The Effect of Clopidogrel and Ticagrelor on Human Adipose Mesenchymal Stem Cell Osteogenic Differentiation Potential: In Vitro Comparative Study

Ticagrelor (TICA) and clopidogrel (CLP) are extensively used antiplatelet drugs that act by antagonizing the P2Y12 receptors that are found on platelets in addition to bone cells. Aim. The purpose of this study was to investigate the effect of clopidogrel and ticagrelor on stem cells osteogenic differentiation in vitro. Methods. Human adipose-derived mesenchymal stem cells (hAd-MSCs) were divided into (1) control group, (2) osteogenic group (osteo group), (3) clopidogrel group (CLP group), and (4) ticagrelor group (TICA group). The osteogenic differentiation potential was determined by mineralization nodule formation using Alizarin Red S staining, measuring ALP enzyme activity by alkaline phosphatase assay. Quantitative determination for osteogenic markers included osteocalcin (OC); runt-related transcription factor 2 (RUNX2) performed using western blot; osteoprotegerin (OPG) using enzyme-linked immunosorbent assay (ELISA) and inflammatory markers; and tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) measured using real-time polymerase chain reaction quantitative (RT-PCR) and ELISA. Results. In comparison to all study groups, the TICA group showed significant increase in the mineralized extracellular matrix, ALP enzyme activity, and bone markers expression as RUNX2 (P < 0.0001), OC, and OPG (P < 0.05). The expression of IL-6 and TNF-α was determined by RT-qPCR and ELISA techniques. TICA and CLP significantly decreased both markers compared to the control group. The TICA group showed statistically significant lower levels of both markers (P < 0.0001) than the CLP and control groups via the ELISA technique. Conclusion. TICA may possess a positive effect on hAd-MSCs osteogenic differentiation compared to CLP.


Introduction
Bone defects that cannot heal spontaneously are termed critical-size bone defects or large bone defects.Tese bone defects can emerge as a consequence of extensive trauma, multiple fractures, infection, tumor, or musculoskeletal disorders [1].Bone healing process is an intricate physiological process that involves multiple cells and cell signaling molecules that interact at the site of fracture to repair bone tissue without scar formation [2].In critical-size bone defects, the typical process of bone healing is insufcient to replenish the lost tissue.Surgical procedures that involve bone grafting are widely used to address bone loss, utilizing autologous, allogeneic, or xenogeneic bone transplantation methods along with synthetic biomaterials.Autologous bone grafting is considered the most reliable approach for reconstructing extensive bone defects [3].Tis process involves transplanting autologous tissue from healthy areas of the body; however, its application is limited by several factors, including the possible large bone volume needed, the associated pain, possible donor site morbidity, prolonged recovery period, and inadequate vascularization [4,5].Synthetic materials designed for bone grafting have been introduced to overcome the disadvantages of autologous bone transplantation of limited bone supply [6].Te primary costs associated with their fabrication prevent the wide application of these graft materials [7].
Adipose tissue-derived mesenchymal stem cells (Ad-MSCs) have the potential to self-renew, exhibit antiinfammatory and immune-modulatory efects, and have the ability to diferentiate into various cell types, including osteoblasts.Furthermore, these stem cells secrete molecules that can initiate or enhance tissue regeneration processes [4].Adipose-derived stem cells (Ad-MSCs) can be obtained from adipose tissue by the liposuction process, which is considered a safe and minimally invasive procedure.Ad-MSCs have been extensively used in ex vivo experiments to test drugs or materials that may possess osteogenic potential, owing to their high osteoinductive potential and osteogenic activities, as well as their greater capacity for proliferation and formation of colonies when compared to stem cells from other sources, such as bone marrow MSCs [8,9].
Clopidogrel is a thienopyridine that inhibits ADPinduced platelet activation via irreversible binding to platelet purinergic receptor P2Y12 [10].Previous research has demonstrated that the P2Y12 receptor is also found on bone cells, including osteoblasts, osteoclasts, and osteocytes, in addition to platelets [11].P2Y12 receptors' impact on bone cell biology and bone homeostasis has been the subject of conficting research.Studies have claimed that P2Y12 receptors promote osteoblastogenesis and prevent the development of osteoclasts, while others have advocated maintaining osteoclast activity.Previous investigations have indicated the signifcance of ADP receptor P2Y12 in the formation and function of osteoclasts, particularly in models of pathological bone loss related to postmenopausal osteoporosis, rheumatoid arthritis, and bone metastases [12,13].Other studies also reported that these antiplatelet drugs decreased postsurgical bone loss, which may be attributed to their anti-infammatory efects [14,15].
Ticagrelor is a newer antiplatelet drug that acts by blocking the P2Y12 receptor and inhibiting cellular adenosine uptake through the equilibrative nucleoside transporter (ENT)-I.Tis mechanism results in increased extracellular adenosine levels.Ticagrelor inhibits osteoclast diferentiation and increases extracellular adenosine levels, thereby stimulating adenosine A 2A receptors (A 2A Rs) [10].
Accordingly, the aim of this study was to investigate the impact of both clopidogrel and ticagrelor on the osteogenic diferentiation potential of human adipose-derived mesenchymal stem cells (hAd-MSCs) by detecting osteogenicrelated markers including osteocalcin (OC), runt-related transcription factor 2 (RUNX2), and osteoprotegerin (OPG).In addition, the drugs' anti-infammatory efect was assessed by measuring infammatory-related markers such as tumor necrosis factor (TNF-α) and interleukin-6 (IL-6).

