Strontium/Birabresib-loaded bioactive glass modulating bone microenvironment for osteoporosis therapy

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 by CCK-8 assay. (3) For immunomodulation, cells were divided into five groups: control, lipopolysaccharide (LPS), LPS+Sr-MBG, LPS+Birabresib, and LPS+Bir@Sr-MBG. After 24 h incubation, immunofluorescence staining for iNOS (M1 marker) and CD206 (M2 marker) was performed; qPCR and ELISA were used to measure expression of IL-1β, IL-6, TNF-α, and IL-4. (4) For osteoclast differentiation, bone marrow macrophages were induced with RANKL and divided into four groups: control, Sr-MBG, Birabresib, and Bir@Sr-MBG. After 5 days, TRAP staining, cytoskeletal staining, and scanning electron microscopy were performed; qPCR was used to measure osteoclast-related genes (CTSK, c-Fos, TRAP, NFATc1). (5) For osteogenic differentiation, rat bone marrow mesenchymal stem cells were cultured in osteogenic medium and divided into five groups: control, LPS, LPS+Sr-MBG, LPS+Birabresib, and LPS+Bir@Sr-MBG. After 7 days, alkaline phosphatase and alizarin red staining were performed; qPCR was used to measure osteogenic genes (ALP, Runx2, OCN, OPN). RESULTS AND CONCLUSION: (1) The drug encapsulation efficiency of Bir@Sr-MBG was 44.82%, drug loading rate was 7.47%, and sustained release of Birabresib was observed for over 168 h. (2) CCK-8 assay showed good cytocompatibility for Birabresib at 0.1-1 μg/mL and Bir@Sr-MBG at 20-200 μg/mL. (3) Immunofluorescence staining showed that Bir@Sr-MBG improved the inflammatory microenvironment by regulating macrophage polarization, with stronger anti-inflammatory effects than Sr-MBG or Birabresib alone. qPCR and ELISA confirmed that Bir@Sr-MBG downregulated pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) and upregulated anti-inflammatory cytokine (IL-4) more effectively than Sr-MBG or Birabresib. (4) TRAP staining, cytoskeletal staining, SEM, and qPCR showed that Bir@Sr-MBG had stronger anti-osteoclast differentiation effects than Sr-MBG or Birabresib. (5) ALP staining, alizarin red staining, and qPCR showed that under inflammatory conditions, Bir@Sr-MBG promoted osteogenic differentiation of rat bone marrow mesenchymal stem cells more effectively than Sr-MBG or Birabresib. (6) These results indicate that Bir@Sr-MBG effectively regulates bone metabolism and improves the bone microenvironment through a dual mechanism, showing significant therapeutic potential for osteoporosis.

1. Introduction

Osteoporosis is a common metabolic bone disease characterized by reduced bone mass and deterioration of bone microarchitecture, leading to increased bone fragility and a significantly elevated risk of fractures, which severely affects the health and quality of life of middle-aged and elderly populations [1-2]. Postmenopausal osteoporosis is the most common type, often insidious in onset after menopause, and has become a major global public health issue, imposing a heavy burden on healthcare systems [3-5].

In normal bone metabolism, bone resorption and bone formation are in dynamic equilibrium. Osteoclasts, derived from the monocyte/macrophage lineage, adhere to the bone surface to form resorption lacunae, secreting acids and proteases to degrade bone matrix. The differentiation and activation of osteoclasts are regulated by cytokines such as receptor activator of nuclear factor-κB ligand (RANKL), interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). Upon binding of RANKL to its receptor, adaptor proteins such as tumor necrosis factor receptor-associated factor 6 (TRAF6) are recruited, activating signaling pathways including mitogen-activated protein kinase (MAPK), nuclear factor-κB (NF-κB), protein kinase B (Akt), c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK), which induce the expression of osteoclast-related genes [6-8]. During bone resorption, osteoblasts secrete osteoprotegerin (OPG) to inhibit osteoclast formation and activity.