There is a critical need to understand pathophysiological mechanisms involved in injury from acute chlorine gas (Cl2) exposure. Limited information is available regarding the time course and mechanisms of injury after acute Cl2 exposure due to a lack of human clinical data and limited fidelity of pre-clinical animal models. We designed and integrated a Cl2 exposure platform to generate and deliver precise concentrations of Cl2 to a microfluidic human airway-on-a-chip microphysiological system. Chemical, biological, structural, and functional airway-on-a-chip responses to Cl2 exposure were characterized across multiple concentrations, exposure times, and post-exposure timepoints. Transcriptomics and metabolomics analyses delineated key molecular, cellular, and physiological pathways involved in the acute response to Cl2 exposure. This work represents a significant advancement towards high-throughput, human-relevant characterization of pulmonary toxicants and medical countermeasure development, addressing critical gaps in toxicology modeling while reducing reliance on animal studies.

INTRODUCTION:Burn injuries present a significant challenge for both military personnel and civilians, accounting for 5%-20% of military casualties and contributing to substantial medical costs and poor clinical outcomes. Medical technologies used in multidomain operations against near-peer adversaries are often heavy, difficult to use, immunogenic, and are not able to support prolonged casualty care in the field. To address these limitations, SiOxMed has developed a novel silica-based fiber (SBF) universal combat matrix with hemostatic and wound repair properties. These studies assess the effects of SBF on immune modulation, extracellular matrix (ECM) remodeling, and cellular proliferation in a porcine full thickness burn model. MATERIALS AND METHODS:Specific pathogen-free Yorkshire pigs were subjected to full-thickness burns using a modified branding iron. Twenty-four hours post-injury, wound beds were debrided before SBF matrix or standard gauze dressing treatment (n = 4 wounds per group). Scheduled bandage changes were followed by euthanasia and necropsy on day 25. Tissue biopsies were collected for histological and immunohistochemical analysis at days 4, 11, 18, and 25 to compare epithelialization, dermal collagen remodeling, and immune modulation among treatment groups. RESULTS:Histological evaluation using H&E (hematoxylin and eosin) and Masson's trichrome staining revealed enhanced rete ridge formation and improved collagen remodeling in SBF-treated wounds compared to control. Immunofluorescent staining for T-cell markers (CD4, CD8) and macrophage markers (CD68) demonstrated a reduced inflammatory response in the SBF group. Ki-67 staining indicated comparable cellular proliferation between SBF-treated and control wounds. CONCLUSIONS:Silica-based fiber treatment improved epidermal rete ridge formation, enhanced ECM collagen remodeling, and reduced inflammatory cell infiltration while maintaining normal cellular proliferation. These findings indicate that SBF not only minimizes inflammation compared to standard gauze treatment but also provides an optimal scaffold environment for tissue regeneration. Given its unique mechanism of action, ultralight weight, stability under ambient conditions, and user-friendly design, SBF shows strong potential for military and civilian applications, particularly in austere prehospital care and mass casualty scenarios.

Bioreactors are used to dynamically condition engineered tissues to achieve the required degree of maturation before in vivo implantation. Integrating sensors and imaging capabilities into bioreactors can help us understand how the culture environment influences tissue maturation and growth. Additionally, this enables the monitoring of tissue constructs and provides critical information for quality control. This study aimed to develop a standardized, self-contained, uniaxial bioreactor module for the clinical manufacturing of tissue constructs; this system would benefit from unidirectional mechanical or electrical stimulation, or both. We achieved this goal by integrating stimulation and sensing components that provide an optimal culture environment and monitoring capabilities to improve tissue manufacturing. The uniaxial bioreactor module included integrated, user-friendly mechanical and electrical stimulations with force measurement to enhance the preconditioning of the engineered tissues. Also, a sensor loop and media exchange system were integrated to monitor the culture environment and cellular metabolites over time, and the camera system above the tissue construct enabled the macroscopic visualization of tissue maturation. Furthermore, the onboard media exchange system was programmed into the module to maintain aseptic culture conditions in the long term. Subsequently, using native skeletal muscle tissue and tissue-engineered skeletal muscle constructs, the performance of the uniaxial bioreactor module was validated for its application in preconditioning and enhancing tissue maturation.

