in-vitro-scratch-assay-to-demonstrate-effects-arsenic-on-skin-cell

<em>In Vitro</em> Scratch Assay to Demonstrate Effects of Arsenic on Skin Cell Migration

Understanding the physiologic mechanisms of wound healing has been the focus of ongoing research for many years. This research directly translates into ...

Northern Arizona University

Varying strategies are currently being evaluated to develop tissue-engineered constructs for the treatment of ischemic heart disease. This study examines an angiogenic and biodegradable cardiac construct seeded with neonatal cardiomyocytes for the treatment of chronic heart failure (CHF).

An electrically coupled tissue-engineered cardiomyocyte scaffold improves cardiac function in rats with chronic heart failure.

Whole slide imaging (WSI) can be used to quantify multiple responses within tissue sections during histological analysis. Feature Analysis on Consecutive Tissue Sections (FACTS®) allows the investigator to perform digital morphometric analysis (DMA) within specified regions of interest (ROI) across multiple serial sections at faster rates when compared with manual morphometry methods. Using FACTS® in conjunction with WSI is a powerful analysis tool, which allows DMA to target specific ROI across multiple tissue sections stained for different biomarkers. DMA may serve as an appropriate alternative to classic, manual, histologic morphometric measures, which have historically relied on the selection of high-powered fields of views and manual scoring (e.g., a gold standard). In the current study, existing preserved samples were used to determine if DMA would provide similar results to manual counting methods. Rodent hearts (n=14, left ventricles) were stained with Masson's trichrome, and reacted for cluster of differentiation 68 (CD-68). This study found no statistical significant difference between a classic, manual method and the use of digital algorithms to perform the similar counts (p=0.38). DMA offers researchers the ability to accurately evaluate morphological characteristics in a reproducible fashion without investigator bias and with higher throughput. 

Validating whole slide digital morphometric analysis as a microscopy tool.

A main goal of tissue engineering is the development of scaffolds that replace, restore and improve injured tissue. These scaffolds have to mimic natural tissue, constituted by an extracellular matrix (ECM) support, cells attached to the ECM, and signaling molecules such as growth factors that regulate cell function. In this study we created electrospun flat sheet scaffolds using different compositions of gelatin and fibrinogen. Smooth muscle cells (SMCs) were seeded on the scaffolds, and proliferation and infiltration were evaluated. Additionally, different concentrations of Transforming Growth Factor-beta2 (TGFβ2) were added to the medium with the aim of elucidating its effect on cell proliferation, migration and collagen production. Our results demonstrated that a scaffold with a composition of 80% gelatin-20% fibrinogen is suitable for tissue engineering applications since it promotes cell growth and migration. The addition of TGFβ2 at low concentrations (≤ 1 ng/ml) to the culture medium resulted in an increase in SMC proliferation and scaffold infiltration, and in the reduction of collagen production. In contrast, TGFβ2 at concentrations >1 ng/ml inhibited cell proliferation and migration while stimulating collagen production. According to our results TGFβ2 concentration has a differential effect on SMC function and thus can be used as a biochemical modulator that can be beneficial for tissue engineering applications.

TGFβ2 differentially modulates smooth muscle cell proliferation and migration in electrospun gelatin-fibrinogen constructs.

Cellular migration assays are useful tools to investigate physiologic events on the bench top. Furthermore, this migration assay can be utilized to investigate wound healing therapeutics (those that encourage or accelerate wound closure) as well as deleterious agents (ones that mitigate or slow wound closure). The current study used an  scratch assay to measure the effects of platelet-rich plasma (PRP) and depleted uranium (DU) in the form of uranyl acetate on cellular migration of human neonatal dermal fibroblasts in an  simulation of wound healing. Data analyses included percent wound closure measured as the distance between cell margins, and rates of wound closure versus untreated controls. The highest doses of PRP (0.063, 0.125%) resulted in 50-65% wound closure after 4-8 hours relative to 38-44% in controls and the low-dose treatment group (0.031%). The high-dose treatments of PRP (0.125, 0.063%) reached 100% wound closure at 12 hours postwound versus 16 hours for controls and the low-dose treatment group (0.031%). Conversely, the higher doses of DU treatments (50 and 100 μM) resulted in <80% closure versus 100% closure in controls after 16 hours, with full closure observed at 20 hours. The highest dose of DU (1,000 μM) resulted in <20% closure versus 100% closure in controls after 16 hours. The use of the described scratch assay serves as a translatable bench-top model that has the potential to predict  outcomes, and in many early studies can help to demonstrate proof-of-concept before moving into complex biological systems.

A Bench-Top  Wound Assay to Demonstrate the Effects of Platelet-Rich Plasma and Depleted Uranium on Dermal Fibroblast Migration.

Coronary artery bypass grafts used to treat coronary artery disease often fail due to compliance mismatch. In this study, we have developed an experimental/computational approach to fabricate an acellular biomimetic hybrid tissue engineered vascular graft composed of alternating layers of electrospun porcine gelatin/polycaprolactone (PCL) and human tropoelastin/PCL blends with the goal of compliance-matching to rat abdominal aorta, while maintaining specific geometrical constraints. Polymeric blends at three different gelatin:PCL (G:PCL) and tropoelastin:PCL (T:PCL) ratios (80:20, 50:50 and 20:80) were mechanically characterized. The stress-strain data was used to develop predictive models, which were used as part of an optimization scheme that was implemented to determine the ratios of G:PCL and T:PCL and the thickness of the individual layers within a tissue engineered vascular graft that would compliance match a target compliance value. The hypocompliant, isocompliant, and hypercompliant grafts had target compliance values of 0.000256, 0.000568 and 0.000880 mmHg-1, respectively. Experimental validation of the optimization demonstrated that the hypercompliant and isocompliant grafts were not statistically significant from their respective target compliance values (p-value=0.37 and 0.89, respectively). The experimental compliance value of the hypocompliant graft was statistically significant than their target compliance value (p-value=0.047). We have successfully demonstrated a design optimization scheme that can be used to fabricate multilayered and biomimetic vascular grafts with targeted geometry and compliance.

Article	Year
An electrically coupled tissue-engineered cardiomyocyte scaffold improves cardiac function in rats with chronic heart failure. The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation\| PubMed ID: 24560982	2014
Validating whole slide digital morphometric analysis as a microscopy tool. Microscopy and microanalysis : the official journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada\| PubMed ID: 25399639	2015
TGFβ2 differentially modulates smooth muscle cell proliferation and migration in electrospun gelatin-fibrinogen constructs. Biomaterials\| PubMed ID: 25453947	2015
A Bench-Top Wound Assay to Demonstrate the Effects of Platelet-Rich Plasma and Depleted Uranium on Dermal Fibroblast Migration. Applied in vitro toxicology\| PubMed ID: 28971114	2016
Computationally optimizing the compliance of multilayered biomimetic tissue engineered vascular grafts. Journal of biomechanical engineering\| PubMed ID: 30778568	2019

Robert S. Kellar

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