Postmortem chondrocyte viability in porcine articular cartilage: Influence of time, temperature, and burial under winter conditions.

The aim of the present study was to investigate the effects of time, temperature, and burial in a natural environment on the viability of chondrocytes in porcine femoral condyles using confocal laser scanning microscopy. Hind trotters from 10 pigs were buried or left unburied. Samples were collected daily and stained with a combination of vital dyes (calcein-AM and ethidium homodimer-1). The chondrocytes showed an intense staining corresponding to their vitality. In the first 3 days, viability decreased slowly and showed no statistical difference between buried and unburied samples. After the first 3 days, it decreased rapidly, with the viability of the buried samples being 66% on day 4, decreasing to 25% on day 8 and to 16% on day 10, while in the unburied samples it decreased to 43% on day 4, 13% on day 8 and 5% on day 10. Our results indicate a time, temperature, and burial dependent decrease in chondrocyte viability and suggest the use of chondrocyte viability as a marker for estimating PMI in both the natural environment and in animals, as well as its potential use in humans.

Dual-mode visual detection strategies of viable pathogens for point-of-care testing.

Here, we introduce a power-free foldable poly(methyl methacrylate) (PMMA) microdevice fully integrating DNA extraction, amplification, and visual detection, realized in novel dual modes - colorimetric and aggregate formation - using 4-Aminoantipyrine (4-AP) for monitoring pathogens. The microdevice contains two parts: reaction and detection zones. A sealing film was utilized to connect the two zones and make the device foldable. The FTA card was deposited in the reaction zone for DNA extraction, followed by loop-mediated isothermal amplification (LAMP) at 65 °C for 45 min. When the detection zone is folded toward the reaction zone, paper discs modified with 4-AP placed in the detection zone are delivered to the reaction zone. Specifically, in the presence of LAMP amplicons, 4-AP is oxidized into antipyrine red or generates aggregates by interacting with copper sulfate, forming copper hybrid nanostructure (Cu-hNs). In the absence of LAMP amplicons, 4-AP is not oxidized and maintains yellow color or fails to form aggregates. Furthermore, we introduced the ethidium homodimer-1 (EthD-1) to identify viable bacteria. EthD-1 penetrated the compromised membranes of nonviable cells and prevented further DNA amplification by intercalating with the DNA. In this way, only samples containing viable cells displayed color change or formed aggregates upon reaction with 4-AP. Using this method, SARS-CoV-2 RNA and Enterococcus faecium were identified by naked eye, with the limit of detection of 103 copies/µL and 102 CFU/mL, respectively, within 60 min. The introduced microdevice can be used for rapidly monitoring viable pathogens and controlling outbreaks of infectious disease in resource-limited settings.

A bioengineering method for modeling alveolar Rhabdomyosarcoma and assessing chemotherapy responses.

Rhabdomyosarcoma (RMS) is the most common pediatric soft-tissue malignant tumor. Treatment of RMS usually includes primary tumor resection along with systemic chemotherapy. Two-dimensional (2D) cell culture systems and animal models have been extensively used for investigating the potential efficacy of new RMS treatments. However, RMS cells behave differently in 2D culture than in vivo, which has recently inspired the adoption of three-dimensional (3D) culture environments. In the current paper, we will describe the detailed methodology we have developed for fabricating a 3D engineered model to study alveolar RMS (ARMS) in vitro. This model consists of a thermally cross-linked collagen disk laden with RMS cells that mimics the structural and bio-chemical aspects of the tumor extracellular matrix (ECM). This process is highly reproducible and produces a 3D engineered model that can be used to analyze the cytotoxicity and autophagy induction of drugs on ARMS cells. The most improtant bullet points are as following:•We fabricated 3D model of ARMS.•The current ARMS 3D model can be used for screening of chemotherapy drugs.•We developed methods to detect apoptosis and autophagy in ARMS 3D model to detect the mechansims of chemotherapy agents.

Designing of spider silk proteins for human induced pluripotent stem cell-based cardiac tissue engineering.

