The therapeutic potential of bone marrow mesenchymal stem cells (BMSCs) in bone tissue engineering (BTE) is compromised by functional decline during conventional two-dimensional (2D) expansion. We hypothesized that chitosan film-based three-dimensional (3D) culture rejuvenates BMSC potency, synergistically promoting angiogenesis and immunomodulation for vascularized bone regeneration. Mouse BMSCs were cultured into spheroids on chitosan films. Their stemness, proliferation, migration, senescence, osteogenic, and proangiogenic potential were compared with 2D cultures. Paracrine effects were evaluated by treating human umbilical vein endothelial cells (HUVECs) and RAW264.7 macrophages with BMSC-conditioned medium (CM). In vivo, gelatin methacryloyl (GelMA) hydrogel-encapsulated 3D-BMSC spheroids were implanted into mouse critical-size cranial defects. Compared to 2D counterparts, 3D-BMSCs exhibited increased stemness, proliferation, migration, delayed senescence, osteogenic differentiation, and enhanced proangiogenic potential. We observed that conditioned medium from 3D-BMSCs (3D-CM) was associated with in vitro angiogenesis and orchestrated a proreparative microenvironment by promoting M2 macrophage polarization and suppressing M1 inflammation. In vivo, GelMA + 3D-BMSC spheroids achieved greater bone regeneration, which was accompanied by a proreparative immune microenvironment and enhanced CD31/EMCN-positive H-type-like vessel formation. This chitosan film-based 3D culture system effectively augments BMSC therapeutic potency, simultaneously enhancing intrinsic cell properties and orchestrating a proregenerative microenvironment, thereby offering a promising experimental platform for critical-sized bone defect repair.
Osteoarthritis (OA) is a progressive, degenerative joint disorder characterized by irreversible loss of articular cartilage, in which NLRP3 inflammasome-mediated chondrocyte pyroptosis and dysregulated Wnt/β-catenin signaling are recognized as central pathological events. This study investigated the potential chondroprotective effects of teriflunomide (TFM) in OA, revealing a repositioning molecular mechanism whereby TFM inhibits NLRP3-dependent pyroptosis through the reactivation of Wnt/β-catenin signaling. In vitro, using immortalized human chondrocyte (HC) cells, TFM markedly inhibited interleukin (IL)-1β-induced NLRP3 inflammasome activation and the subsequent pyroptosis relative to that of vehicle-treated control cells. Concurrently, TFM restored Wnt/β-catenin signaling, as indicated by the recovered expression of β-catenin, c-Myc, and cyclin D1. Furthermore, TFM suppressed IL-1β-induced reactive oxygen species generation and speck formation by apoptosis-associated speck-like protein containing a CARD, thus promoting the functional recovery of chondrocytes. These in vitro results were further corroborated in a rat model of OA (n = 6 per group). TFM administration (30 mg/kg, every other day for 4 weeks) improved joint morphology, alleviated synovitis, and restored cartilage thickness and proteoglycan content relative to those observed in vehicle-treated animals. Collectively, these findings suggest that TFM exerts chondroprotective effects in preclinical models by reactivating Wnt/β-catenin signaling and inhibiting NLRP3-dependent pyroptosis. TFM warrants further investigation as a potential candidate agent for the treatment of patients with OA.
The placental barrier plays a critical role in protecting the fetus from xenobiotics and regulating the maternal-fetal exchange. However, conventional trophoblast cell lines and animal models often fail to accurately recapitulate the key structural and functional features of the human placental barrier. In this study, a novel organ-on-a-chip (OOC) model was developed that integrated human JEG-3 trophoblast organoids with human umbilical vein endothelial cells under combined fluid shear stress and mechanical strain. This configuration supports the self-assembly of a three-dimensional, functional placental barrier. The engineered tissue exhibited essential physiological characteristics, including syncytiotrophoblast marker expression, human chorionic gonadotropin secretion, and enhanced glucose transport activity. This model was used to assess the transbarrier transport of several flavonoid compounds. The results indicated that permeation occurred primarily via passive diffusion, with permeability differences governed mainly by physicochemical properties, such as lipophilicity, rather than molecular size. This integrated OOC platform establishes a biomimetic human placental model that provides a reliable tool for evaluating placental permeability and the potential toxicity risks of complex compounds.
Osteoporosis-associated bone fractures are a leading cause of disability in the elderly population. Developing effective therapeutic strategies to enhance bone repair under osteoporotic conditions remains a major clinical challenge. Increasing evidence indicates that aberrant lineage commitment of mesenchymal stem/stromal cells (MSCs) resident in bone marrow contributes to osteoporosis-related bone loss. However, incomplete understanding of the regulatory mechanisms governing MSC differentiation has limited the development of efficient therapeutic approaches. In this study, we identified micro-RNA-423 (miR-423) as a negative regulator of osteogenic differentiation, and demonstrated that inhibition of miR-423 significantly enhanced osteoblast differentiation of MSCs. To enable in vivo delivery of the miR-423 inhibitor for bone repair, MSC-derived exosomes (MSC-Exo) were used as a delivery vehicle, generating the Exo-miR-423 inhibitor construct. These exosomes were subsequently incorporated into an apatite-coated poly(lactic-co-glycolic acid) scaffold to form an Exo-miR-423 inhibitor/scaffold complex. Implantation of this complex significantly promoted bone healing in a calvarial defect model in ovariectomized mice. Collectively, these findings demonstrate a promising miRNA-modulated, exosome-based tissue engineering strategy for enhancing bone defect and fracture repair under osteoporotic conditions, and highlight its potential for further optimization and translational application.
