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1. Notch signaling and fluid shear stress in regulating osteogenic differentiation

   https://doi.org/10.3389/fbioe.2022.1007430  

   Zhao, Yuwen; Richardson, Kiarra; Yang, Rui; Bousraou, Zoe; Lee, Yoo Kyoung; Fasciano, Samantha; Wang, Shue (October 2022, Frontiers in Bioengineering and Biotechnology)

   Osteoporosis is a common bone and metabolic disease that is characterized by bone density loss and microstructural degeneration. Human bone marrow-derived mesenchymal stem cells (hMSCs) are multipotent progenitor cells with the potential to differentiate into various cell types, including osteoblasts, chondrocytes, and adipocytes, which have been utilized extensively in the field of bone tissue engineering and cell-based therapy. Although fluid shear stress plays an important role in bone osteogenic differentiation, the cellular and molecular mechanisms underlying this effect remain poorly understood. Here, a locked nucleic acid (LNA)/DNA nanobiosensor was exploited to monitor mRNA gene expression of hMSCs that were exposed to physiologically relevant fluid shear stress to examine the regulatory role of Notch signaling during osteogenic differentiation. First, the effects of fluid shear stress on cell viability, proliferation, morphology, and osteogenic differentiation were investigated and compared. Our results showed shear stress modulates hMSCs morphology and osteogenic differentiation depending on the applied shear and duration. By incorporating this LNA/DNA nanobiosensor and alkaline phosphatase (ALP) staining, we further investigated the role of Notch signaling in regulating osteogenic differentiation. Pharmacological treatment is applied to disrupt Notch signaling to investigate the mechanisms that govern shear stress induced osteogenic differentiation. Our experimental results provide convincing evidence supporting that physiologically relevant shear stress regulates osteogenic differentiation through Notch signaling. Inhibition of Notch signaling mediates the effects of shear stress on osteogenic differentiation, with reduced ALP enzyme activity and decreased Dll4 mRNA expression. In conclusion, our results will add new information concerning osteogenic differentiation of hMSCs under shear stress and the regulatory role of Notch signaling. Further studies may elucidate the mechanisms underlying the mechanosensitive role of Notch signaling in stem cell differentiation. 

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2. Modulation of acoustofluidic parameters to assess effect on molecular loading in human T cells.

   https://doi.org/10.1121/10.0007543  

   Centner, Connor; Moore, John; Baxter, Mary; Vinseiro Figueira, Mariana; Long, Zachary; Belott, Clinton; Menze, Michael; Bates, Paula; Yaddanapudi, Kavitha; Kopechek, Jonathan. (October 2021, The journal of the Acoustical Society of America)

   T-cell therapies are rapidly emerging for treatment of cancer and other diseases but are limited by inefficient non-viral delivery methods. Acoustofluidic devices are in development to enhance non-viral delivery to cells. The effect of acoustofluidic parameters, such as channel geometry, on molecular loading in human T cells was assessed using 3D-printed acoustofluidic devices. Devices with rectilinear channels (1- and 2-mm diameters) were compared directly with concentric spiral channel geometries. Intracellular delivery of a fluorescent dye (calcein, 100 lg/ml) was evaluated in Jurkat T cells using flow cytometry after ultrasound treatment with cationic microbubbles (2.5% v/v). B-mode ultrasound pulses (2.5 MHz, 3.8 MPa output pressure) were generated by a P4-1 transducer on a Verasonics Vantage ultrasound system. Cell viability was assessed using propidum iodine staining (10 lg/ml). Intracellular molecular delivery was significantly enhanced with acoustofluidic treatment in each channel geometry, but treatment with the 1-mm concentric spiral geometry further enhanced delivery after acoustofluidic treatment compared to both 1- and 2-mm rectilinear channels (ANOVA p < 0.001, n ¼ 6/group). These results indicate that 3Dprinted acoustofluidic devices enhance molecular delivery to T cells, and channel geometry modulates intracellular loading efficiency. This approach may offer advantages to improve manufacturing of T cell therapies. 