Materials made of recombinant spider silk proteins are promising candidates for cardiac tissue engineering, and their suitability has so far been investigated utilizing primary rat cardiomyocytes. Herein, we expanded the tool box of available spider silk variants and demonstrated for the first time that human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes attach, contract, and respond to pharmacological treatment using phenylephrine and verapamil on explicit spider silk films. The hiPSC-cardiomyocytes contracted for at least 14 days on films made of positively charged engineered Araneus diadematus fibroin 4 (eADF4(κ16)) and three different arginyl-glycyl-aspartic acid (RGD)-tagged spider silk variants (positively or negatively charged and uncharged). Notably, hiPSC-cardiomyocytes exhibited different morphologies depending on the spider silk variant used, with less spreading and being smaller on films made of eADF4(κ16) than on RGD-tagged spider silk films. These results indicate that spider silk engineering is a powerful tool to provide new materials suitable for hiPSC-based cardiac tissue engineering.

Viable cryopreserved umbilical tissue (vCUT) reduces post-operative adhesions in a rabbit abdominal adhesion model.

Post-operative adhesions, a common complication of surgery, cause pain, impair organ functionality, and often require additional surgical interventions. Control of inflammation, protection of injured tissue, and rapid tissue repair are critical for adhesion prevention. Adhesion barriers are biomaterials used to prevent adhesions by physical separation of opposing injured tissues. Current adhesion barriers have poor anti-inflammatory and tissue regenerative properties. Umbilical cord tissue (UT), a part of the placenta, is inherently soft, conforming, biocompatible, and biodegradable, with antimicrobial, anti-inflammatory, and antifibrotic properties, making it an attractive alternative to currently available adhesion barriers. While use of fresh tissue is preferable, availability and short storage time limit its clinical use. A viable cryopreserved UT (vCUT) "point of care" allograft has recently become available. vCUT retains the extracellular matrix, growth factors, and native viable cells with the added advantage of a long shelf life at -80 °C. In this study, vCUT's anti-adhesion property was evaluated in a rabbit abdominal adhesion model. The cecum was abraded on two opposing sides, and vCUT was sutured to the abdominal wall on the treatment side; whereas the contralateral side of the abdomen served as an internal untreated control. Gross and histological evaluation was performed at 7, 28, and 67 days post-surgery. No adhesions were detectable on the vCUT treated side at all time points. Histological scores for adhesion, inflammation, and fibrosis were lower on the vCUT treated side as compared to the control side. In conclusion, the data supports the use of vCUT as an adhesion barrier in surgical procedures.

Rat hepatocyte culture model of macrosteatosis: effect of macrosteatosis induction and reversal on viability and liver-specific function.

BACKGROUND & AIMS: A common cause of liver donor ineligibility is macrosteatosis. Recovery of such livers could enhance donor availability. Living donor studies have shown diet-induced reduction of macrosteatosis enables transplantation. However, cadaveric liver macrosteatotic reduction must be performed ex vivo within hours. Towards this goal, we investigated the effect of accelerated macrosteatosis reduction on hepatocyte viability and function using a novel system of macrosteatotic hepatocytes. METHODS: Hepatocytes isolated from lean Zucker rats were cultured in a collagen sandwich, incubated for 6 days in fatty acid-supplemented medium to induce steatosis, and then switched for 2 days to medium supplemented with lipid metabolism promoting agents. Intracellular lipid droplet size distribution and triglyceride, viability, albumin and urea secretion, and bile canalicular function were measured. RESULTS: Fatty acid-supplemented medium induced microsteatosis in 3 days and macrosteatosis in 6 days, the latter evidenced by large lipid droplets dislocating the nucleus to the cell periphery. Macrosteatosis significantly impaired all functions tested. Macrosteatosis decreased upon returning hepatocytes to standard medium, and the rate of decrease was 4-fold faster with supplemented agents, yielding 80% reduction in 2 days. Viability of macrosteatosis reduced hepatocytes was similar to control lean cells. Accelerated macrosteatotic reduction led to faster recovery of urea secretion and bile canalicular function, but not of albumin secretion. CONCLUSIONS: Macrosteatosis reversibly decreases hepatocyte function and supplementary agents accelerate macrosteatosis reduction and some functional restoration with no effect on viability. This in vitro model may be useful to screen agents for macrosteatotic reduction in livers before transplantation.