Mechano-Activated Cell Therapy for Accelerated Diabetic Wound Healing.

Chronic diabetic wounds are a significant global healthcare challenge. Current strategies, such as biomaterials, cell therapies, and medical devices, however, only target a few pathological features and have limited efficacy. A powerful platform technology combining magneto-responsive hydrogel, cells, and wireless magneto-induced dynamic mechanical stimulation (MDMS) is developed to accelerate diabetic wound healing. The hydrogel encapsulates U.S. Food and Drug Administration (FDA)-approved fibroblasts and keratinocytes to achieve ∼3-fold better wound closure in a diabetic mouse model. MDMS acts as a nongenetic mechano-rheostat to activate fibroblasts, resulting in ∼240% better proliferation, ∼220% more collagen deposition, and improved keratinocyte paracrine profiles via the Ras/MEK/ERK pathway to boost angiogenesis. The magneto-responsive property also enables on-demand insulin release for spatiotemporal glucose regulation through increasing network deformation and interstitial flow. By mining scRNAseq data, a mechanosensitive fibroblast subpopulation is identified that can be mechanically tuned for enhanced proliferation and collagen production, maximizing therapeutic impact. The "all-in-one" system addresses major pathological factors associated with diabetic wounds in a single platform, with potential applications for other challenging wound types.

A wireless and battery-free wound infection sensor based on DNA hydrogel.

The confluence of wireless technology and biosensors offers the possibility to detect and manage medical conditions outside of clinical settings. Wound infections represent a major clinical challenge in which timely detection is critical for effective interventions, but this is currently hindered by the lack of a monitoring technology that can interface with wounds, detect pathogenic bacteria, and wirelessly transmit data. Here, we report a flexible, wireless, and battery-free sensor that provides smartphone-based detection of wound infection using a bacteria-responsive DNA hydrogel. The engineered DNA hydrogels respond selectively to deoxyribonucleases associated with pathogenic bacteria through tunable dielectric changes, which can be wirelessly detected using near-field communication. In a mouse acute wound model, we demonstrate that the wireless sensor can detect physiologically relevant amounts of Staphylococcus aureus even before visible manifestation of infection. These results demonstrate strategies for continuous infection monitoring, which may facilitate improved management of surgical or chronic wounds.

A flexible multiplexed immunosensor for point-of-care in situ wound monitoring.

Chronic wounds arise from interruption of normal healing due to many potential pathophysiological factors. Monitoring these multivariate factors can provide personalized diagnostic information for wound management, but current sensing technologies use complex laboratory tests or track a limited number of wound parameters. We report a flexible biosensing platform for multiplexed profiling of the wound microenvironment, inflammation, and infection state at the point of care. This platform integrates a sensor array for measuring inflammatory mediators [tumor necrosis factor-α, interleukin-6 (IL-6), IL-8, and transforming growth factor-ß1], microbial burden (Staphylococcus aureus), and physicochemical parameters (temperature and pH) with a microfluidic wound exudate collector and flexible electronics for wireless, smartphone-based data readout. We demonstrate in situ multiplexed monitoring in a mouse wound model and also profile wound exudates from patients with venous leg ulcers. This technology may facilitate more timely and personalized wound management to improve chronic wound healing outcomes.

Pulpal upregulation of connexin 43 during pulpitis.

OBJECTIVES: Connexins are building blocks of membranous channels that form gap junctions and hemichannels. These channels are essential portals for information exchange and coordination during inflammation. Pathologic levels of these conduits may result in excessive inflammation and collateral destruction. This study aimed to analyse temporospatial levels of connexin 43 (Cx43) during pulpitis in extracted human teeth and in a rodent model. A specific interest was directed at the pulpal stroma as it is conserved during vital pulp therapy. MATERIALS AND METHODS: Pulpal tissues were attained from human extracted teeth of various pulpal inflammatory stages and fixed for cryosections. Pulpal exposures were created in bilateral maxillary molars in Sprague-Dawley rats. Rats were sacrificed at days 1 to 5 post-exposure. Immunofluorescence histology was performed to detect Cx43, markers for inflammation, and cell death. Immunofluorescent levels in the pulpal stroma at 3 sites (wound/near/far) were matched to pulpal condition (human) or days post-exposure (rodent). RESULTS: Cx43 upregulation was observed with increased severity of pulpitis both in humans and rodent model. The upregulation appeared to be global and included distant regions. Elevated levels of neutrophils were present in advanced pulpitis. Apoptosis and necroptosis seem to be upregulated in human samples as Cx43 levels rose. CONCLUSIONS: We observed a disseminated upregulation of Cx43 throughout the pulpal stroma as inflammation became advanced. This observation may facilitate cell death signal transfer or represent overt levels of purinergic signalling that leads to pro-inflammatory conditions. CLINICAL RELEVANCE: Cx43 downregulation may represent a potential therapeutic approach to enable resolution of pulpal inflammation.