PDF file - 813K, Secretion of angiogenic cytokines by O-ASC, BM-MSC, SC-ASC and WI38.

Supplementary Figure 2 from Fibroblasts Isolated from Common Sites of Breast Cancer Metastasis Enhance Cancer Cell Growth Rates and Invasiveness in an Interleukin-6–Dependent Manner

PDF file - 5585K, Double staining for GFP and TGF-β detects BM-hMSCs-GFP near tumor cells expressing TGF-β in intracranial tumors.

Fig. S1. Sustained adrenergic signaling increases nerve counts in tumors. Fig. S2. Characterization of tumoral innervation. Fig. S3. NE induces BDNF expression. Fig. S4. NE-induced BDNF expression is mediated by ADRB3/Epac/Jnk. Fig. S5. BDNF increases nerve counts. Fig. S6. Adrenergic-mediated mTrkB activation leads to increased in vivo tumor nodule counts. Table S1. Alteration in pathways associated with neuronal growth and function after NE treatment (HeyA8 and SKOV3ip1 cells). Table S2. Association of Clinicopathologic variables with BDNF protein expression. Table S3. Association of Clinicopathologic variables with Nerve Counts.

Supplementary Figure Legends 1-7 from Human Bone Marrow–Derived Mesenchymal Stem Cells for Intravascular Delivery of Oncolytic Adenovirus Δ24-RGD to Human Gliomas

PDF file - 29856K, Assays of STR polymorphism using twelve unique markers in RCS, RAT2, C6, GSC001 low, GSC001 high cells.

PDF file - 1271K, Proliferation effects of knocking down TGFβRII or CD105 in BM-hMSCs.

Supplementary Figure 1 from Fibroblasts Isolated from Common Sites of Breast Cancer Metastasis Enhance Cancer Cell Growth Rates and Invasiveness in an Interleukin-6–Dependent Manner

PDF file - 281K, Hec1a secrete IL8 and CXCL1.

PDF file - 139K, Endometrial Hec1a cancer cell proliferation in co-culture with OSC, MSC, ASC and WI38 fibroblasts.

PDF file - 57K, Primer sequences for STR markers.

Supplementary Figure 3 from Fibroblasts Isolated from Common Sites of Breast Cancer Metastasis Enhance Cancer Cell Growth Rates and Invasiveness in an Interleukin-6–Dependent Manner

The human airways are complex structures with important interactions between cells, extracellular matrix (ECM) proteins and the biomechanical microenvironment. A robust, well-differentiated in vitro culture system that accurately models these interactions would provide a useful tool for studying normal and pathological airway biology. Here, we report the development and characterization of a physiologically relevant air–liquid interface (ALI) 3D airway ‘organ tissue equivalent’ (OTE) model with three novel features: native pulmonary fibroblasts, solubilized lung ECM, and hydrogel substrate with tunable stiffness and porosity. We demonstrate the versatility of the OTE model by evaluating the impact of these features on human bronchial epithelial (HBE) cell phenotype. Variations of this model were analyzed during 28 days of ALI culture by evaluating epithelial confluence, trans-epithelial electrical resistance, and epithelial phenotype via multispectral immuno-histochemistry and next-generation sequencing. Cultures that included both solubilized lung ECM and native pulmonary fibroblasts within the hydrogel substrate formed well-differentiated ALI cultures that maintained a barrier function and expressed mature epithelial markers relating to goblet, club, and ciliated cells. Modulation of hydrogel stiffness did not negatively impact HBE differentiation and could be a valuable variable to alter epithelial phenotype. This study highlights the feasibility and versatility of a 3D airway OTE model to model the multiple components of the human airway 3D microenvironment.

CARS imaging of ex vivo ovarian cancer samples

Supplementary Figure 4 from Fibroblasts Isolated from Common Sites of Breast Cancer Metastasis Enhance Cancer Cell Growth Rates and Invasiveness in an Interleukin-6–Dependent Manner

PDF file - 678K, O-ASC do not co-localize with CD31 positive endothelial cells.

PDF file - 1.0MB, Expression of IL8 / CXCL1 receptors in mesenchymal cells.