Voltage-dependent volume regulation controls epithelial cell extrusion and morphology.

Epithelial cells work collectively to provide a protective barrier, yet also turn over rapidly by cell death and division. If the number of dying cells does not match those dividing, the barrier would vanish, or tumors can form. Mechanical forces and the stretch-activated ion channel (SAC) Piezo1 link both processes; stretch promotes cell division and crowding triggers cell death by initiating live cell extrusion1,2. However, it was not clear how particular cells within a crowded region are selected for extrusion. Here, we show that individual cells transiently shrink via water loss before they extrude. Artificially inducing cell shrinkage by increasing extracellular osmolarity is sufficient to induce cell extrusion. Pre-extrusion cell shrinkage requires the voltage-gated potassium channels Kv1.1 and Kv1.2 and the chloride channel SWELL1, upstream of Piezo1. Activation of these voltage-gated channels requires the mechano-sensitive Epithelial Sodium Channel, ENaC, acting as the earliest crowd-sensing step. Imaging with a voltage dye indicated that epithelial cells lose membrane potential as they become crowded and smaller, yet those selected for extrusion are markedly more depolarized than their neighbours. Loss of any of these channels in crowded conditions causes epithelial buckling, highlighting an important role for voltage and water regulation in controlling epithelial shape as well as extrusion. Thus, ENaC causes cells with similar membrane potentials to slowly shrink with compression but those with reduced membrane potentials to be eliminated by extrusion, suggesting a chief driver of cell death stems from insufficient energy to maintain cell membrane potential.

Early mechanical selection of cell extrusion and extrusion signaling in cancer.

Epithelial cells use the process of extrusion to promote cell death while preserving a tight barrier. To extrude, a cell and its neighbors contract actin and myosin circumferentially and basolaterally to seamlessly squeeze it out of the epithelium. Recent research highlights how early apical pulsatile contractions within the extruding cell might orchestrate contraction in three dimensions so that a cell extrudes out apically. Along with apical constrictions, studies of ion channels and mathematical modeling reveal how differential contraction between cells helps select specific cells to extrude. In addition, several studies have offered new insights into pathways that use extrusion to eliminate transformed cells or cause an aberrant form of extrusion that promotes cell invasion.

Biomimetic coatings and negative pressure wound therapy independently limit epithelial downgrowth around percutaneous devices.

Biomimetic material coatings and negative pressure wound therapy (NPWT) have been shown independently to limit the epithelial downgrowth rates in percutaneous devices. It was therefore hypothesized that these techniques, in combination, could further limit the clinically observed epithelial downgrowth around these devices. In this study, we evaluated the efficacy of two biomimetic coatings, collagen and hydroxyapatite (HA), to prevent downgrowth when used with continuous NPWT. Using an established single-stage surgical protocol, collagen (n = 10) and HA (n = 10) coated devices were implanted subdermally on the back of hairless guinea pigs. Five animals from each group were subjected to continuous ~90 mmHg NPWT. Four weeks post-implantation, animals were sacrificed, and the devices and surrounding tissues were harvested, processed, and downgrowth was computed and compared to historical porous titanium coated controls. Data showed a significant reduction in downgrowth in NPWT treated animals (p ≤ 0.05) when compared to the untreated porous titanium controls. HA coated devices, without the NPWT treatment, also showed significantly decreased downgrowth compared to the untreated porous titanium controls.

Peri-prosthetic tissue reaction to discontinuation of negative pressure wound therapy around porous titanium percutaneous devices.

Negative pressure wound therapy (NPWT) has been reported to limit epithelial downgrowth, one of the failure mechanisms of percutaneous devices. In a previous study, when NPWT was applied for 4 weeks (NPWT Group) to porous coated titanium percutaneous devices, downgrowth (5 ± 4%; mean ± one SD) was significantly reduced compared to untreated controls (Untreated Group) (16 ± 6%; p ≤ 0.01). However, it was unclear whether this beneficial effect was sustained when NPWT was discontinued. In order to test this, porous coated titanium percutaneous devices were implanted into 6 hairless guinea pigs. Post-surgery, animals received 4 weeks of NPWT treatment followed by 4 weeks of no treatment (Discontinued Group). At necropsy, the devices and surrounding tissues were harvested and processed. Quantitative downgrowth measurements and qualitative analyses of tissue characteristics were performed, and compared to historical controls (NPWT and Untreated Groups). The Discontinued Group, at 8 weeks, had significantly more downgrowth than the NPWT Group at 4 weeks (23 ± 3% vs. 5 ± 4%; p ≤ 0.01). At 8 weeks, the Discontinued Group qualitatively appeared to exhibit reduced numbers of blood vessels and increased degree of fibrosis compared to the NPWT Group at 4 weeks. This study suggests that NPWT will only be an effective treatment for limiting downgrowth if used continuously. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2018. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 564-572, 2019.

Negative pressure wound therapy limits downgrowth in percutaneous devices.

Maintenance of a soft tissue seal around percutaneous devices is challenged by the downgrowth of periprosthetic tissues-a gateway to potential infection. As negative pressure wound therapy (NPWT) is used clinically to facilitate healing of complex soft tissue pathologies, it was hypothesized that NPWT could limit downgrowth of periprosthetic tissues. To test this hypothesis, 20 hairless guinea pigs were randomly assigned into four groups (n = 5/group). Using a One-Stage (Groups 1 and 3) or a Two-Stage (Groups 2 and 4) surgical procedure, each animal was implanted with a titanium-alloy subdermal device porous-coated with commercially pure, medical grade titanium. Each subdermal device had a smooth titanium-alloy percutaneous post. The One-Stage procedure encompassed insertion of a fully assembled device during a single surgery. The Two-Stage procedure involved the implantation of a subdermal device during the first surgery, and then three weeks later, insertion of a percutaneous post. Groups 1 and 2 served as untreated controls and Groups 3 and 4 received NPWT. Four weeks postimplantation of the post, the devices and surrounding tissues were harvested, and histologically evaluated for downgrowth. Within the untreated control groups, the Two-Stage surgical procedure significantly decreased downgrowth (p = 0.027) when compared with the One-Stage procedure. Independent of the surgical procedures performed, NPWT significantly limited downgrowth (p ≤ 0.05) when compared with the untreated controls.