Depth-sensitive Raman spectroscopy for skin wound evaluation in rodents.

Raman spectroscopy has demonstrated great potential for skin wound assessment. Given that biochemical changes in wound healing is depth dependent as the skin is a layered structure, depth sensitive Raman spectroscopy could enhance the power of Raman spectroscopy in this application. Considering the critical importance of rodent studies in the field of skin wound assessment, it is necessary to develop and validate a system that can perform depth sensitive measurements in rat skin with a proper target depth range. In this manuscript, we report the design, optimization and evaluation of a new snapshot depth-sensitive Raman instrument for rat skin measurements. The optical design and optimization process are presented first. The depth sensitive measurement performance is characterized on both ex vivo porcine skin with a gradient of layer thickness and ex vivo rat skin samples with wounds. The statistical analysis of the measured Raman spectra demonstrates the feasibility of differentiation between the wound edge and healthy skin. Moreover, the accuracy of classification improves monotonically as more data from new depths are used, which implies that each depth offers additional information useful for classification. This instrument demonstrates the ability to perform snapshot depth sensitive Raman measurements from rat skin, which paves the way towards in vivo preclinical studies of rat skin wounds.

The Potential Impact of Connexin 43 Expression on Bcl-2 Protein Level and Taxane Sensitivity in Head and Neck Cancers-In Vitro Studies.

The poor prognosis of head and neck squamous cell carcinoma (HNSCC) is partly due to the lack of reliable predictive markers. Connexin 43 (Cx43) protein and its cell-communication channels have been assigned tumor suppressor functions while the anti-apoptotic Bcl-2 (B-cell lymphoma-2) protein has been associated with negative prognostic significance in cancer. This study aimed to test the role of Cx43 protein on Bcl-2 expression, tumor progression and response to taxane-based treatment in HNSCC. Human papillomavirus (HPV) negative HNSCC cell lines were tested for paclitaxel sensitivity through measuring apoptosis induction, cell viability and changes in Cx43 and Bcl-2 levels using flow cytometry, cell viability assay, immunocytochemistry and western blot. Inhibition of Cx43 expression using siRNA increased Bcl-2 protein levels in SCC25 (tongue squamous cell carcinoma) cells, while forced Cx43 expression reduced Bcl-2 levels and supported paclitaxel cytotoxicity in FaDu (hypopharynx squamous cell carcinoma) cells. In vitro results were in line with protein expression and clinicopathological features tested in tissue microarray samples of HNSCC patients. Our data demonstrate that elevated Cx43 and reduced Bcl-2 levels may indicate HNSCC sensitivity to taxane-based treatments. On the contrary, silencing of the Cx43 gene GJA1 (gap junction protein alpha-1) can result in increased Bcl-2 expression and reduced paclitaxel efficiency. Clinical tumor-based analysis also confirmed the inverse correlation between Cx43 and Bcl-2 expression.

The impact of ischemia-reperfusion injuries on skin resident murine dendritic cells.

Pressure ulcers are a chronic problem for patients or the elderly who require extended periods of bed rest. The formation of ulcers is due to repeated cycles of ischemia-reperfusion (IR), which initiates an inflammatory response. Advanced ulcers disrupt the skin barrier, resulting in further complications. To date, the immunological aspect of skin IR has been understudied, partly due to the complexity of the skin immune cells. Through a combination of mass cytometry, confocal imaging and intravital multiphoton imaging, this study establishes a workflow for multidimensionality single cell analysis of skin myeloid cell responses in the context of IR injury with high spatiotemporal resolution. The data generated has provided us with previously uncharacterized insights into the distinct cellular behavior of resident dendritic cells (DCs) and recruited neutrophils post IR. Of interest, we observed a drop in DDC numbers in the IR region, which was subsequently replenished 48h post IR. More importantly, in these cells, we observe an attenuated response to repeated injuries, which may have implications in the subsequent wound healing process.

