BMP-2 plays a powerful regulatory role in bone and cartilage formation and repair, making it a pivotal component in bone tissue engineering. Physiologically, BMP-2 levels in human natural bone are low, approximately 1–2 ng per gram of cortical bone, markedly contrasting with the initial therapeutic doses for long bone defects (10–12 mg) [26]and the currently recommended concentration (1.5 µg/kg) [27]. To date, there exists no standardized dosage for BMP-2, and in clinical practice, achieving optimal osteogenic effects often requires dosages and concentrations significantly exceeding normal physiological levels. However, studies have revealed that the osteogenic efficacy of BMP-2 is not strongly correlated with dosage; instead, maintaining a specific concentration for sustained release proves more crucial [5, 28]. Conversely, the administration of BMP-2 in large doses and high concentrations can lead to adverse reactions, including local inflammation, hematoma formation, ectopic bone formation, and potentially tumorigenesis [29, 33]. To attenuate the dosage of BMP-2 while ensuring consistent and sustained drug concentrations, current strategies emphasize investigating drug synergies and controlled-release technologies. Howard et al. [34] utilized self-assembling layer-by-layer film deposition to incorporate a modest BMP-2 dosage (0.5 µg) into films for rat cranial defect repair. Their findings demonstrated that BMP-2 sustained release over 30 days yielded superior osteogenic effects compared to shorter release durations (2, 4, and 14 days) at the same dosage, validating the impact of BMP-2 release kinetics on therapeutic outcomes. Despite various approaches employed to reduce BMP-2 dosage, it still exceeds physiological levels in native bone. Studies have validated the osteogenic enhancement of BMP-2 by growth factors such as Vascular Endothelial Growth Factor (VEGF), basic Fibroblast Growth Factor (FGF), and Platelet-Rich Plasma (PRP), which exhibit synergistic effects when combined with BMP-2 [35–38]. Compared to growth factors, small molecule drugs like DEX, simvastatin, hydroxycholesterol, and estrogen can also enhance the osteogenic action of BMP-2 [10, 11, 39–42], and they are more readily available and less expensive. Studies indicate that the effect of DEX on bone may be related to the concentration used, with low concentrations (< 10− 8 M) having an osteogenic effect [15, 43]and high concentrations (> 10− 6 M) having an inhibitory effect [14, 17]. For different species, cell lines, and culturing methods, the concentrations of BMP-2 and DEX used also vary, usually lower in in vitro experiments. Based on previous related studies [13, 15, 16, 44], this experiment used low concentrations of BMP-2 (100 ng/mL) combined with low concentrations of DEX (10− 8 M) to study their osteogenic effects on MC3T3-E1 cells in vitro.
MC3T3-E1 cells are a cell line widely used in osteogenesis research. Under osteogenic induction conditions, they can differentiate into osteoblasts and express osteogenesis-related specific genes such as Alkaline Phosphatase (ALP), OCN, and Bone Morphogenetic Proteins (BMP). Moreover, MC3T3-E1 cells have strong proliferative capabilities and can be passaged indefinitely, overcoming the limitations of primary cells, such as poor proliferative ability, long culture periods, and weak transfection capability. As precursors to osteoblasts, they can simulate the entire process of osteogenesis and are commonly used in studies on bone biomaterials and bone regeneration [45]. In this experiment, MC3T3-E1 cells, which were subjected to predetermined concentrations of BMP-2 and DEX induction, exhibited no notable decrease in cell count after 24 hours of culture compared to the control group, as observed under an inverted and fluorescence microscope. Notably, cells maintained distinct contours and exhibited robust spreading, indicative of heightened cell viability. The preservation of cellular morphology, including intact cytoskeletal structures, further suggested optimal cellular function. Additionally, the experimental group displayed healthy cytoskeletal integrity, signifying cellular robustness, alongside notable cytoplasmic protrusions, indicative of MC3T3-E1 cell differentiation towards osteoblasts. The cell survival rate assessed by the CCK-8 assay showed that after 5 days of culture, the survival rate in all groups was above 90% compared to the control group, indicating that the selected concentrations of BMP-2 and DEX have no significant cytotoxic effects.
