The Impact of Curcumin on Bone Osteogenic Promotion of MC3T3 Cells under High Glucose Conditions and Enhanced Bone Formation in Diabetic Mice

: Diabetic osteoporosis (DOP) is characterized by impaired bone microstructure and reduced bone density resulting from high glucose levels. Curcumin (CURC) is extensively applied in the treatment of inﬂammation-associated diseases. However, the e ﬀ ect of curcumin on bone metabolism in diabetic osteoporosis is unclear. Therefore, this study investigated the optimal concentration of curcumin on enhancing osteogenesis in diabetic osteoporosis. Osteoblasts were treated with a high or low concentration of curcumin under a series of concentrations of high-glucose conditions. Type 2 diabetic mice were intervened with curcumin. Cell proliferation, apoptosis, and osteogenesis-related gene expressions were evaluated by CCK-8, ﬂow cytometry, and real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). Bone formation was evaluated by histological staining. The ﬁndings revealed that curcumin suppressed apoptosis and enhanced proliferation and osteogenesis-related gene expressions of osteoblasts under high glucose concentrations ( p < 0.05). The histological sections displayed reduced bone destruction and increased the growth rate of trabecular bone and the bone density of diabetic mice treated with curcumin, compared to diabetic mice. These results showed that curcumin could reverse the harmful e ﬀ ects of diabetic osteoporosis in a dose-dependent manner, and 10 µ mol / L was regarded as the optimal concentration, which supports the potential use of curcumin for bone regeneration under high glucose concentrations.


Introduction
Diabetes mellitus is characterized by hyperglycemia caused by decreased insulin sensitivity or insulin deficiency [1], which has been regarded as a significant risk factor that threatens human health. Based on the statistical reports from the International Diabetes Federation (IDF), approximately 451 million diabetic patients suffered from diabetic complications in 2017 [2]. More seriously, the number is predicted to significantly increase to 590 million by 2035. Among various diabetic complications, diabetic osteoporosis (DOP) results in low bone mass, impaired bone microstructure, and reduced bone mineral density (BMD) [3,4]. Research has demonstrated a more than 60% higher incidence of

. Cell Proliferation
Cell viability was determined by a Cell Counting Kit-8 (Beyotime, Shanghai, China). The MC3T3-E1 cells were seeded at 2 × 10 3 cells per well in 96-well culture plates for 24, 48, and 72 h. At the above time points, the cells were washed twice with PBS solution, and 250 µL fresh culture medium with 25 µL CCK-8 reagent was sequentially added to each sample. After incubation for 2 h at 37 • C, 100 µL medium was transferred to a 96-well plate and measured at 450 nm by using a micro-plate reader (Infinite M200, Tecan, Männedorf, Switzerland).

Cell Apoptosis
Annexin V-fluoroisothiocyanate (FITC)/propidium iodide (PI) double staining (Dojindo, Japan) was used to determine cell apoptosis. Cells were seeded at 1 × 10 6 cells per well in 6-well plates and cultured in growth media for 24 h. Subsequently, the medium was replaced with fresh growth medium containing different concentrations of glucose and curcumin as described above. After 48 h, cells were stained with PI and Annexin V-FITC in a dark room for 15 min, according to manufacturer instructions, and analyzed by flow cytometry (Cytomics FC 500 MCL, Beckman Coulter, Brea, CA, USA).

Experimental Animals and Grouping
The protocol was approved by the Ethical Committee and the Laboratory Animal Center of China medical university, Shenyang, Liaoning, China. Eighteen 10-week-old specific pathogen-free (SPF) male mice from the Jackson Laboratory (Bar Harbor, ME, USA) were maintained at a constant temperature (22-25 • C) in a 12 h light/dark cycle and fed with a standard laboratory diet and water.
Six non-diabetic BLKS/jdb/m male mice were selected as the control group (db/m, n = 6, weight: 18.4-10.7 g). Twelve spontaneous diabetic BLKS/jdb/db male mice were randomly separated into the diabetic group (db/db, n = 6, weight: 35.9-40.9 g) and the curcumin-treated diabetic group (db/db + C, n = 6, weight: 35.7-40.3 g). Each group was kept in the same cage. The curcumin-treated group was intragastrically given a dose of 200 mg/kg/d curcumin for 10 weeks, and the control group and diabetic group were given equivalent volumes of solution (normal saline + 0.1% DMSO) for 10 weeks. Curcumin was dissolved in DMSO, then diluted with normal saline to make the content of DMSO 0.1% and administered daily by gavage. All groups were sacrificed at 20 weeks, and mandibles (3 mm × 3 mm) were harvested and fixed in 4% paraformaldehyde solution for 7 days at 4 • C in the dark. The Block sections were sequentially dehydrated in ascending serial concentrations of ethanol, cleared with xylene twice, and embedded in poly(methyl methacrylate). Serial sections with a thickness of 70-80 µm were cut parallel to the coronal plane and prepared for fluorescence microscopy and histological staining. All analyses of in vivo experiments were made by an experienced pathologist (blind to the treatments of the mice) in order to characterize any changes.

