CQ-SLNp treatment is likely mediated through the synergistic actions of BMP-6 and RUNX2 upregulation and Wnt/β-catenin pathway activation ^{6, 57, 58}. These pathways are known to be pivotal for the initiation and progression of osteoblast differentiation, as well as for the maturation of osteocytes, providing a mechanistic basis for the observed enhancement in osteogenesis ⁵⁹. The utilization of nanoparticles in therapies focused on regenerating bone has the potential to address limitations in treatments for bone related disorder ⁶⁰. Nanotechnology offers applications in engineering bone tissue, including targeted delivery of molecules and growth factors scaffold construction and cell labeling when compared to free supplementation of drug ⁶¹. These applications can augment the differentiation process of bone cells and support tissue regeneration ⁶². In relation to osteoporosis combining nanomaterials within a scaffold holds promise for localized and long-term treatment options ⁶³. Overall incorporating nanoparticles into bone regeneration therapies presents an approach with potential, for improving treatment outcomes.

5. Conclusion

This research emphasizes the potential of using lipid nanoparticles made from C. quadrangularis (CQ SLNp) to enhance the process of bone formation in human mesenchymal stem cells (hMSCs) through increased bioavailability and bio efficacy. By improving differentiation of osteocyte and increasing the expression of markers for bone formation CQ-SLNp proves to be more effective compared to traditional methods used for bone development, such as C. quadrangularis extracts alone. These findings highlight the novel and efficient approach that CQ-SLNp offers for regenerating bones, which was considered as a step ahead towards addressing conditions like failure in targeted drug delivery or loss of bioavailability. Further studies are necessary to optimize the formulation of CQ-SLNp, for its application thus advancing medicine and the field of bone tissue engineering.

Conflict of Interest Statement: The authors declare that there is no conflict of interest.

Author Contribution: NA conducted the analysis and drafted the original manuscript. P.S-B developed the methodology. AA re-vised the manuscript. P.S-B and AA reviewed, edited, and approved the final manuscript.

Data Availability: The data presented in this study are available on request from the corresponding author.

Acknowledgement: This research was funded by the Researchers Supporting Project number (RSP2024R178), King Saud University, Riyadh, Saudi Arabia.

References