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1. Wound Healing Evaluation of Zinc Particle-Embedded Nanofibrous Scaffolds through Protein Expression Profiling

   https://doi.org/10.1021/acsabm.6c00391  

   Moody, Alexis; Bhattarai, Narayan (September 2026, ACS Applied Bio Materials)

   Understanding how the components of bioactive scaffolds regulate coordinated inflammatory and regenerative signaling is critical for the development of advanced wound healing materials. Here, zinc metal particle-embedded polycaprolactone (PZ) electrospun nanofiber scaffolds were evaluated as an immunomodulatory platform that directs protein expression to influence the wound healing process. Scaffolds containing 0, 0.5, and 1 wt % Zn were assessed during fibroblast monoculture, macrophage monoculture, and fibroblast-macrophage coculture models under sustained inflammatory stimulation. Zn incorporation enhanced cell viability and metabolic activity while maintaining low cytotoxicity across all culture systems. Fibroblasts cultured on Zn-containing scaffolds exhibited reduced inflammatory signaling, suppressed COX-2 expression, and increased secretion of proangiogenic and pro-proliferative factors such as EGF, bFGF, and VEGF. Macrophages displayed restrained inflammatory activation, accompanied by elevated growth factor output, consistent with a pro-healing phenotype. Coculture studies revealed that Zn scaffolds selectively dampened inflammatory amplification while preserving cooperative angiogenic signaling, evidenced by reduced IL-6 and COX-2 expression. Altogether, these results establish Zn-embedded PCL scaffolds as a tunable platform for directing inflammatory and regenerative signaling relevant to wound healing applications. 

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2. Modulating TRPV4 Channel Activity in Pro-Inflammatory Macrophages within the 3D Tissue Analog

   https://doi.org/10.3390/biomedicines12010230  

   Babaniamansour, Parto; Jacho, Diego; Niedzielski, Skyler; Rabino, Agustin; Garcia-Mata, Rafael; Yildirim-Ayan, Eda (January 2024, Biomedicines)

   Investigating macrophage plasticity emerges as a promising strategy for promoting tissue regeneration and can be exploited by regulating the transient receptor potential vanilloid 4 (TRPV4) channel. The TRPV4 channel responds to various stimuli including mechanical, chemical, and selective pharmacological compounds. It is well documented that treating cells such as epithelial cells and fibroblasts with a TRPV4 agonist enhances the Ca2+ influx to the cells, which leads to secretion of pro-inflammatory cytokines, while a TRPV4 antagonist reduces both Ca2+ influx and pro-inflammatory cytokine secretion. In this work, we investigated the effect of selective TRPV4 modulator compounds on U937-differentiated macrophages encapsulated within three-dimensional (3D) matrices. Despite offering a more physiologically relevant model than 2D cultures, pharmacological treatment of macrophages within 3D collagen matrices is largely overlooked in the literature. In this study, pro-inflammatory macrophages were treated with an agonist, 500 nM of GSK1016790A (TRPV4(+)), and an antagonist, 10 mM of RN-1734 (TRPV4(−)), to elucidate the modulation of the TRPV4 channel at both cellular and extracellular levels. To evaluate macrophage phenotypic alterations within 3D collagen matrices following TRPV4 modulator treatment, we employed structural techniques (SEM, Masson’s trichrome, and collagen hybridizing peptide (CHP) staining), quantitative morphological measures for phenotypic assessment, and genotypic methods such as quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC). Our data reveal that pharmacological modulation of the macrophage TRPV4 channel alters the cytoskeletal structure of macrophages and influences the 3D structure encapsulating them. Moreover, we proved that treating macrophages with a TRPV4 agonist and antagonist enhances the expression of pro- and anti-inflammatory genes, respectively, leading to the upregulation of surface markers CD80 and CD206. In the TRPV4(−) group, the CD206 gene and CD206 surface marker were significantly upregulated by 9- and 2.5-fold, respectively, compared to the control group. These findings demonstrate that TRPV4 modulation can be utilized to shift macrophage phenotype within the 3D matrix toward a desired state. This is an innovative approach to addressing inflammation in musculoskeletal tissues. 

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3. Synergetic role of TRPV4 inhibitor and mechanical loading on reducing inflammation

   https://doi.org/10.3389/fimmu.2024.1456042  

   Babaniamansour, Parto; Jacho, Diego; Rabino, Agustin; Garcia-Mata, Rafael; Yildirim-Ayan, Eda (January 2025, Frontiers in Immunology)

