Abstract
There is a high incidence of failure and recurrence for chronic skin wounds following conventional therapies. To promote healing, the use of skin substitutes containing living cells as wound dressings has been proposed. The aim of this study was to produce a scaffold-free cell-based bilayered tissue-engineered skin substitute (TES) containing living fibroblasts and keratinocytes suitable for use as wound dressing, while considering production time, handling effort during the manufacturing process, and stability of the final product. The self-assembly method, which relies on the ability of mesenchymal cells to secrete and organize connective tissue sheet sustaining keratinocyte growth, was used to produce TESs. Three fibroblast-seeding densities were tested to produce tissue sheets. At day 17, keratinocytes were added onto 1 or 3 (reference method) stacked tissue sheets. Four days later, TESs were subjected either to 4, 10, or 17 days of culture at the air–liquid interface (A/L). All resulting TESs were comparable in terms of their histological aspect, protein expression profile and contractile behavior in vitro. However, signs of extracellular matrix (ECM) digestion that progressed over culture time were noted in TESs produced with only one fibroblast-derived tissue sheet. With lower fibroblast density, the ECM of TESs was almost completely digested after 10 days A/L and lost histological integrity after grafting in athymic mice. Increasing the fibroblast seeding density 5 to 10 times solved this problem. We conclude that the proposed method allows for a 25-day production of a living TES, which retains its histological characteristics in vitro for at least two weeks.
Highlights
Chronic and non-healing wounds, including mixed and venous lower-extremity ulcers, are common health conditions that reduce patients’ quality of life and account for an important economic burden for the health care system [1,2,3]
The self-assembly method to produce tissue-engineered skin substitute (TES) referred to as Self-Assembled Skin Substitute (SASS)-3 in a previous report [28] was chosen over the self-assembly approach originally presented by our team in 1999 [27] because a high quality tissue can be obtained in a shorter time, without using a material-based scaffold
This method involves the stacking of 3 fibroblast-derived tissue sheets (FSs) obtained by culturing 4 × 103 fibroblasts/cm2 with ascorbic acid to reconstruct a dermis
Summary
Chronic and non-healing wounds, including mixed and venous lower-extremity ulcers, are common health conditions that reduce patients’ quality of life and account for an important economic burden for the health care system [1,2,3]. Treatments with proven effectiveness for chronic venous leg ulcers include compressive products (bandages and stockings) and oral pentoxifylline [4]. These treatments are associated with high percentages of recurrence and healing failure [2,5]. Large or lasting ulcers are known as being more difficult to manage [6,7,8]. In those circumstances, autograft or tissue flap harvested from an uninjured donor site on the patient may be applied to definitively close the wound. The harvesting procedure is painful, and can result in scarring, which can yield non-optimal aesthetical results [9,10]
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