In Vitro Procedures
(1) Ethical Statement.Te study protocol of this research has obtained approval from the Research Ethics Committee at Cairo University's Faculty of Pharmacy with the license number PT (2941, 3/2021).

Isolation and Culture of Human
Adipose-Derived (hAd-) MSCs.Te hAd-MSCs were derived from lipoaspirate obtained with informed consent from patients aged 28-40 years (n � 3) who were free from any underlying medical conditions.Lipoaspirate was collected by a plastic surgeon in two sterile plastic bottles (100 ml/bottle) that were immediately transferred to the laboratory under sterile conditions within 2 hours of the surgery.To isolate the stromal vascular fraction, the lipoaspirate was washed with sterile phosphate-bufered saline (PBS) multiple times.After that, the tissue underwent enzymatic digestion using 0.1% collagenase A in PBS containing 1% bovine serum albumin (BSA) for 60 minutes at 37 °C with intermittent shaking.Te digested lipoaspirate was then washed with Dulbecco's modifed Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and centrifuged at 600 g for 10 minutes.Te resulted cell pellet was resuspended in PBS and fltered through a 200 mm mesh to eliminate blood and any residual tissue debris.Te solution was centrifuged again, and the supernatant was resuspended in DMEM/F12 media containing 10% FBS and 1% streptomycin/penicillin. (2) Determination of the hAd-MSCs Multilineage Diferentiation Potential.To evaluate the multilineage diferentiation capacity of the isolated hAd-MSCs in vitro, cells from the fourth passage were cultured at a concentration of 5 × 10 4 cells/mL in a 24-well plate.Multilineage diferentiation was performed using the commercially available human mesenchymal stem cell functional identifcation kit, which included osteogenic, adipogenic, and chondrogenic diferentiation media.Te diferentiation processes for each lineage were carried out following the instructions provided by the manufacturer.Noninduced hAd-MSCs cultured in complete growth medium (10% FBS-DMEM) served as control.After approximately 21 days of diferentiation, adipogenesis was determined by the appearance of lipid droplets, which were assessed using Oil-Red staining.For osteogenic diferentiation, staining by Alizarin Red-S was performed to detect extracellular matrix rich in calcium.Lastly, for chondrogenic diferentiation, staining was performed using Alcian blue to confrm the production of sulfated proteoglycans [18].

