Osteoarthritis (OA) is a degenerative joint disease characterized by global joint dysfunction, including cartilage degradation, subchondral bone alterations, and joint inflammation (Chen et al., 2024a, Kloppenburg et al., 2025). Its incidence is primarily influenced by aging, gender, obesity, and increased joint injuries (Du et al., 2022, Kang and Im, 2013). Traditional treatments for osteoarthritis (OA) can only delay disease progression, while conventional drug delivery systems are associated with numerous side effects. Currently, pharmacological therapy is widely used in the management of OA. Oral administration is the most convenient therapeutic route, however its poor intra-articular drug accumulation leads to deficient targeting and raises the risk of complications (Tian et al., 2017). Transdermal therapy can relieve osteoarthritis-related pain but fails to deliver satisfactory therapeutic outcomes (Chen and Gao, 2016). Compared with oral and transdermal routes, IA injection can directly deliver drugs to the joint cavity and provide higher local therapeutic drug concentrations, thereby enhancing therapeutic efficacy (Assi et al., 2023). However, conventional IA injectable drug solutions are rapidly cleared from the joint cavity. Given that OA is a chronic disease, frequent injections are required to maintain effective drug concentrations, which increases the risk of local infections and compromises patient compliance. Therefore, developing sustained-release formulations for IA injection holds significant clinical and scientific value (Yuan et al., 2019).

In recent years, microspheres have emerged as promising carriers for OA treatment, with poly(lactic-co-glycolic acid) (PLGA) being the most widely used biodegradable polymer due to its excellent biocompatibility and biodegradability (Kim et al., 2021). Microspheres are spherical microparticles with diameters ranging from 1 to 1000 μm, typically composed of a polymeric matrix in which drug is dispersed as individual molecules or small clusters. Microspheres have excellent biocompatibility, biodegradability, non-toxicity, and non-immunogenicity (Lin et al., 2024, Su et al., 2021). Since the launch of Lupron Depot® in 1989, approximately 20 long-acting injectable PLGA microspheres have been available on the market (Liu et al., 2025). Several publications have focused on the development of PLGA microparticles for the treatment of OA. Semee Seon et al. developed dexamethasone (DXA)-loaded microspheres by combining poly(lactic-co-glycolic acid) (PLGA) with Pluronic F127, achieving low initial drug burst release and long-term sustained release (Seon et al., 2024). Meanwhile, Zhang et al. designed and developed a multifunctional injectable PLGA microsphere. These microspheres can adhere to cartilage defects, recruit synovial mesenchymal stem cells (SMSCs), and stimulate their differentiation into chondrocytes after intra-articular injection (Liang et al., 2020, Zhang et al., 2024). Zilretta® (Flexion Therapeutics, Inc.), an FDA-approved IA formulation, encapsulates triamcinolone acetonide (TrA) crystals in a PLGA matrix via spray-drying, verifying the feasibility of PLGA microspheres for OA therapy (Maudens et al., 2018). Notably, the drug existing form within the PLGA matrix is a key factor regulating drug release behavior, but this has not been systematically explored in existing MLX-loaded microsphere studies. PLGA microspheres prepared via methods like emulsion-solvent evaporation and spray drying involve multiple intercoupled variables. This coupling hinders precise control of drug encapsulation efficiency and distribution. In this study, by controlling other variables, the type of organic solvent was varied to prepare microspheres with different drug existence forms via the microfluidic technology.

Meloxicam (MLX) is a non-steroidal anti-inflammatory drug (NSAID) with potent analgesic and anti-inflammatory activities for OA treatment (Macasoi et al., 2024). However, its oral administration is associated with gastrointestinal adverse reactions, including dyspepsia, gastric ulcers, bleeding, and even perforation. Thus, PLGA-based MLX sustained-release microspheres (MLX-MS) for IA injection offer enhanced safety—they can achieve long-term anti-inflammatory effects while reducing gastrointestinal side effects of NSAIDs (Chen et al., 2022). In a previous study, Pei et al.,2023 utilized the oil-in-water (O/W) emulsion method to prepare MLX-MS, where MLX and PLGA were dissolved in a mixture of dimethyl sulfoxide (DMSO) and dichloromethane (DCM). The MLX-MS exhibited a significant initial burst release (2 days to release 30%), with no further drug release observed over the subsequent 27 days. Subsequently, they used a modified solid-in-oil-in-water (S/O/W) emulsion method to prepare the microspheres: meloxicam and Mg(OH)2 were dispersed into DCM to form a solid-in-oil (S/O) suspension, then the suspension was transferred into polyvinyl alcohol (PVA) solution followed by emulsification. The results showed that the formulation containing 12 mg of Mg(OH)2 achieved 40% drug release within 1 day and 77.6% within 7 days. Although this method effectively increased drug release in the later phase, the resulting MLX-MS still suffered from an initial burst release problem in the early stage.

Herein, to achieve tailored and optimal sustained release, we prepared various MLX-MS by the method of cosolvent combination or micronized drug—with the core goal of investigating how three distinct drug existing forms (microcrystal, particle and molecular state) distributed within the PLGA matrix regulate drug release behavior (Bee et al., 2018). Scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS) was used to characterize the surface and internal morphologies of MLX-MS. X-ray diffraction (XRD) and infrared spectroscopy (IR) confirmed the existence and distribution of MLX in the three targeted forms within the PLGA matrix. We systematically examined the in vitro drug release kinetics and degradation behavior of the microspheres, and further explored their in vivo release characteristics and degradation processes. Furthermore, via Micro-CT imaging and tissue section staining, we comprehensively evaluated the repair efficacy and therapeutic safety of MLX-MS on osteoarthritic lesions from both morphological and histological perspectives.