Low-dose acetaminophen induces early disruption of cell-cell tight junctions in human hepatic cells and mouse liver

Dysfunction of cell-cell tight junction (TJ) adhesions is a major feature in the pathogenesis of various diseases. Liver TJs preserve cellular polarity by delimiting functional bile-canalicular structures, forming the blood-biliary barrier. In acetaminophen-hepatotoxicity, the mechanism by which tissue cohesion and polarity are affected remains unclear. Here, we demonstrate that acetaminophen, even at low-dose, disrupts the integrity of TJ and cell-matrix adhesions, with indicators of cellular stress with liver injury in the human hepatic HepaRG cell line, and primary hepatocytes. In mouse liver, at human-equivalence (therapeutic) doses, dose-dependent loss of intercellular hepatic TJ-associated ZO-1 protein expression was evident with progressive clinical signs of liver injury. Temporal, dose-dependent and specific disruption of the TJ-associated ZO-1 and cytoskeletal-F-actin proteins, correlated with modulation of hepatic ultrastructure. Real-time impedance biosensing verified in vitro early, dose-dependent quantitative decreases in TJ and cell-substrate adhesions. Whereas treatment with NAPQI, the reactive metabolite of acetaminophen, or the PKCα-activator and TJ-disruptor phorbol-12-myristate-13-acetate, similarly reduced TJ integrity, which may implicate oxidative stress and the PKC pathway in TJ destabilization. These findings are relevant to the clinical presentation of acetaminophen-hepatotoxicity and may inform future mechanistic studies to identify specific molecular targets and pathways that may be altered in acetaminophen-induced hepatic depolarization

Chitosan microchannel scaffolds for tendon tissue engineering characterized using optical coherence tomography.

Tendon tissue engineering requires the generation of a uniaxially orientated collagen type I matrix with several organization scales that confer mechanical functionality upon the tendon. A combination of factors in a dose- and time-dependent manner, such as growth factors and mechanical environment, may be the key to an in vitro-engineered tendon. To define the progress of tissue development within a scaffold, on-line systems need to be applied to monitor the newly generated matrix. To address this challenge, we designed a new porous chitosan scaffold with microchannels (diameter: 250 microm), which allows primary porcine tenocytes to proliferate in a bundle-like structure. The cell proliferation and extracellular matrix (ECM) production within the microchannels were successfully assessed under sterile conditions using optical coherence tomography (OCT). A semi-quantitative method that calculated the microchannel occupation ratio (the degree of cell proliferation and tissue turnover based on the total backscattered intensity in the microchannels) was developed. We further investigated the effect of different culture conditions on tendon cell matrix formation. Using a perfusion bioreactor, we demonstrated how fluid flow can increase (p < 1e(3)) ECM production within the microchannels significantly more than static culture. Our study illustrates how using a guiding scaffold in combination with the fast and non-destructive assessment of the microstructure using OCT allows discrimination between the parameters affecting the production and the organization of the ECM

In-depth imaging and quantification of degenerative changes associated with Achilles ruptured tendons by polarization-sensitive optical coherence tomography.

The objective of this study was to develop a method based on polarization-sensitive optical coherent tomography (PSOCT) for the imaging and quantification of degenerative changes associated with Achilles tendon rupture. Ex vivo PSOCT examinations were performed in 24 patients. The study involved samples from 14 ruptured Achilles tendons, 4 tendinopathic Achilles tendons and 6 patellar tendons (collected during total knee replacement) as non-ruptured controls. The samples were imaged in both intensity and phase retardation modes within 24 h after surgery, and birefringence was quantified. The samples were fixed and processed for histology immediately after imaging. Slides were assessed twice in a blind manner to provide a semi-quantitative histological score of degeneration. In-depth micro structural imaging was demonstrated. Collagen disorganization and high cellularity were observable by PSOCT as the main markers associated with pathological features. Quantitative assessment of birefringence and penetration depth found significant differences between non-ruptured and ruptured tendons. Microstructure abnormalities were observed in the microstructure of two out of four tendinopathic samples. PSOCT has the potential to explore in situ and in-depth pathological change associated with Achilles tendon rupture, and could help to delineate abnormalities in tendinopathic samples in vivo