Assessment of Clopidogrel (CLP) and Ticagrelor (TICA)
Osteogenic Efect on hAd-MSCs.To determine CLP and TICA concentrations that should be used in our study, a viability assay (MTT assay) was performed using serial dilutions of both drugs (Supplementary Data A).Te concentration of 1.8 μM of TICA showed 100% cell viability (not cytotoxic) and was used for both drugs throughout the study experiments.
For induction of osteogenic diferentiation, cells were seeded in a 6-well plate at a density of 30 × 10 4 cells/well.After reaching 70% confuency, the medium was replaced with osteogenic diferentiation medium, which composed of α-MEM, 10% FBS, dexamethasone 100 nM, ascorbic acid-2phosphate 200 uM, and β-glycerophosphate 10 mM [19].Cells were divided into 4 groups: cells cultured in osteogenic medium with 1.8 μM clopidogrel [14] (CLP group), cells cultured in osteogenic medium with 1.8 μM ticagrelor (TICA group), cells cultured in normal culture medium (DMEM) served as negative control (control group), and cells cultured in osteogenic medium only served as positive control (osteo group).Because all drugs were dissolved in dimethyl sulfoxide (DMSO), this solvent was added to the drugs and control media at a dilution of 1 : 10,000 [10].Both osteogenic induction medium and basal culture medium were refreshed every three days for 3 weeks.

Osteogenic Assays
(1) Alizarin Red S Assay.At the end of the osteogenic differentiation period, the presence of calcifed mineralized nodules was detected and quantifed among the study groups.Te culture medium was removed, and the cells were fxed with a 10% formaldehyde solution at room temperature for 15 minutes.To eliminate any nonadherent cells, the plates were washed three times with phosphate-bufered saline (PBS).Subsequently, the cells were stained with a 20% Alizarin Red S solution (pH 4.2) for 30 minutes in the dark at room temperature.Following staining, the cells were rinsed three times with PBS to remove any excess stain, and they were imaged using an inverted light microscope (Labomed, USA).Te Alizarin Red staining was then solubilized with a 10% glacial acetic acid solution, and the produced color was measured using a benchtop microplate reader at 405 nm [20].All samples were performed in triplicate (n � 3), and the experiment was repeated three times.
(2) Alkaline Phosphatase (ALP) Activity Assay.To evaluate the alkaline phosphatase enzyme (ALP) activity, the monolayers of cells were rinsed twice with PBS, followed by an additional wash with 1 mL of alkaline phosphatase bufer (ALPB).Subsequently, each well was treated with 1 mL of ALPB and an equal volume of para-nitrophenylphosphate (p-NPP) that had been precooled to 4 °C.Immediately after adding the p-NPP solution, 50 μL aliquots were collected from each well and mixed with an equal volume of sodium hydroxide (NaOH) to stop the enzyme-substrate reaction.Tis sampling procedure was repeated every minute for a total of 10 minutes per well.In this assay, the ALP enzyme converts colorless p-NPP to yellow-color p-nitrophenolate (p-NP), which was measured by using a spectrophotometer at 405 nm.Te rate of p-nitrophenolate accumulation (absorbance) was plotted for each well against time, and the initial rate of the reaction, indicating the reaction rate, was determined by calculating the slope of the curve in each Advances in Pharmacological and Pharmaceutical Sciences experimental group [21].All samples were performed in triplicate (n � 3), and the experiment was repeated three times.

