**1. 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 bone fracture in diabetic patients than that of unaffected patients [5–7]. More importantly, the delayed

union of bone fracture after surgery in clinic severely affects patients' physical function and even mental health.

The traditional method for the therapy of type 2 diabetes mellitus is to inject insulin to reduce blood glucose levels, but therapy has hardly promoted bone formation [8]. Polypeptides, hormones, and genes are also used locally as bioactive molecules to enhance bone formation. However, the instable status, risk of an immunological inflammatory response, and the high cost of these molecules need to be carefully studied [9–11].

Recent investigations have focused on natural components that have no related side effects and promote osteoimmunomodulation at low cost [12]. Curcumin (CURC), derived from the plant *Curcuma longa*, is a bioactive component of turmeric with the ability to modulate the immune system [13]. Although curcumin has poor water solubility and low bioavailability and stability, some studies have confirmed that drug carriers such as proteins, polymeric particles, and polylactic-glycolic acid copolymer (PLGA) microspheres can effectively solve this problem and give full play to its antioxidant, anti-inflammatory, and anti-hyperglycemic properties [14–18]. Natural or chemically modified curcumin could upregulate insulin sensitivity and reduce glucose and glycosylated hemoglobin levels, which has great potential as an alternative therapeutic option for diabetes mellitus and its complications [19,20]. More importantly, it is reported that curcumin could suppress osteoclast activities by inhibiting the expression of transcription factor AP-1 [21]. Moreover, curcumin, in combination with insulin, inhibits alveolar bone loss of experimental periodontitis in diabetic rats [22].

Although several studies have reported the effect of curcumin on diabetic bone formation [23,24], there is a lack of scientific information on the concentration of curcumin and its toxicity. The concentration of glucose in vitro has also not been systematically investigated. Thus, it is important to confirm the optimal concentration of curcumin and its effect on osteogenesis under a series of high glucose concentrations.

Therefore, our study investigated the optimal concentration of curcumin, its toxicity, and osteogenic effect on osteogenesis of osteoblasts under a series of glucose concentrations in vitro. The bone density and growth rate of type 2 diabetic mice in the presence of curcumin were evaluated by histological sections.

#### **2. Materials and Methods**

#### *2.1. In Vitro Analysis*

#### 2.1.1. Cell Culture

Mouse osteoblast precursor MC3T3 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in α-MEM (Hyclone Laboratories, Inc., Logan, UT, USA) containing 10% fetal bovine serum (FBS) (Gemini Bio-Products, West Sacramento, CA, USA) and 1% streptomycin (Hyclone). Osteogenic differentiation medium contained 10% FBS, 1% streptomycin, 1% dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), 50 μg/mL L-ascorbic acid (Sigma-Aldrich), and 10 mmol/L β-sodium glycerophosphate (Sigma-Aldrich.) and was used for osteogenic differentiation tests. The cells were cultured at 37 ◦C with 5% CO2. The culture medium was replaced every 2 days until the cells reached 80%–100% confluence. Experiment groups were divided based on the concentrations of curcumin and glucose, which are listed in Table 1.


**Table 1.** Experiment groups and abbreviations.
