The blood-brain barrier (BBB) is a specialized brain endothelial barrier structure that regulates the highly selective transport of molecules under continuous blood flow. Recently, various types of BBB-on-chip models have been developed to mimic the microenvironmental cues that regulate the human BBB drug transport. However, technical difficulties in complex microfluidic systems limit their accessibility. Here, we propose a simple and easy-to-handle microfluidic device integrated with a cell culture insert to investigate the functional regulation of the human BBB endothelium in response to fluid shear stress (FSS). Using currently established immortalized human brain microvascular endothelial cells (HBMEC/ci18), we formed a BBB endothelial barrier without the substantial loss of barrier tightness under the relatively low range of FSS (0.1–1 dyn/cm2). Expression levels of key BBB transporters and receptors in the HBMEC/ci18 cells were dynamically changed in response to the FSS, and the effect of FSS reached a plateau around 1 dyn/cm2. Similar responses were observed in the primary HBMECs. Taking advantage of the detachable cell culture insert from the device, the drug efflux activity of P-glycoprotein (P-gp) was analyzed by the bidirectional permeability assay after the perfusion culture of cells. The data revealed that the FSS-stimulated BBB endothelium exhibited the 1.9-fold higher P-gp activity than that of the static culture control. Our microfluidic system coupling with the transwell model provides a functional human BBB endothelium with secured transporter activity, which is useful to investigate the bidirectional transport of drugs and its regulation by FSS.

Articular cartilage is crucial for joint function but its avascularity limits intrinsic repair, leading to conditions like osteoarthritis (OA). Chondromodulin-I (Cnmd) has emerged as a key molecule in cartilage biology, with potential implications for OA therapy. Cnmd is primarily expressed in cartilage and plays an important role in chondrocyte proliferation, cartilage homeostasis, and the blocking of angiogenesis. In vivo and in vitro studies on Cnmd, also suggest an involvement in bone repair and in delaying OA progression. Its downregulation correlates with OA severity, indicating its potential as a therapeutic target. Further research is needed to fully understand the mode of action of Cnmd and its beneficial implications for managing OA. This comprehensive review aims to elucidate the molecular characteristics of Cnmd, from its expression pattern, role in cartilage maintenance, callus formation during bone repair and association with OA.

In vitro vessel-mimicking models have been spotlighted as a powerful tool for investigating cellular behaviours in vascular development and diseases. However, it is still challenging to create micro-scale tubular tissues while mimicking the structural features of small arteries. Here, we propose a 3D culture model of small vascular tissue using a self-folding graphene-based porous film. Vascular endothelial cells were encapsulated within the self-folding film to create a cellular construct with a controlled curvature radius ranging from 10 to 100 μm, which is comparable to the size of a human arteriole. Additionally, vascular endothelial cells and smooth muscle cells were separately co-cultured on the inner and outer surfaces of the folded film, respectively. The porous wall worked as a permeable barrier between them, affecting the cell–cell communications like the extracellular layer in the artery wall. Thus, the culture model recapitulates the structural features of a small artery and will help us better understand intercellular communications at the artery wall in physiological and pathological conditions.

Proper connections between cartilaginous and muscular primordia through tendinous/ligamentous primordia are essential for musculoskeletal integration. Herein, we report a novel double-reporter mouse model for investigating this process via fluorescently visualising scleraxis (Scx) and SRY-box containing gene 9 (Sox9) expression. We generated ScxTomato transgenic mice and crossed them with Sox9EGFP knock-in mice to obtain ScxTomato;Sox9EGFP mice. Deep imaging of optically cleared double-reporter embryos at E13.5 and E16.5 revealed previously unknown differences in the dynamic interactions between cartilaginous and tendinous/ligamentous primordia in control and Scx-deficient mice. Tendon/ligament maturation was evaluated through simultaneous detection of fluorescence and visualisation of collagen fibre formation using second harmonic generation imaging. Lack of deltoid tuberosity in Scx-deficient mice caused misdirected muscle attachment with morphological changes. Loss of Scx also dysregulated progenitor cell fate determination in the chondrotendinous junction, resulting in the formation of a rounded enthesis rather than the protruding enthesis observed in the control. Hence, our double-reporter mouse system, in combination with loss- or gain-of-function approaches, is a unique and powerful tool that could be used to gain a comprehensive understanding of musculoskeletal integration.

Runt-related transcription factor 2 (RUNX2) is essential for skeletogenesis, and mutations in its gene cause cleidocranial dysplasia (CCD), an autosomal dominant skeletal disorder. The evolutionarily conserved 128-amino acid Runt homology domain (RHD) of human RUNX2 is essential for DNA binding and heterodimerization, and serves as a mutation hotspot associated with severe CCD phenotypes. To elucidate the functional impact of pathogenic RHD mutations in vivo, we generated 2 novel mouse lines: one carrying a missense mutation, c.695G>A, p.Arg232Gln (p.R232Q), corresponding to the human RUNX2 c.674G>A, p.Arg225Gln (p.R225Q), and the other harboring a frameshift mutation, c.697_698delGA, p.Glu233Thrfs*9 (p.E233Tfs*9), causing a premature stop codon. Homozygous Runx2R232Q/R232Q and Runx2E233Tfs*9/E233Tfs*9 mice lacked membranous ossification, whereas heterozygous Runx2R232Q/+ and Runx2E233Tfs*9/+ mice displayed typical CCD-like skeletal features, including an open anterior fontanelle and clavicle hypoplasia. Unexpectedly, heterozygotes carrying pathogenic RHD mutations developed small root-like protrusions, mostly 1 but rarely 2, at the pulp chamber floor of 3-rooted maxillary first molars during furcation, revealing a previously unrecognized dental phenotype. Dual luciferase assays showed that p.R232Q almost completely lost transactivation of the osteocalcin enhancer/promoter. Immunostaining showed that wild-type Runx2 was robustly expressed in osteoblasts and hypertrophic chondrocytes during bone formation, while the p.R232Q mutant Runx2 in Runx2R232Q/R232Q mice exhibited reduced expression in hypertrophic chondrocytes and partially impaired nuclear localization. These abnormalities led to defective osteoblast differentiation and chondrocyte maturation. Thus, our mutant mouse model provides a valuable in vivo platform to study CCD pathogenesis, mechanisms of tooth root furcation, and therapeutic interventions targeting dysfunctional RHD.

This paper describes a fabrication method for an in vitro branched vascular model using ECM-derived materials. This method helps prevent deformation of water-soluble templates in the sacrificial molding method.