Determination of Osteogenic-Related Markers
(1) Assessment of RUNX2 and OC Using Western Blot Analysis.Bone markers, including RUNX2 and OC, were determined using western blot analysis.Total protein extraction was done using the ready-prepared TM protein extraction kit (total protein) according to the manufacturer's guidelines.Te lysed pellets from all diferent groups were treated with sodium dodecyl sulfate (SDS) sample bufer to obtain the total cellular protein, which was subsequently heated at 95 °C for 5 minutes at pH 6.8.Te protein concentrations were determined using the Bradford Protein Assay Kit.20 μg of proteins were then separated on a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difuoride membrane (PVDF).Te blot was run for 7 min at 25 V to allow protein bands transfer from gel to membrane using Bio-Rad Trans-Blot Turbo.After blocking with PVDF Blocking Reagent containing 20 mM Tris pH 7.5, 150 mM NaCl, 3% BSA, and 0.1% Tween 20, the membranes were incubated overnight at 4 °C with a 1 : 1000 dilution of RUNX2 primary antibody and a 1 : 500 dilution of OC primary antibody in tris-bufered saline with Tween 20 (TBST) bufer.Subsequently, the membranes were incubated with a horseradish peroxidase-(HRP-) conjugated secondary antibody solution (Goat anti-human IgG-HRP-1 mg Goat mAb) with a dilution of 1 : 5000 for 1 hour at room temperature.Te blot was then washed multiple times with TBST before the protein bands were visualized on a photographic flm (Sigma-Aldrich) [22].Each sample was performed in triplicate (n � 3).
(2) Determination of OPG Using ELISA Assay.Osteoprotegerin (OPG), a marker for bone metabolism, was quantifed using the enzyme-linked immunosorbent assay (ELISA) with a commercially available kit.Te ELISA procedure was carried out following the manufacturer's instructions.Te ELISA microplate was precoated with the primary antibody specifc to each marker.After adding samples or standards, a biotinylated detection antibody specifc for OPG was added.Avidin-Horseradish Peroxidase (HRP) conjugate was added successively to each well and incubated.Ten, wells were washed and the substrate was added for 15 minutes.Te enzyme-substrate reaction is terminated by the addition of sulfuric acid solution.Te assay demonstrated a sensitivity of 4.69 pg/mL for detecting OPG; the intra-assay and interassay coefcients of variation for the ELISA tests were found to be below 10% and 12%, respectively.Absorbance measurements at a wavelength of 450 nm were performed using a UVN-340 ASYS Hitech GmbH microplate reader [23].All samples were performed in triplicate (n � 3), and the experiment was repeated three times.

Assessment of Clopidogrel (CLP) and Ticagrelor (TICA)
Anti-Infammatory Efects on hAd-MSCs Te obtained results were presented as fold change using the 2 −ΔΔCt method and normalized to the reference gene β-actin.
A negative control that contains all the components of the reaction except for the cDNA was included in each PCR run to detect contamination or nonspecifc amplifcation.Te primer sequences used for amplifcation were assessed for specifcity by NCBI Primer-BLAST and are provided in Table 1.

Determination of Proinfammatory Markers Using
ELISA.Te proinfammatory markers, TNF-α and IL-6, were further quantifed using the enzyme-linked immunosorbent assay (ELISA) with a commercially available kit.Te assessment of proinfammatory markers was performed after incubating the cells with 1.8 μM TICA and 1.8 μM CLP for 7 days without osteogenic induction [25].Te ELISA procedure was carried out following the manufacturer's instructions as previously mentioned, using biotinylated detection antibody specifc for TNF-α and IL-6.Te assay demonstrated a sensitivity of 0.011 pg/ mL for TNF-α and IL-6 levels.Absorbance measurements at 450 nm were performed using a UVN-340 ASYS Hitech GmbH microplate reader.All samples were performed in triplicate (n � 3), and the experiment was repeated three times.

Statistical Analysis.
All experiments were conducted in triplicate (n � 3) and repeated in three independent runs.Statistical analysis was performed using the one-way ANOVA test, followed by pair-wise Tukey's post hoc comparisons using GraphPad Prism v8.1.0software (GraphPad Software, San Diego, CA, USA).Te results are expressed as the mean ± standard deviation (SD).P values less than 0.05 were considered statistically signifcant.

Isolation of Human Adipose-Derived Mesenchymal Stem
Cells (hAd-MSCs).Te isolated hAd-MSCs started to grow as adherent cells, attaining the characteristic fbroblast-like shape of the MSCs. Figure 1(a) shows the hAd-MSCs fve days after isolation (passage 0, P0).Te cells continue to grow, attaining the same shape until passage 4 (P4) (Figure 1(b)).All experiments were done using cells in P4.