Polyfluorochrome Sequential Labeling of Bone
Bone formation was assessed by sequential labeling with Calcein and tetracycline (Sigma-Aldrich), which were deposited at the active site of mineralization. The fluorochromes were administered by intraperitoneal injection in the following sequence: calcein (20 mg/kg) was added to 2% NaHCO 3 buffer and given to three 19-week-old mice randomly selected from each group (n = 9). Four days later, the same mice were injected with tetracycline (30 mg/kg) in 0.9% normal saline. The sections were observed by fluorescence microscopy.

Histological Analysis
The osteogenesis effect of curcumin treatment in diabetic mice was evaluated by Masson's Trichrome staining (Beyotime). The sections were preserved in well-prepared Weigert's hematoxylin solution for 10 min. After rinsing with running tap water and acetified water, the slides were firstly stained by Masson Fuchsin Acid Complex for 5 min, then immersed in 2% glacial acetic acid solution, 1% phosphomolybdic acid, 2% aniline blue, and 0.2% acetic acid in sequence. Finally, sections were fixed into neutral gum for microscopy observation.

Statistical Analysis
GraphPad Prism 5.0 software (San Diego, CA, USA) was utilized to perform statistical analysis. The quantitative data were depicted as mean ± standard deviation (n = 3). One-way ANOVA and multi-way ANOVA were used to calculate the statistical significance among glucose-treated groups and curcumin-treated groups, respectively. Statistically significant differences between each two groups at each time point were evaluated using the t-test. A p value of less than 0.05 was considered statistically significant.

Results and Discussion
Previous studies have demonstrated high glucose inhibits proliferation and differentiation of osteoblasts [25]. Curcumin promotes osteogenic differentiation of osteoblasts [26]. The concentration of curcumin at 15 µmol/L showed a stronger osteogenic effect than that at 10 µmol/L, whereas it was not statistically significant, and the concentration of curcumin at 25 µmol/L showed obvious cytotoxicity [27,28]. Therefore, we grouped within the concentration range of 10µmol/l in our study. In addition, curcumin can induce cancerous cell apoptosis in specific doses and times through different pathways. However, it does not have a significant effect on normal cells in the same doses/times, which suggests that sensitivity of cells to curcumin varies under different conditions [28]. Therefore, the protective effect of curcumin on high glucose-induced apoptosis and the optimal concentration of curcumin in promoting osteogenesis in diabetic osteoporosis under different glucose concentrations are still unclear. Our findings proved that curcumin effectively alleviates high glucose-induced negative effects on osteoblasts, its optimal concentration, and osteogenic impact on osteogenesis of osteoblasts under a series of glucose concentrations.

Effect of Curcumin on the Viability of MC3T3 Cells in High-Glucose Conditions
The proliferation of MC3T3 treated by curcumin under different concentrations of glucose was investigated by using a CCK-8 kit at 24, 48, and 72 h. No differences could be found among each group at 24 h. Cell viability in the control group increased most up to 48 h, and was significantly higher than that in the other groups (p < 0.05). The high-glucose group had the lowest cell viability, indicating that a higher concentration of glucose severely affected cell viability. When curcumin was introduced, cell viabilities in different glucose groups significantly increased (p < 0.05), whereas there were no differences between low and high concentrations of curcumin treatment. At 72 h, the cell viability in the curcumin-treated groups was significantly higher than that in the glucose groups (p < 0.05; Figure 1). Three concentrations of glucose were selected in present study. At 16.5 mmol/L glucose, there was a negative influence on cell viability. No differences were found on the effect of concentrations of curcumin among curcumin-treated groups.
Coatings 2020, 10, x FOR PEER REVIEW 5 of 11 concentration of curcumin in promoting osteogenesis in diabetic osteoporosis under different glucose concentrations are still unclear. Our findings proved that curcumin effectively alleviates high glucose-induced negative effects on osteoblasts, its optimal concentration, and osteogenic impact on osteogenesis of osteoblasts under a series of glucose concentrations.

