Medium flow rate regulates viability and barrier function of engineered skin substitutes in perfusion culture

Perfusion culture of engineered tissues improves mass transfer of nutrients and provides flow-mediated mechanical stimulation to the developing constructs, thereby improving their anatomy and physiology in vitro. In this study, the responses to medium flow rate of engineered skin substitutes (ESS) incubated in perfusion at the air-liquid interface were investigated. ESS fabricated with autologous keratinocytes, fibroblasts, and collagen-glycosaminoglycan (GAG) sponges were incubated for 21 days at the air-liquid interface in a custom-built recirculating bioreactor system at flow rates of 5, 15, and 50 mL/min (n = 8 per condition). ESS were evaluated in vitro using histology, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, bromodeoxyuridine (BrdU) incorporation, and surface hydration. ESS incubated at 5 and 15 mL/min had histological organization comparable with that of control ESS incubated in static conditions. ESS incubated at 50 mL/min displayed a disorganized epidermal substitute and, at later time points in culture, showed greater degradation of the dermal scaffold. Cell viability measured using MTT assay was significantly higher in ESS incubated at 5 mL/min than in static controls at day 14 (mean +/- standard error of the mean 1.63 +/- 0.11 vs 1.30 +/- 0.14, p < 0.05) and day 21 (1.66 +/- 0.12 vs 1.11+/- 0.15, p < 0.05) of culture. Viability of ESS incubated at 15 mL/min was comparable with that of controls. ESS incubated at 50 mL/min had significantly lower viabilities than controls at all time points. Results of BrdU incorporation data showed that, although ESS incubated at 5 and 15 mL/min were comparable with controls, those incubated at 50 mL/min had fewer proliferating keratinocytes per high-power field than controls (2.77 +/- 0.48 vs 28.1 +/- 0.78, p < 0.05). ESS incubated at 5 mL/min had surface hydration comparable with that of controls, whereas those incubated at 15 mL/min and 50 mL/min had significantly higher surface hydration than static controls at all time points. ESS incubated at a 5 mL/min flow rate and transplanted onto full-thickness wounds on athymic mice demonstrated wound healing comparable with that of controls. From these results, it can be concluded that perfusion culture of ESS at lower flow rates increases cell viability and maintains an epidermal barrier suitable for grafting, whereas higher flow rates lead to deterioration of ESS anatomy and physiology in vitro.     

A role for vitamin D and the vitamin D receptor in keloid disorder

Keloids are disfiguring fibroproliferative lesions that can occur in susceptible individuals following any skin injury. They are extremely challenging to treat, with relatively low response rates to current therapies and high rates of recurrence after treatment. Although several distinct genetic loci have been associated with keloid formation in different populations, there has been no single causative gene yet identified and the molecular mechanisms guiding keloid development are incompletely understood. Further, although it is well known that keloids are more commonly observed in populations with dark skin pigmentation, the basis for increased keloid risk in skin of colour is not yet known. Because individuals with dark skin pigmentation are at higher risk for vitamin D deficiency, the role of vitamin D in keloid pathology has gained interest in the keloid research community. A limited number of studies have found lower serum vitamin D levels in patients with keloids, and reduced expression of the vitamin D receptor (VDR) in keloid lesions compared with uninjured skin. Vitamin D has documented anti-inflammatory, anti-proliferative and pro-differentiation activities, suggesting it may have a therapeutic role in suppression of keloid fibrosis. Here we review the evidence supporting a role for vitamin D and VDR in keloid pathology.     

An improved human skin explant culture method for testing and assessing personal care products

Background

Cultured human skin models have been widely used in the evaluation of dermato-cosmetic products as alternatives to animal testing and expensive clinical testing. The most common in vitro skin culture approach is to maintain skin biopsies in an airlifted condition at the interface of the supporting culture medium and the air phase. This type of ex vivo skin explant culture is not, however, adequate for the testing of cleansing products, such as shampoos and body washes. One major deficiency is that cleansing products would not remain confined on top of the epidermis and have a high chance of running off toward the dermal side, thus compromising the experimental procedure and data interpretation.

Materials and Methods

Here, we describe an improved ex vivo method for culturing full-thickness human skin for the effective testing and evaluation of skin care products by topical application.

Results

This newly developed ex vivo human skin culture method has the ability to maintain healthy skin tissues for up to 14 days in culture. Importantly, the model provides a quick and safe way to evaluate skin care products at different time points after single or repetitive topical applications using a combined regimen of leave-on and wash-off. We found that the results obtained using the new skin culture method are reproducible and consistent with the data collected from clinical testing.

Conclusion

Our new ex vivo skin explant method offers a highly efficient and cost-effective system for the evaluation and testing of a variety of personal care products and new formulations.