Understanding the physiologic mechanisms of wound healing has been the focus of ongoing research for many years. This research directly translates into changes in clinical standards used for treating wounds and decreasing morbidity and mortality for patients. Wound healing is a complex process that requires strategic cell and tissue interaction and function. One of the many critically important functions of wound healing is individual and collective cellular migration. Upon injury, various cells from the blood, surrounding connective, and epithelial tissues rapidly migrate to the wound site by way of chemical and/or physical stimuli. This migration response can largely dictate the outcomes and success of a healing wound. Understanding this specific cellular function is important for translational medicine that can lead to improved wound healing outcomes. Here, we describe a protocol used to better understand cellular migration as it pertains to wound healing, and how changes to the cellular environment can significantly alter this process. In this example study, dermal fibroblasts were grown in media supplemented with fetal bovine serum (FBS) as monolayer cultures in tissue culture flasks. Cells were aseptically transferred into tissue culture treated 12-well plates and grown to 100% confluence. Upon reaching confluence, the cells in the monolayer were vertically scratched using a p200 pipet tip. Arsenic diluted in culture media supplemented with FBS was added to individual wells at environmentally relevant doses ranging 0.1-10 μM. Images were captured every 4 hours (h) over a 24 h period using an inverted light microscope to observe cellular migration (wound closure). Images were individually analyzed using image analysis software, and percent wound closure was calculated. Results demonstrate that arsenic slows down wound healing. This technique provides a rapid and inexpensive first screen for evaluation of the effects of contaminants on wound healing.

Coronary artery bypass grafts used to treat coronary artery disease (CAD) 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 (TEVG) 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 were 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 TEVG 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 values of the hypocompliant graft were 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.

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 in vitro 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 in vitro 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 in vivo outcomes, and in many early studies can help to demonstrate proof-of-concept before moving into complex biological systems.

Chronic wounds in patients suffering from type II diabetes mellitus (DMII) where wounds remain open with a complicated pathophysiology, healing, and recovery process is a public health concern. Normal wound healing plays a critical role in wound closure, restoration of mechanical properties, and the biochemical characteristics of the remodeled tissue. Biological scaffolds provide a tissue substitute to help facilitate wound healing by mimicking the extracellular matrix (ECM) of the dermis. In the current study an electrospun biomimetic scaffold, wound healing device (WHD), containing tropoelastin (TE) and collagen was synthesized to mimic the biochemical and mechanical characteristics of healthy human skin. The WHD was compared to a commercially available porcine small intestinal submucosa (SIS) matrix that has been used in both partial and full-thickness wounds, Oasis® Wound Matrix. Using a diabetic murine model C57BKS.Cg-m+/+Leprdb/J mice (db/db) wound closure rates, histochemistry (CD31 and CD163), qPCR (GAPDH, TNF-α, NOS2, ARG1 and IL10), and mechanical testing of treated wound sites were evaluated. The WHD in a splinted, full thickness, diabetic murine wound healing model demonstrated skin organ regeneration, an enhanced rate of wound closure, decreased tissue inflammation, and a stronger and more durable remodeled tissue that more closely mimics native unwounded skin compared to the control device.