Effect of Curcumin on the Viability of MC3T3 Cells in High-Glucose Conditions
The proliferation of MC3T3 treated by curcumin under different concentrations of glucose was investigated by using a CCK-8 kit at 24, 48, and 72 h. No differences could be found among each group at 24 h. Cell viability in the control group increased most up to 48 h, and was significantly higher than that in the other groups (p < 0.05). The high-glucose group had the lowest cell viability, indicating that a higher concentration of glucose severely affected cell viability. When curcumin was introduced, cell viabilities in different glucose groups significantly increased (p < 0.05), whereas there were no differences between low and high concentrations of curcumin treatment. At 72 h, the cell viability in the curcumin-treated groups was significantly higher than that in the glucose groups (p < 0.05; Figure 1). Three concentrations of glucose were selected in present study. At 16.5 mmol/L glucose, there was a negative influence on cell viability. No differences were found on the effect of concentrations of curcumin among curcumin-treated groups. Figure 1. Effects of curcumin on viability of MC3T3 cells in high-glucose conditions. Cell viability was measured using the CCK8 assay after culturing for 24, 48, and 72 h, separately. The bar chart shows the mean optical density (OD) values (n = 3). *p < 0.05 vs the control group; #p < 0.05 vs the medium glucose group; ▲p < 0.05 vs the high-glucose group.

Effect of Curcumin on Apoptosis of MC3T3 Cells in High-Glucose Conditions
Flow cytometry showed that curcumin treatment had an effect on glucose-induced apoptosis in MC3T3 cells (Figure 2A). The first-quadrant cells were necrotic and consisted of late apoptotic cells, which could be simultaneously stained with Annexin V-FITC and PI. The second-quadrant cells were PI-negative. In the third quadrant, normal living cells were not stained with Annexin V-FITC or PI. The fourth-quadrant cells were only stained with Annexin V-FITC, indicating early apoptotic cells. The early apoptosis rates of the control and glucose groups were 9.03% ± 0.67%, 14.70% ± 0.26%, and 18.70% ± 0.61%, and the curcumin-treated groups were 13.30% ± 0.10%, 12.00% ± 0.85%, 16.80% ± 0.20%, and 13.77% ± 0.21%. Glucose significantly increased the rate of apoptosis (p < 0.05), whereas treatment with both low and high doses of curcumin significantly decreased the apoptosis rate for cells under different concentrations of glucose (p < 0.05). In the high glucose group, the apoptosis rate in the high concentration of the curcumin group was significantly lower than that in the low concentration of the curcumin group (p < 0.05; Figure 2B), whereas there were no differences in the medium glucose group. It can be concluded that treatment with curcumin significantly protected cells from hyperglycemia-triggered apoptosis of MC3T3 cells under high-glucose Figure 1. Effects of curcumin on viability of MC3T3 cells in high-glucose conditions. Cell viability was measured using the CCK8 assay after culturing for 24, 48, and 72 h, separately. The bar chart shows the mean optical density (OD) values (n = 3). *p < 0.05 vs the control group; #p < 0.05 vs the medium glucose group; p < 0.05 vs the high-glucose group.

Effect of Curcumin on Apoptosis of MC3T3 Cells in High-Glucose Conditions
Flow cytometry showed that curcumin treatment had an effect on glucose-induced apoptosis in MC3T3 cells (Figure 2A). The first-quadrant cells were necrotic and consisted of late apoptotic cells, which could be simultaneously stained with Annexin V-FITC and PI. The second-quadrant cells were PI-negative. In the third quadrant, normal living cells were not stained with Annexin V-FITC or PI. The fourth-quadrant cells were only stained with Annexin V-FITC, indicating early apoptotic cells. The early apoptosis rates of the control and glucose groups were 9.03% ± 0.67%, 14.70% ± 0.26%, and 18.70% ± 0.61%, and the curcumin-treated groups were 13.30% ± 0.10%, 12.00% ± 0.85%, 16.80% ± 0.20%, and 13.77% ± 0.21%. Glucose significantly increased the rate of apoptosis (p < 0.05), whereas treatment with both low and high doses of curcumin significantly decreased the apoptosis rate for cells under different concentrations of glucose (p < 0.05). In the high glucose group, the apoptosis rate in the high concentration of the curcumin group was significantly lower than that in the low concentration of the curcumin group (p < 0.05; Figure 2B), whereas there were no differences in the medium glucose group. It can be concluded that treatment with curcumin significantly protected cells from hyperglycemia-triggered apoptosis of MC3T3 cells under high-glucose conditions. This was shown in a dose-dependent manner under a high concentration of glucose, but not under a low concentration of glucose, under which a low concentration of curcumin can achieve an optimal protective effect on cells. This expands on previous observations that curcumin mitigates hyperglycemia-related clinical symptoms [29,30].
Coatings 2020, 10, x FOR PEER REVIEW 6 of 11 conditions. This was shown in a dose-dependent manner under a high concentration of glucose, but not under a low concentration of glucose, under which a low concentration of curcumin can achieve an optimal protective effect on cells. This expands on previous observations that curcumin mitigates hyperglycemia-related clinical symptoms [29,30].