Enterococcus faecalis Modulates Immune Activation and Slows Healing During Wound Infection.

Enterococcus faecalis is one of the most frequently isolated bacterial species in wounds yet little is known about its pathogenic mechanisms in this setting. Here, we used a mouse wound excisional model to characterize the infection dynamics of E faecalis and show that infected wounds result in 2 different states depending on the initial inoculum. Low-dose inocula were associated with short-term, low-titer colonization whereas high-dose inocula were associated with acute bacterial replication and long-term persistence. High-dose infection and persistence were also associated with immune cell infiltration, despite suppression of some inflammatory cytokines and delayed wound healing. During high-dose infection, the multiple peptide resistance factor, which is involved in resisting immune clearance, contributes to E faecalis fitness. These results comprehensively describe a mouse model for investigating E faecalis wound infection determinants, and suggest that both immune modulation and resistance contribute to persistent, nonhealing wounds.

Connexins in endothelial barrier function - novel therapeutic targets countering vascular hyperpermeability.

Prolonged vascular hyperpermeability is a common feature of many diseases. Vascular hyperpermeability is typically associated with changes in the expression patterns of adherens and tight junction proteins. Here, we focus on the less-appreciated contribution of gap junction proteins (connexins) to basal vascular permeability and endothelial dysfunction. First, we assess the association of connexins with endothelial barrier integrity by introducing tools used in connexin biology and relating the findings to customary readouts in vascular biology. Second, we explore potential mechanistic ties between connexins and junction regulation. Third, we review the role of connexins in microvascular organisation and development, focusing on interactions of the endothelium with mural cells and tissue-specific perivascular cells. Last, we see how connexins contribute to the interactions between the endothelium and components of the immune system, by using neutrophils as an example. Mounting evidence of crosstalk between connexins and other junction proteins suggests that we rethink the way in which different junction components contribute to endothelial barrier function. Given the multiple points of connexin-mediated communication arising from the endothelium, there is great potential for synergism between connexin-targeted inhibitors and existing immune-targeted therapeutics. As more drugs targeting connexins progress through clinical trials, it is hoped that some might prove effective at countering vascular hyperpermeability.

Near-infrared light-sensitive liposomes for enhanced plasmid DNA transfection.

Near-infrared (NIR) light-responsive liposomes are attractive carriers for targeted and controlled drug delivery to the superficial organ or tissue (e.g., skin). This work describes the development of NIR-responsive liposomes by incorporating gold nanostars within liposomes composed of Phospholipon 90 g and cholesterol. Following cellular delivery, photothermal effect around the gold nanostar upon NIR stimulation induces microcavitation and liposome phase transition which consequently triggers the release of encapsulated molecules. Taking GFP plasmid as an example, we demonstrate enhanced gene transfection into fibroblasts following NIR treatment.

Controlled release of drugs in electrosprayed nanoparticles for bone tissue engineering.

Generating porous topographic substrates, by mimicking the native extracellular matrix (ECM) to promote the regeneration of damaged bone tissues, is a challenging process. Generally, scaffolds developed for bone tissue regeneration support bone cell growth and induce bone-forming cells by natural proteins and growth factors. Limitations are often associated with these approaches such as improper scaffold stability, and insufficient cell adhesion, proliferation, differentiation, and mineralization with less growth factor expression. Therefore, the use of engineered nanoparticles has been rapidly increasing in bone tissue engineering (BTE) applications. The electrospray technique is advantageous over other conventional methods as it generates nanomaterials of particle sizes in the micro/nanoscale range. The size and charge of the particles are controlled by regulating the polymer solution flow rate and electric voltage. The unique properties of nanoparticles such as large surface area-to-volume ratio, small size, and higher reactivity make them promising candidates in the field of biomedical engineering. These nanomaterials are extensively used as therapeutic agents and for drug delivery, mimicking ECM, and restoring and improving the functions of damaged organs. The controlled and sustained release of encapsulated drugs, proteins, vaccines, growth factors, cells, and nucleotides from nanoparticles has been well developed in nanomedicine. This review provides an insight into the preparation of nanoparticles by electrospraying technique and illustrates the use of nanoparticles in drug delivery for promoting bone tissue regeneration.