Flow Cytometry Characterization.
Te results of fow cytometry showed that the isolated cells were positive for MSC surface markers including CD73, CD105, and CD90 and negative for hematopoietic markers including HLA-DR, CD34, and CD45 (Figure 2).Te percentages are presented relative to the proper expression of mesenchymal stem cell surface markers, indicating that isolated hAd-MSCs were a homogenous population of cells expressing MSC markers, while lacking hematopoietic markers fulflling the criteria of MSC characterization.

3.4.1.
Alizarin Red S Assay.Osteogenic diferentiation was confrmed by staining of calcium deposits (mineralized nodules) using Alizarin Red.After applying the osteogenic induction medium, the cells were observed regularly for morphological changes.Te undiferentiated control hAd-MSCs were cultured in DMEM-F12 medium and showed no Alizarin Red labeling (Figure 4(a)).In the study groups, the calcium deposits started to accumulate at the end of the third week of osteogenic diferentiation.Te staining became denser, and multiple isolated mineralized extracellular nodules appeared (Figures 4(b)-4(d)).Statistical analysis showed signifcant increase in stain density between groups, which was measured by using a spectrophotometer after stain solubilization (Figure 4(e)).Te results showed that the TICA group possessed the highest signifcant increase in absorbance (stain density) among the other study groups.Te mean absorbance values were 0.0586 ± 0.0008, 0.140 ± 0.0032, 0.116 ± 0.0008, and 0.1197 ± 0.0035; P < 0.0001 in the control, TICA, CLP, and osteo group, respectively.Nonsignifcant diference was observed in the CLP group compared to the osteo group (P � 0.3880).

Alkaline Phosphatase (ALP) Activity Assay.
Alkaline phosphatase (ALP) enzyme activity was analyzed in the study groups after osteogenic diferentiation.Results showed that, after culturing hAd-MSCs in osteogenic medium for 21 days, an increase in the ALP enzyme activity was observed in all study groups compared to the control group (Figure 5(a)).Statistical analysis was done by calculating the slope of each curve, demonstrating the initial rate of the reaction (p-NP accumulation).Te control group possessed the lowest rate of accumulation, while the TICA group showed the highest rate.Te ALP enzyme level increased signifcantly (P < 0.0001) in all groups in comparison with the control group as shown in Figure 5(b).Statistical analysis showed that ALP levels increased by 5 folds for the TICA group, 4.3 folds for CLP and 2.4 folds for the osteo group compared to the control group.CLP group revealed nonsignifcant diference (P � 0.9993) in comparison to the osteo group while the TICA group showed statistically signifcant increase in the ALP enzyme activity compared to all the study groups.

Te Expression of Osteogenic-Related Genes OC and RUNX2 Using Western Blot.
To assess the protein levels of OC and RUNX2 in the four experimental groups, western blot analysis was conducted.OC expression showed a statistically signifcant increase (P < 0.05) in the TICA group by 3 folds, 1.9 folds for the CLP group, and 2.2 folds for the osteo group in comparison with the control group (Figure 6(b)).Te expression of RUNX2 was signifcantly increased (P < 0.0001) by 4 folds for the TICA group, 2.7 folds for the CLP group, and 2.8 folds for the osteo group when compared to the control group (Figure 6(c)).Te CLP group showed nonsignifcant diference in the expression of OC and RUNX2 (P � 0.1451; P � 0.7592), when compared to the osteo group.On the other hand, the TICA group revealed statistically signifcant increase in the expression of OC and RUNX2 compared to all the study groups.Te full western blot images of Figure 6(a) were provided as Supplementary Data B.