Effect of Curcumin on Osteogenic Differentiation of MC3T3 Cells in Glucose Conditions
The expressions of osteogenesis-related genes Runx2, Opn, and Col-1 in MC3T3 cells related to different concentrations of glucose and curcumin were evaluated using real-time quantitative PCR (RT-qPCR). Runx2 is crucial for osteoblastogenesis, regulates the differentiation, maturation, and bone formation of osteoblasts, and activates the expression of other osteogenic genes, such as Col-1 during early stages and Opn during late stages [31,32]. Col-1 is an important organic component of the bone matrix and the most crucial extracellular protein in bone, which initially provides a structural framework for inorganic deposition [33]. Opn is a phosphorylated glycoprotein secreted by osteoblasts and can promote biomineralization and bone remodeling [18]. In addition, the specific binding of Opn to Col-1 may naturally localize Opn, influencing the adhesion, differentiation, and function of osteoblasts [34]. In the present study, the expressions of Runx2, Opn, and Col-1 genes were significantly downregulated in the high glucose-treated group and were significantly lower than those of the control and medium glucose-treated groups (p < 0.05), indicating that a higher concentration of glucose severely affected expression of osteogenesis-related genes. When curcumin was introduced, the expressions of Runx2, Opn, and Col-1 genes were significantly upregulated at different concentrations of glucose (p < 0.05). The expressions of Runx2 and Col-1 genes in the high curcumin-treated group were significantly higher than those in the low curcumin-treated group (p < 0.05), whereas there was no significant difference in the expression of Opn between the low and high curcumin-treated groups (Figure 3). The results demonstrated that the osteogenic effect of curcumin was dose-dependent in the early stage of osteogenesis, but not in the late stage. It is possible that curcumin might predominantly upregulate the expression of Runx2, which in turn enhanced Col-1 expression in precursor osteoblasts, whereas Opn has a unique regulation mechanism, which is different from the combination of Col-1 and Runx2. It is interesting to further investigate the mechanism of curcumin upregulating Runx2, Opn, and Col-1 expressions in osteoblasts and the relationship between the expression of each gene. The current results indicated that curcumin could induce differentiation of MC3T3 cells from pre-osteoblasts to osteoblasts in different concentrations of glucose in a dose-dependent manner. Curcumin could serve as an effective method to recover glucose-suppressed osteogenic differentiation in precursor osteoblasts. In addition, 10 µmol/L curcumin was found to be an effective concentration that could promote osteogenic differentiation of MC3T3 under different concentrations of glucose.

Curcumin Treatment Improved Alveolar Bone Formation in Diabetic Mice
Diabetes mellitus (DM) is a significant risk factor for osteoporosis. The proliferation and differentiation of osteoblasts in the alveolar bone of diabetic patients remains highly relevant in the daily work of dentists, as they can impact treatment for many oral diseases such as dental implants and oral surgery. Hyperglycemia is often displayed in diabetic patients and has a negative effect on alveolar bone reconstruction [35].
Previous studies have suggested a positive effect of curcumin on bone formation in diabetes and diabetes-related periodontitis [22]. However, few studies have investigated the effectiveness of natural curcumin on alveolar bone formation in diabetic osteoporosis, and there is also a lack of investigations into cortical bone and cancellous bone.

