*3.3. E*ff*ect of Curcumin on Osteogenic Di*ff*erentiation 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.

**Figure 3.** *Cont*.

**Figure 3.** Effects of curcumin on osteogenic differentiation of MC3T3 cells in glucose conditions. Expressions of (**A**) Runx2, (**B**) Opn, and (**C**) Col-1 were measured by reverse transcription quantitative polymerase chain reaction (RT-qPCR; *n* = 3). The results are shown as a relative expression level of the target gene using glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) as the inner reference gene, and analyzed by 2-ΔΔCt. Data are shown as mean ± SD. \**p* < 0.05.

## *3.4. 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.

**Figure 4.** Evaluation of the osteogenic effect of curcumin on bone formation in vivo. (**A**) Fluorescent microscopy images of alveolar bone in non-diabetic (control), 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.