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3. Mesenchymal Stem/Stromal Cells-Derived Exosomal Micro-RNA Delivery Enhances Bone Repair in Osteoporotic Conditions

   Mandal, Subhasis; Kim, Lauren; Kang, Minjee; Elamin, Iram; Rao, Meghna; Elsayed, Ishraga S; Xu, Changlu; Aghaloo, Tara L; Lee, Min; Fan, Jiabing (July 2026, Tissue engineering Part A)

   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 microRNA-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 employed as a delivery vehicle, generating Exo–miR-423 inhibitor construct. These exosomes were subsequently incorporated into an apatite-coated poly(lactic-co-glycolic acid) (PLGA) 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 (OVX) 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. 

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4. Precision treatment of viral pneumonia through macrophage-targeted lipid nanoparticle delivery

   https://doi.org/10.1073/pnas.2314747121  

   Zhao, Gan; Xue, Lulu; Geisler, Hannah C; Xu, Junchao; Li, Xinyuan; Mitchell, Michael J; Vaughan, Andrew E (February 2024, Proceedings of the National Academy of Sciences)

   Macrophages are integral components of the innate immune system, playing a dual role in host defense during infection and pathophysiological states. Macrophages contribute to immune responses and aid in combatting various infections, yet their production of abundant proinflammatory cytokines can lead to uncontrolled inflammation and worsened tissue damage. Therefore, reducing macrophage-derived proinflammatory cytokine release represents a promising approach for treating various acute and chronic inflammatory disorders. However, limited macrophage-specific delivery vehicles have hindered the development of macrophage-targeted therapies. In this study, we screened a pool of 112 lipid nanoparticles (LNPs) to identify an optimal LNP formulation for efficient siRNA delivery. Subsequently, by conjugating the macrophage-specific antibody F4/80 to the LNP surface, we constructed MacLNP, an enhanced LNP formulation designed for targeted macrophage delivery. In both in vitro and in vivo experiments, MacLNP demonstrated a significant enhancement in targeting macrophages. Specifically, delivery of siRNA targeting TAK1, a critical kinase upstream of multiple inflammatory pathways, effectively suppressed the phosphorylation/activation of NF-kB. LNP-mediated inhibition of NF-kB, a key upstream regulator in the classic inflammatory signaling pathway, in the murine macrophage cell line RAW264.7 significantly reduced the release of proinflammatory cytokines after stimulation with the viral RNA mimic Poly(I:C). Finally, intranasal administration of MacLNP-encapsulated TAK1 siRNA markedly ameliorated lung injury induced by influenza infection. In conclusion, our findings validate the potential of targeted macrophage interventions in attenuating inflammatory responses, reinforcing the potential of LNP-mediated macrophage targeting to treat pulmonary inflammatory disorders. 

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5. Functional and analytical recapitulation of osteoclast biology on demineralized bone paper

   https://doi.org/10.1038/s41467-023-44000-9  

   Park, Yongkuk; Sato, Tadatoshi; Lee, Jungwoo (December 2023, Nature Communications)

   Abstract: Osteoclasts are the primary target for osteoporosis drug development. Recent animal studies revealed the crucial roles of osteoblasts in regulating osteoclastogenesis and the longer lifespans of osteoclasts than previously thought with fission and recycling. However, existing culture platforms are limited to replicating these newly identified cellular processes. We report a demineralized bone paper (DBP)-based osteoblast culture and osteoclast assay platform that replicates osteoclast fusion, fission, resorption, and apoptosis with high fidelity and analytical power. An osteoid-inspired DBP supports rapid and structural mineral deposition by osteoblasts. Coculture osteoblasts and bone marrow monocytes under biochemical stimulation recapitulate osteoclast differentiation and function. The DBP-based bone model allows longitudinal quantitative fluorescent monitoring of osteoclast responses to bisphosphonate drug, substantiating significantly reducing their number and lifespan. Finally, we demonstrate the feasibility of humanizing the bone model. The DBP-based osteo assay platforms are expected to advance bone remodeling-targeting drug development with improved prediction of clinical outcomes. 

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