Te Expression of OPG Protein Levels in the hAD-MSCs
Using ELISA.A signifcant increase in OPG protein concentration was observed between groups with the highest Advances in Pharmacological and Pharmaceutical Sciences percentage in the TICA group as shown in Figure 7. Te OPG mean values were 0.395 ± 0.025, 2.055 ± 0.195, 1.3 ± 0.13, and 1.405 ± 0.045 (P < 0.05) in the control, TICA, CLP, and osteo groups, respectively.Te CLP group showed no statistical signifcant diference (P � 0.7149) compared to the osteo group; however, the TICA group demonstrated a statistical signifcant increase in sOPG level in comparison to all the study groups.

Te Expression of Proinfammatory Markers (TNF-α and IL-6
) Levels in the hAD-MSCs.Te mRNA expression of proinfammatory markers (IL-6 and TNF-α) was frst evaluated among the three groups via RT-qPCR.Te results showed that the expression of IL-6 was statistically significant lower in both TICA and CLP groups compared to the control group (Figure 8(a)), with mean values (1.039 ± 0.039, 0.399 ± 0.024, and 0.453 ± 0.074; P < 0.0001) in the control, TICA, and CLP groups, respectively.Similar results were observed for TNF-α expression; the relative expression of TNF-α was signifcantly lower in both the TICA and CLP groups in comparison with the control group (Figure 8(b)), with mean values (1.002 ± 0.002, 0.301 ± 0.087, and 0.406 ± 0.038; P < 0.0001) in the control, TICA, and CLP groups, respectively.Regarding both infammatory markers expression, the TICA group showed lower expression compared to the CLP group; however, it was not statistically signifcant (P > 0.05).Tese results were confrmed by measuring these proinfammatory markers on protein levels among the study groups using ELISA assay, as previously mentioned.Te concentration of TNF-α showed a signifcant decrease in the TICA group compared to the control and CLP groups, while the CLP group showed nonsignifcant diference (P � 0.548) compared to the control