   Resolution of inflammation is essential for normal tissue healing and regeneration, with macrophages playing a key role in regulating this process through phenotypic changes from a pro-inflammatory to an anti-inflammatory state. Pharmacological and mechanical (mechanotherapy) techniques can be employed to polarize macrophages toward an anti-inflammatory phenotype, thereby diminishing inflammation. One clinically relevant pharmacological approach is the inhibition of Transient Receptor Potential Vanilloid 4 (TRPV4). This study investigates the effects of various mechanical loading amplitudes (0%, 3%, and 6%) and TRPV4 inhibition (10 µM RN-1734) on the phenotypic commitments of pro-inflammatory (M1) macrophages within three-dimensional (3D) collagen matrices. M1 macrophages exposed to 3% mechanical strain exhibited upregulated pro-inflammatory responses, including increased pro-inflammatory gene expression and enhanced proteolytic activity within the extracellular matrix. TRPV4 inhibition partially mitigated this inflammation. Notably, 6% mechanical strain combined with TRPV4 inhibition suppressed Mitogen-Activated Protein Kinase (MAPK) expression, leading to reduced pro-inflammatory gene expression and increased anti-inflammatory markers such as CD206. Gene expression analysis further demonstrated significant reductions in pro-inflammatory gene expression and a synergistic promotion of anti-inflammatory phenotypes under TRPV4 inhibition at 6% mechanical strain. Surface protein analysis via immunohistochemistry confirmed these phenotypic shifts, highlighting changes in the expression of CD80 (pro-inflammatory) and CD206 (anti-inflammatory) markers, alongside F-actin and nuclear staining. This research suggests that TRPV4 inhibition, combined with specific mechanical loading (6%), can drive macrophages toward an anti-inflammatory state, thereby may promote inflammation resolution and tissue repair. 

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4. Injectable Cell-Laden Nanofibrous Matrix for Treating Annulus Fibrosus Defects in Porcine Model: An Organ Culture Study

   https://doi.org/10.3390/life12111866  

   Roebke, Evan; Jacho, Diego; Eby, Oliver; Aldoohan, Sulaiman; Elsamaloty, Haitham; Yildirim-Ayan, Eda (November 2022, Life)

   Lower back pain commonly arises from intervertebral disc (IVD) failure, often caused by deteriorating annulus fibrosus (AF) and/or nucleus pulposus (NP) tissue. High socioeconomic cost, quality of life issues, and unsatisfactory surgical options motivate the rapid development of non-invasive, regenerative repair strategies for lower back pain. This study aims to evaluate the AF regenerative capacity of injectable matrix repair strategy in ex vivo porcine organ culturing using collagen type-I and polycaprolactone nanofibers (PNCOL) with encapsulated fibroblast cells. Upon 14 days organ culturing, the porcine IVDs were assessed using gross optical imaging, magnetic resonance imaging (MRI), histological analysis, and Reverse Transcriptase quantitative PCR (RT-qPCR) to determine the regenerative capabilities of the PNCOL matrix at the AF injury. PNCOL-treated AF defects demonstrated a full recovery with increased gene expressions of AF extracellular matrix markers, including Collagen-I, Aggrecan, Scleraxis, and Tenascin, along with anti-inflammatory markers such as CD206 and IL10. The PNCOL treatment effectively regenerates the AF tissue at the injury site contributing to decreased herniation risk and improved surgical outcomes, thus providing effective non-invasive strategies for treating IVD injuries. 

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5. TSG-6 Activated MSC-derived Extracellular Vesicles Present Altered micro-RNA Contents and Ameliorate the Inflammatory Phenotype of Macrophages in Vitro

   https://doi.org/10.1007/s10753-025-02398-y  

   Martinez-Zalbidea, Iker; Rzasa, Alyssa; Puvanesarajah, Varun; Hitzl, Wolfgang; Wuertz‑Kozak, Karin (December 2026, Inflammation)

   Abstract Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have shown promising immunomodulatory properties; however, strategies to enhance their therapeutic potential remain limited. Here, we employed CRISPR activation of the gene TSG-6 in MSCs to evaluate the impact of elevated TSG-6 on EV cargo and immunomodulatory function in an in vitro macrophage model. CRISPR-mediated gene activation was confirmed by RT-qPCR, demonstrating more than an 1800 fold increase in TSG-6 mRNA compared to controls. EVs were isolated from TSG-6 overexpressing MSCs and thoroughly characterized by nanoparticle tracking analysis, transmission electron microscopy, and Western blot, confirming their typical size distribution, morphology, and surface markers. Small RNA sequencing of these EVs revealed 15 differentially expressed miRNAs relative to EVs from control MSCs. When THP-1–derived macrophages were stimulated with LPS and treated with TSG-6-overexpressing MSC-EVs (Standard dosage: 1000 particle/cell,n = 11; Alternative dosages: 500, 1000, or 2000 particles/cell,n = 6), a marked reduction in pro-inflammatory cytokine gene expression (IL-1β, CCL2, CXCL10, and TNF-α) and secreted protein levels (CCL2, TNF-α, CXCL1, and MIP-3α) was observed. Taken together, these findings demonstrate that CRISPR-based TSG-6 activation reprograms MSC-EV miRNA cargo (as well as their protein cargo, as previously shown), which can boost their anti-inflammatory effects. These findings underscore the promise of CRISPR-activation as a novel platform for boosting the bioactive properties of MSC-EVs and enhancing immunotherapeutic efficacy. 

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