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.

Arsenic, a naturally occurring environmental contaminant, is harmful to humans at elevated concentrations. Increased levels of arsenic in the environment occur as a result of human activities and from natural geologically sourced leaching into ground and surface water. These sources pose an exposure risk above the USEPA standard to individuals whose food and water sources become contaminated. Arsenic exposure negatively impacts organ function and increases the risk for developing pathologies, including cancer. Some of the effects of arsenic on cancer translate to normal cell function in wound healing. To evaluate whether arsenic influences wound healing, an in vitro scratch assay was employed to study the effects of arsenic on cellular migration, which is a key component in the normal wound-healing process. In this study, skin cells were exposed to environmentally relevant concentrations of arsenic, and wound closure was evaluated. Results indicated that arsenic significantly decreased the rate of cellular migration in the scratch assay when compared with controls. In addition, estradiol, which has been shown to positively influence cellular and tissue processes involved in wound healing, reversed the slowing effects of arsenic on wound closure. These results suggest that arsenic contamination may inhibit, and estrogen may provide a therapeutic benefit for individuals with arsenic-contaminated wounds.

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.

Platelet Rich Plasma (PRP) is an autologous clinical treatment that has been demonstrated to expedite wound healing through the delivery of platelets at a minimum concentration that is three times that of whole blood. PRP is currently used in a variety of clinical applications, including but not limited to: orthodontics, osteogenic care, orthopedics, acute and chronic wounds and various cosmetic applications. Although the treatment has been investigated in many regards, there is a lack of exploration into means of activation through the use of electrospun (e-spun) collagen scaffold technology that allows for activation in situ, or directly in the wound. In this study, this combination therapy (PRP and e-spun collagen scaffolds) was created and investigated first using an in-vitro assay and then in an in-vivo murine full-thickness wound model. In the current study, a standardized double centrifuge protocol was utilized for the creation of PRP. Flow cytometry and manual counts were performed in order to quantify the degree of platelet concentration. Manual counts yielded a mean platelet count for whole blood and PRP 1 µL smears at 10.08 ± 3.09 platelets/μl and 878.76 ± 156.28829.7 platelets, respectively (p < 0.01). Flow cytometry reported a mean platelet count of 426,461 ± 47,394 for the processed PRP and a whole blood platelet count of 35,480 ± 6,463.75 (p < 0.01), supporting that the currently described method produces clinically defined PRP. Light and scanning electron microscopy were utilized to determine if electrospun collagen scaffolds were an activator of platelets. Analysis was performed using morphological features of the platelets as the activation measurement. Upon application to an electrospun scaffold, platelets presented with an activated state in which the discoid morphology was no longer present, but was replaced with extended pseudopods and blebs. The morphology of non-activated PRP remained discoid and smooth. The change in morphology was indicative of activation and validated that the scaffolds serve as an in-situ platelet activator. An in-vitro scratch assay was utilized as an outcome measure to test the potency and effectiveness of the resulting PRP. The scratch assay has been commonly used as a cellular migration assessment, or in the current study, as a high throughput means to observe in-vitro wound closure. Varying concentrations of PRP were created and applied to a scratched (wounded) fibroblast monolayer in order to determine its effects on the wound. A concentration of 0.25% PRP was determined to have the highest percent closure at hour 12 compared to the control that did not close until hour 16. An in-vivo murine full-thickness wound model was used to demonstrate the effectiveness of the PRP and collagen scaffold treatment in a biological model. Post wound creation (96 hours) the wound closure percentage of the PRP/electrospun collagen treatment was 100% compared to the control at 86.076 ± 11.2806% (p=0.01), supporting the use of a combination therapy over standardized care. There was...