Discussion
Healing large bone defects is considered a signifcant challenge in the medical feld and can impede therapeutic interventions.While bone has inherent regenerative capabilities, pathologic or extensive fractures can hinder the natural healing process due to factors such as inadequate blood supply, infections in the bone or surrounding tissues, and other causes, leading to delayed repair [10].Traditional bone grafting methods have limitations, necessitating the exploration of alternative approaches for addressing bone defects [26].Tere are various surgical and pharmaceutical options available, but treating such conditions is challenging due to the complex processes involved in bone healing [10].
Limited research has been conducted on the osteogenic potential of antiplatelet drugs as TICA and CLP, and conficting fndings are found in the literature.Some studies have shown that CLP inhibits osteoblastic activity and bone regeneration [27], while others suggest that both TICA and CLP inhibit osteoclast diferentiation and enhance osteoblastic activity in vitro [10][11][12][13][14].In our study, we aimed to compare the osteogenic efects of CLP and TICA using a 1.8 μM drug concentration, which showed no cytotoxicity on stem cells after performing a viability assay (MTT) using serial dilutions of both drugs.In addition, this dose was previously investigated by Comibra et al. [14] on human bone marrow-derived MSCs using CLP and reported that the drug had a positive efect on MSCs.As far as we know, this is the frst study to examine and compare the osteogenic efects of both antiplatelet agents on hAd-MSCs.
In the present study, the osteogenic potential of TICA and CLP was determined on hAd-MSCs by several assays, including Alizarin Red S assay, ALP assay, bone markers expression measurement via ELISA, and western blot, in addition to detecting proinfammatory markers using RT-qPCR and ELISA technique.
Alizarin Red S is an in vitro assay used to detect osteogenic activity of cultured cells and quantify the produced  8 Advances in Pharmacological and Pharmaceutical Sciences calcifed nodules.Our results stated that the amount of calcium-rich extracellular matrix was not signifcantly different between the CLP and osteo groups (cells cultured in osteogenic medium only), while the TICA group showed a signifcantly higher amount of calcium-rich extracellular matrix compared to both groups.Alizarin Red S assay results were further supported by the ALP assay which was employed to determine alkaline phosphatase activity, a key enzyme for osteoblast diferentiation and mineralization, calorimetrically.Aligning with the Alizarin assay results, ALP activity showed the same pattern as the Alizarin assay results, and the TICA group showed an increase in enzyme activity compared to the CLP and osteo groups.Mediero et al. [10] investigated the local efects of TICA and CLP Advances in Pharmacological and Pharmaceutical Sciences in vitro and in vivo on bone defects.Te in vitro study showed that very low doses of both drugs (nM) inhibited osteoblast diferentiation, while higher doses (μM) had no negative efect on the osteogenic diferentiation of the cells using the Alizarin Red S assay.Te study also reported an increase in ALP protein expression in both groups in vivo using immunohistochemistry.
In contrast our results, Syberg et al. [27] studied the role of CLP in bone homeostasis in vitro and in vivo using a 10 μM dose of CLP on rat calvarial-derived osteoblast.Te study showed a signifcant decrease in ALP activity in vitro using ALP colorimetric assay and a signifcant decrease in ALP expression in vivo using RT-qPCR.Tis is not consistent with the results obtained from our study, which showed that CLP did not possess a signifcant reduction in ALP activity in comparison to the osteo group; this could be attributed to the diference in the doses used in both studies.
In the current study, the expression of diferent osteogenic markers was measured, including osteocalcin (OC), a protein produced by osteoblasts [28], and RUNX2, a transcription factor essential for osteoblasts diferentiation and proliferation, using western blot.In addition, osteoprotegerin (OPG), a soluble decoy receptor that inhibits osteoclast diferentiation and activation [23], was assessed using the ELISA technique.Our fndings revealed a signifcant increase in the expression levels of OC, RUNX2, and OPG in the TICA group compared to both the CLP and osteo groups.Our results agreed with Mediero et al. [10] who examined the expression of RUNX2 and OPG in vitro after the application of CLP and TICA.Te study reported that TICA signifcantly increased RUNX2 expression, while CLP showed a slight but nonsignifcant increase in the level of RUNX2.Regarding OPG expression, a signifcant increase was observed using both drugs; however, TICA showed a higher level than CLP.Syberg et al. [27] investigated the efect of CLP when applied to rat calvarial-derived osteoblasts using a 25 μM dose.Te study revealed that CLP decreased the expression of OC, while the expression of RUNX2 was unafected.Te diferences in results among this study and our fndings, as mentioned before, may originate from diferences in the doses of the drug used.
Upon thorough research in the literature, previous clinical and experimental studies were found investigating the efect of CLP on bone metabolism; however, these studies showed controversies regarding the drug efect.Lillis et al. [29] investigated the efect of perioperative use of CLP orally on bone healing in rabbit calvarial bone defects.Te study reported that continued perioperative oral use of CLP did not possess a negative bone efect but promoted fracture healing.
A cohort clinical study by Jørgensen et al. [12] investigated the relation between CLP administration and fracture incidence, in which all individuals had prescribed CLP during the years 1996-2008 in Denmark.Te study results revealed a dose-dependent association between fractures risk and CLP usage.In comparison to controls, low-dose exposure was associated with a lower fracture risk, while higher exposure was associated with an increased risk of fracture.Te study concluded that further research is required in order to investigate the potential efect of the antiplatelet drugs on bone metabolism in vivo.
Another recent study was conducted by Lv et al. [30] who collected data from the National Health and Nutrition Examination Survey (NHANES) and studied the association between osteoporosis or osteopenia using diferent antiplatelet drugs.Te study showed that antiplatelet drugs including CLP were associated with osteoporosis in female subjects but not in males and recommended that further studies are needed to confrm the results.
Te expression of proinfammatory markers such as IL-6 and TNF-α was determined in our study using RT-qPCR and ELISA.Our fndings revealed that the expression of both proinfammatory markers decreased signifcantly in the TICA and CLP groups in comparison to the control group.TICA showed a slight lower expression than CLP, which was statistically nonsignifcant.Te results were further confrmed by measuring the aforementioned proinfammatory markers on protein level using an ELISA assay.Te results showed that the TICA and CLP groups decreased both markers compared to the control group; moreover, the TICA group showed decreased levels of TNF-α compared to the CLP group.In line with our results, Coimbra et al. [31] investigated the anti-infammatory efect of CLP and aspirin on periodontitis in a rat model and showed that both drugs signifcantly reduced the levels of IL-6 and TNF-α.A clinical study conducted by Tomas et al. [32] investigated the effects of TICA and CLP on immune responses.Te results revealed that TICA signifcantly decreased IL-6 and TNF-α, while CLP signifcantly reduced TNF-α with a nonsignifcant decrease in IL-6.
In the light of the current study results, using a low concentration of TICA on hAd-MSCs showed signifcant increase in the mineralized extracellular matrix, ALP enzyme activity, and bone markers expression as OC, RUNX2, and OPG, in addition to its anti-infammatory efect through the reduction of IL-6 and TNF-α expression.In contrast, CLP did not show a signifcant positive impact on the bone metabolic markers; however, the drug possessed a signifcant anti-infammatory efect using the same respective dose.Te big controversies found in the literature about the CLP efect on bone metabolism and the results of our study suggest that the drug efect on bone metabolism is most probably dose dependent; the dose of CLP used in this study possessed no signifcant osteogenic potential.
In conclusion, the study showed that CLP did not possess osteogenic potential at the low dose used, compared to TICA which may possess a positive efect on hAd-MSC osteogenic diferentiation.Tese results could pave the way for in vivo studies and further investigations on bone regeneration.

