Laboratory Research
Dysregulation and impairment in the process of normal wound healing may lead to chronic wounds. A number of skin substitutes are available to cover, support, and treat chronic wounds; however, the mechanism of these products remains largely unknown. In this study, we characterized key extracellular matrix (ECM) components, markers of activated keratinocytes, cellular proliferation, and key cytokines were also evaluated of a bi-layered living cellular construct (BLCCº) using immunofluorescence (IF) staining. Furthermore, a porcine in vivo diabetic delayed wound healing model was utilized to evaluate the impact of single or multiple applications of BLCC treatments.
Methods:
Structural assessments for BLCCs were made using hematoxylin and eosin (H&E) staining. To evaluate components relevant to normal skin, IF staining was completed for key ECM proteins (pro-collagen I, collagen I, collagen III, collagen IV, fibronectin, and laminin), markers of activated keratinocytes (K19), proliferation (Ki67), and key cytokines (VEGF and TGF-β). To assess the impact of BLCCs on wound healing, a porcine-derived BLCC was developed and utilized in a diabetic delayed wound healing model. After chemical induction of diabetes, full-thickness wounds were created and then either left untreated (controls) or treated with single or multiple treatments of porcine BLCC.
Results:
H&E staining of BLCCs showed stratified epidermis and dermal layer separated by basement membrane, mimicking normal skin. IF staining showed pro-collagen I, collagen I, collagen III, fibronectin, and TGF-β expression in the dermal layer. Both collagen IV and laminin were represented in the basement membrane. K19 expression was located within keratinocytes, along with Ki67 and VEGF in the epidermis. A single treatment of BLCC in the in vivo model resulted in significantly greater wound healing on day 17. Multiple treatments of BLCCs resulted in statistically greater wound healing at days 10, 13, and 17; rapid wound closure was also improved.
Discussion:
In sum, BLCC contains complex components and ECM proteins that are consistent with normal skin. Furthermore, treatment with BLCC accelerated wound healing in an in vivo model, which was primarily driven by re-epithelialization.
Trademarked Items: º Apligraf®, Organogenesis, Canton, MA
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