Abstract: BACKGROUND: Existing treatments can effectively reduce fracture risk in patients with osteoporosis, but their effectiveness is limited in patients with concurrent inflammatory diseases (such as rheumatoid arthritis) or severe postmenopausal osteoporosis. Therefore, the development of novel therapeutic strategies with both anti-inflammatory and anti-osteoclast properties is of great clinical significance.  
OBJECTIVE: To develop an innovative Sr²⁺ and bromodomain inhibitor Birabresib-loaded nanocomposite material (Bir@Sr-MBG) and characterize their cytocompatibility and in vitro immunomodulatory, anti-osteoclast differentiation, and osteoclast differentiation-promoting effects. 
METHODS: (1) Strontium-bioactive glass (Sr-MBG) was synthesized using a modified microemulsion-assisted sol-gel method. Birabresib was loaded into the mesoporous structure of Sr-MBG using an optimized solution adsorption method. The resulting material, designated Bir@Sr-MBG, was characterized for drug encapsulation efficiency, drug loading rate, and in vitro drug release. (2) Primary mouse bone marrow macrophages were cultured with different concentrations of birabresib or Bir@Sr-MBG, and cytocompatibility was assessed using the CCK-8 assay. (3) Primary mouse bone marrow macrophages were divided into five intervention groups: the control group received no intervention; the lipopolysaccharide group received lipopolysaccharide (to induce an inflammatory model); the Sr-MBG group received lipopolysaccharide + 10 mg/mL Sr-MBG; the birabresib group received lipopolysaccharide + 10 mg/mL birabresib, and the Bir@Sr-MBG group received lipopolysaccharide + 10 mg/mL Bir@Sr-MBG. After 24 hours of incubation, the expression levels of inducible nitric oxide synthase (a marker of M1 macrophages) and CD206 (a marker of M2 macrophages) were detected by immunofluorescence staining. The mRNA expression levels of interleukin-1β, interleukin-6, tumor necrosis factor-α, and interleukin-4 were detected by qPCR. The levels of interleukin-1β, interleukin-6, tumor necrosis factor-α, and interleukin-4 in the cell supernatant were detected by ELISA. (4) After osteoclastogenesis induction, primary mouse bone marrow macrophages were divided into four intervention groups: the control group received no intervention; the Sr-MBG group received 5 mg/mL Sr-MBG; the birabresib group received 0.5 mg/mL birabresib, and the Bir@Sr-MBG group received 5 mg/mL Bir@Sr-MBG. After 5 days of intervention, tartrate-resistant acid phosphatase staining and cytoskeleton staining were performed, followed by scanning electron microscopy observation. qPCR was used to measure the mRNA expression of osteoclastogenesis-related genes (cathepsin K, proto-oncogene Fos, tartrate-resistant acid phosphatase, and activated T-cell nuclear factor 1). (5) Adherent rat bone marrow mesenchymal stem cells were cultured in osteogenic induction medium and divided into five intervention groups: the control group received no intervention; the lipopolysaccharide group received lipopolysaccharide; the Sr-MBG group received lipopolysaccharide plus 10 mg/mL Sr-MBG; the birabresib group received lipopolysaccharide plus 0.5 mg/mL birabresib, and the Bir@Sr-MBG group received lipopolysaccharide plus 10 mg/mL Bir@Sr-MBG. On day 7 of culture, cells were stained with alkaline phosphatase and Alizarin red. qPCR was used to analyze the mRNA expression of osteogenic-related genes (alkaline phosphatase, RUNT-related transcription factor 2, osteocalcin, and osteopontin). 
RESULTS AND CONCLUSION: (1) The drug encapsulation efficiency and drug loading efficiency of Bir@Sr-MBG were 44.82% and 7.47%, respectively. Bir@Sr-MBG sustainedly released birabresib for over 168 hours. (2) CCK-8 assays showed that both 0.1–1 μg/mL birabresib and 20–200 μg/mL Bir@Sr-MBG exhibited good cytocompatibility. (3) Immunofluorescence staining revealed that Bir@Sr-MBG improved the inflammatory microenvironment by regulating macrophage polarization, and the material exhibited stronger anti-inflammatory properties than Sr-MBG and birabresib. qPCR and ELISA revealed that Bir@Sr-MBG downregulated the expression of proinflammatory cytokines (interleukin-1β, interleukin-6, and tumor necrosis factor-α) and upregulated the expression of anti-inflammatory cytokines (interleukin-4) more than Sr-MBG and birabresib. (4) Tartrate-resistant acid phosphatase staining, cytoskeleton staining, scanning electron microscopy, and qPCR revealed that Bir@Sr-MBG exhibited a stronger ability to inhibit osteoclast differentiation than Sr-MBG and birabresib. (5) Alkaline phosphatase staining, Alizarin red staining, and qPCR revealed that under inflammatory conditions, Bir@Sr-MBG promoted osteogenic differentiation of rat bone marrow mesenchymal stem cells more effectively than Sr-MBG and birabresib. (6) These results suggest that Bir@Sr-MBG effectively regulates bone metabolism and improves the bone microenvironment through a dual mechanism of action, demonstrating significant therapeutic potential for osteoporosis. 

Key words: osteoporosis, mesoporous bioactive glass, Sr2?, immune regulation, osteoclast differentiation, osteoblast differentiation, biomaterial