This experiment focuses on achieving effective osteogenesis while mitigating the dosage and adverse reactions associated with BMP-2, accomplished through its synergistic action with DEX. Jiang F et al., [46] found that low doses of BMP-2 did not promote new bone formation but triggered M1 macrophages to release IL-1β. The addition of DEX promoted osteogenesis by inhibiting M1 polarization and enhancing BMP-2 induced differentiation of Mesenchymal Stem Cells (MSCs). Similarly, Li L and colleagues [47] developed a nano-particle-embedded electrospun fiber scaffold for dual delivery of BMP-2 and DEX, showing strong induction of osteoblast differentiation due to the synergistic action of BMP-2 and DEX, with dual-drug-loaded nano-fiber scaffolds demonstrating the best repair effects. In line with these findings, our experiments validated the enhanced osteogenic effect of low-concentration DEX on low-concentration BMP-2 through various assays including ALP activity, in vitro mineralization, and PCR of osteogenesis-related genes. Alkaline Phosphatase (ALP) is one of the marker enzymes for osteoblast maturation and an early marker of cellular osteogenesis, reflecting the activity and function of osteoblasts. Studies have shown ALP expression within 3 days of osteoblast culture, peaking at 14 days [48]. Our experiment observed ALP expression at days 7 and 14, with the Control and BMP-2(-)DEX(+) groups showing weaker expression and only slight increases over time, indicating weak osteogenic differentiation in basic medium and DEX alone. The BMP-2(+)DEX(+), BMP-2(+)DEX(-), and Osteoinductive groups showed increased ALP activity over time, with the BMP-2(+)DEX(+) group displaying the strongest activity at all tested times, indicating a synergistic effect of combined BMP-2 and DEX use.
Alizarin Red Staining (ARS) detected the formation of calcium nodules after osteogenic differentiation. By day 14, calcium nodule formation commenced, reaching its peak between days 21 and 28, serving as a critical late-stage marker of osteogenesis. Through ARS and quantitative analysis at days 14 and 21, the Control and BMP-2(-)DEX(+) groups exhibited only a mild increase in staining over time, with limited calcium deposition. Conversely, the BMP-2(+)DEX(+), BMP-2(+)DEX(-), and Osteoinductive groups displayed progressively deeper staining and increased calcium nodules, with the BMP-2(+)DEX(+) group exhibiting the most pronounced calcium deposition, thereby further confirming the enhancing effect of DEX on BMP-2's osteogenic action. The expression of genes such as ALP, Runx2, Col-1, and OCN served as crucial markers of osteoblastic bone function. ALP, Runx2, and Col-1 were expressed within the first three days post-osteogenic induction, while OCN expression became detectable after 3 days, increasing over time [14]. On day 7 of osteogenic induction in MC3T3-E1 cells, the expression of osteogenesis-related genes ALP, Runx2, Col-1, and OCN was evident, with minimal expression observed in the Control and BMP-2(-)DEX(+) groups, and the most robust expression observed in the BMP-2(+)DEX(+) group, surpassing that of BMP-2 or DEX used alone. This genetic evidence confirms that the combined use of BMP-2 and DEX can promote heightened expression of osteogenic genes, demonstrating a synergistic effect.
The BMPs-Smad pathway is the classical signaling pathway for BMP-2-induced osteogenic differentiation. BMP-2 first binds to type I and II transmembrane receptors to form a heterodimer, activating the Smad complex to form phosphorylated Smad1/5/8, which then regulates the expression of downstream Runx2 to promote osteogenic differentiation [22, 24]. The p38-MAPK pathway is one of the non-classical signaling pathways for BMP-2-induced osteogenic differentiation, where phosphorylated p38 can also regulate osteogenic differentiation through RUNX2 [25, 49]. Studies have shown that DEX is hydrophobic and mainly enters cells by binding to cell membrane receptors, forming an endocytic vesicle for cellular entry, and then releasing in the cytoplasm 15. Lu MF et al., [50] studied the expression of Smad3 in the osteogenic process of MC3T3-E1 cells under the concentration of 10 − 7 M DEX, showing that this concentration of DEX significantly inhibited Smad-stimulated ALP activity, thereby inhibiting osteogenesis. Gan Qi et al., [51] prepared a gelatin sponge scaffold loaded with BMP-2 and DEX to study the osteogenic activity of the dual drugs, finding that 100 nM DEX could significantly enhance BMP-2 induced ALP activity and ALP mRNA expression in C2C12 cells through a RUNX2-dependent pathway compared to the use of BMP-2 or DEX alone. Thus, DEX can inhibit or enhance osteogenesis through the BMPs-Smad pathway, potentially related to its concentration.
Western blot analysis of Smad protein expression within the BMP-2 signaling pathway revealed that DEX alone did not augment Smad1/5/8 levels, whereas BMP-2 alone promoted their elevation. Furthermore, the addition of DEX further enhanced Smad1/5/8 expression, suggesting potential synergy via the BMPs-Smad pathway in the MC3T3-E1 cell line. Conversely, analysis of p38 expression indicated that DEX alone did not significantly impact p38 levels, while BMP-2 alone elevated them. Interestingly, the combined use of BMP-2 and DEX did not further increase p38 expression, implying that their synergistic action may not involve the p38-MAPK signaling pathway. Therefore, the inferred mechanism of synergistic action between BMP-2 and DEX (as depicted in Fig. 10) suggests that BMP-2 binds to transmembrane receptors, forming a heterodimer and activating the BMPs-Smads signaling pathway, while DEX enhances BMP-2's osteogenic effect through this pathway.