Curcumin Treatment Improved Alveolar Bone Formation in Diabetic Mice
Diabetes mellitus (DM) is a significant risk factor for osteoporosis. The proliferation and differentiation of osteoblasts in the alveolar bone of diabetic patients remains highly relevant in the daily work of dentists, as they can impact treatment for many oral diseases such as dental implants and oral surgery. Hyperglycemia is often displayed in diabetic patients and has a negative effect on alveolar bone reconstruction [35].
Previous studies have suggested a positive effect of curcumin on bone formation in diabetes and diabetes-related periodontitis [22]. However, few studies have investigated the effectiveness of natural curcumin on alveolar bone formation in diabetic osteoporosis, and there is also a lack of investigations into cortical bone and cancellous bone.
The present study evaluated cortical and cancellous bones by fluorescent double-labeling and Masson staining, respectively. The results revealed that significant differences in alveolar bone loss were observed in curcumin-treated groups when compared with diabetic mice. A general observation from fluorescence images ( Figure 4A) was that curcumin treatment significantly enhanced alveolar cortical bone continuity and thickness when compared to diabetic mice, which was similar to that of non-diabetic mice. A general observation from Masson staining ( Figure 4C) was that curcumin treatment extremely enhanced alveolar cancellous bone density and mineralization in diabetic mice, which is similar to that of non-diabetic mice. Curcumin treatment significantly enhanced the trabecular bone formation rate compared to diabetic mice (1.60 ± 0.11 um/d (diabetic) vs. 0.55 ± 0.05 um/d (curcumin), p < 0.05), and there was no significant difference observed when compared to the non-diabetic group (1.18 ± 0.11 um/d (non-diabetic)) ( Figure 4B). These results show that curcumin treatment could improve alveolar bone structure in diabetes, and this is in agreement with the results of osteogenesis of MC3T3-E1 cells in response to curcumin treatment in vitro.
Diabetic hyperglycemia can trigger excessive production of ROS and increase oxidative stress, which leads to damaged macromolecular substances (nucleic acids and lipids), induction of cell apoptosis, and inhibition of osteogenic differentiation [36]. Bone protective effects of curcumin on diabetic mice may be explained by reducing the production of ROS induced by high glucose, enhancing antioxidant defense, and even further regulating the osteoimmunological RANK/RANKL/OPG pathway, which inhibits the expression of RANKL and promotes the expression of OPG in osteoblasts, results in an increase in bone formation and a decrease in bone resorption, thus offsetting the negative effects caused by high-glucose conditions. However, the specific mechanism remains to be confirmed by further research.
Coatings 2020, 10, x FOR PEER REVIEW 9 of 11 RANK/RANKL/OPG pathway, which inhibits the expression of RANKL and promotes the expression of OPG in osteoblasts, results in an increase in bone formation and a decrease in bone resorption, thus offsetting the negative effects caused by high-glucose conditions. However, the specific mechanism remains to be confirmed by further research. , diabetic, and curcumin-treated groups, respectively. Alveolar cortical bone continuity and thickness improved in the db/db + C group compared with that in the db/db group, which was similar to that of the db/m group. (B) The bar chart shows the rate of trabecular formation. *p < 0.05. (C) Masson staining images of alveolar bone in non-diabetic (control), diabetic, and curcumin-treated groups, respectively. Alveolar cancellous bone density and mineralization improved in the db/db + C group compared with that in the db/db group, which was similar to that of the db/m group.

Conclusions
The results demonstrated that curcumin could protect cells from high glucose-mediated cytotoxicity, promote osteogenic differentiation of mouse precursor MC3T3 cells in a dose-dependent manner, and significantly improve alveolar bone formation in type 2 diabetic mice. These findings provide new insights into the pathogenesis of diabetes-related osteoporosis and indicate that curcumin may be valuable for prevention and inhibition of diabetes-related osteoporosis, providing a theoretical basis for the clinical treatment of alveolar bone disease in diabetic patients.   , diabetic, and curcumin-treated groups, respectively. Alveolar cortical bone continuity and thickness improved in the db/db + C group compared with that in the db/db group, which was similar to that of the db/m group. (B) The bar chart shows the rate of trabecular formation. *p < 0.05. (C) Masson staining images of alveolar bone in non-diabetic (control), diabetic, and curcumin-treated groups, respectively. Alveolar cancellous bone density and mineralization improved in the db/db + C group compared with that in the db/db group, which was similar to that of the db/m group.

Conclusions
The results demonstrated that curcumin could protect cells from high glucose-mediated cytotoxicity, promote osteogenic differentiation of mouse precursor MC3T3 cells in a dose-dependent manner, and significantly improve alveolar bone formation in type 2 diabetic mice. These findings provide new insights into the pathogenesis of diabetes-related osteoporosis and indicate that curcumin may be valuable for prevention and inhibition of diabetes-related osteoporosis, providing a theoretical basis for the clinical treatment of alveolar bone disease in diabetic patients.