Figure 3 :
Figure 3: Multilineage diferentiation of hAd-MSCs.(a) Adipogenesis was confrmed by oil droplets staining with Oil-Red compared to the control (b), (c) osteogenesis was confrmed by staining of mineralized nodules by Alizarin Red-S staining compared to the control (d), and (e) chondrogenesis was confrmed by staining proteoglycans with Alcian blue staining compared to the control (f ).Magnifcation: 100×; scale bar: 250 μm.

Figure 4 :
Figure 4: Calcium deposition, as a fnal product of the osteogenic diferentiation process, was assessed using Alizarin Red S staining on day 21 (Mag.100×).Scale bar: 250 μm.(a) Control, (b) ticagrelor (TICA), (c) clopidogrel (CLP), (d) osteogenic medium, and (e) absorbance values (after stain solubilization) reported as mean ± SD of three independent experiments.* P signifcant diference of the respective group compared to the control.# P signifcant diference of the respective group compared to the osteo group.$ P signifcant diference of the respective group compared to the CLP group.Data are expressed as mean ± SD of three independent experiments.

Figure 5 :Figure 6 :
Figure 5: ALP activity after 21 days of osteogenic induction.(a) Representative kinetic profle of ALP assay demonstrating accumulation of the yellow p-nitrophenolate product over time among the diferent groups.(b) Rate of accumulation of the yellow p-NP was determined by calculating the slope of each reaction.Te values reported are the means ± SD of three independent experiments.* P signifcant diference of the respective group compared to the control (cells in normal medium).# P signifcant diference of the respective group compared to the osteo group (osteogenic medium).$ P signifcant diference of the respective group compared to the CLP group.P values <0.0001 are statistically signifcant.Data are expressed as mean ± SD of three independent experiments.

Figure 7 :Figure 8 :
Figure7: Osteoprotegerin (OPG) protein expression among the four groups after 21 days using ELISA.* P signifcant diference of the respective group compared to the control (cells in normal medium).# P signifcant diference of the respective group compared to the osteo group (osteogenic medium).$ P signifcant diference of the respective group compared to the CLP group.P values <0.05 are statistically signifcant.Te data are expressed as mean ± SD of three independent experiments.
in fnal volume of 20 μL per sample.Te PCR thermal cycling parameters for mRNA were 2 min at 50 °C, 30 s at 95 °C, 35 cycles of 95 °C for 10 s, and 60 °C for 40 s.

Table 1 :
Sequence for primers used in RT-qPCR.