Development of the mechanical properties of engineered skin substitutes after grafting to full-thickness wounds

Abstract

Engineered skin substitutes (ESSs) have been reported to close full-thickness burn wounds but are subject to loss from mechanical shear due to their deficiencies in tensile strength and elasticity. Hypothetically, if the mechanical properties of ESS matched those of native skin, losses due to shear or fracture could be reduced. To consider modifications of the composition of ESS to improve homology with native skin, biomechanical analyses of the current composition of ESS were performed. ESSs consist of a degradable biopolymer scaffold of type I collagen and chondroitin-sulfate (CGS) that is populated sequentially with cultured human dermal fibroblasts (hF) and epidermal keratinocytes (hK). In the current study, the hydrated biopolymer scaffold (CGS), the scaffold populated with hF dermal skin substitute (DSS), or the complete ESS were evaluated mechanically for linear stiffness (N/mm), ultimate tensile load at failure (N), maximum extension at failure (mm), and energy absorbed up to the point of failure (N-mm). These biomechanical end points were also used to evaluate ESS at six weeks after grafting to full-thickness skin wounds in athymic mice and compared to murine autograft or excised murine skin. The data showed statistically significant differences (p <0.05) between ESS in vitro and after grafting for all four structural properties. Grafted ESS differed statistically from murine autograft with respect to maximum extension at failure, and from intact murine skin with respect to linear stiffness and maximum extension. These results demonstrate rapid changes in mechanical properties of ESS after grafting that are comparable to murine autograft. These values provide instruction for improvement of the biomechanical properties of ESS in vitro that may reduce clinical morbidity from graft loss.

Fig. 1

Representative test specimen and loading curve. (a) A dog-bone-shaped sample placed between grips and gauze for uniaxial failure testing. Markers were placed on the surface to facilitate strain analysis if needed (scale = mm). (b) The load-extension curve of an ESS graft depicting where the structural properties of an ESS graft were extracted from, including the ultimate load at failure (N), the maximum extension at failure (mm), the linear stiffness (N/mm) (red-line), and the energy absorbed up to failure (N-mm) (gray area under the curve).

Fig. 2

Sample histology. Representative histological sections show the progression of engineered skin substitute (ESS) remodeling from (a) an acellular collagen-glycosaminoglycan sponge (CGS), (b) dermal skin substitute (DSS) cultured in vitro two weeks, (c) ESS cultured in vitro two weeks, (d) grafted ESS six weeks post implantation on athymic mice, and compared to (e) mouse autograft, and (f) native mouse skin. Scale bar = 0.5 mm.

Fig. 3

Grafted ESS and autograft postsurgery. (a) At two weeks postsurgery the grafted ESS is still visible. (b) At six weeks postsurgery the grafted ESS margins are less distinguishable from the surrounding skin. The autograft shows better integration and a better appearance at (c) two weeks that (d) continues to improve so that at six weeks it is difficult to find the margins. Scale is in cm.

Fig. 4

Averaged load-displacement curves. The averaged sample responses (filled circles) are depicted prior to the point of failure of the first sample in the group. The corresponding standard deviation is indicated by the shaded region. The addition of fibroblasts (DSS) and coculture of fibroblasts and keratinocytes (ESS) increased the stiffness and UTL of the construct before failure occurred. Substantial improvements in mechanical properties developed in six-week grafted ESS. The grafted ESS and autograft were both stiffer and less compliant than the native mouse skin.

Fig. 5

Structural property boxplots. (a) Linear stiffness, (b) ultimate tensile load, (c) energy, (d) maximum extension for CGS (n = 11), DSS (n = 6), ESS (n = 23), grafted ESS (GESS, n = 5), autograft (AG, n = 8), and intact murine skin (n = 13). Shaded area corresponds to in vivo samples collected from athymic mice at six weeks after grafting. a indicates significant difference (p <0.05) between in vitro group with CGS, b indicates significant difference (p < 0.05) between ESS and DSS. c indicates significant difference (p < 0.05) between grafted ESS and ESS. d indicates significant difference (p < 0.05) between grafted ESS and autograft. e indicates significant difference (p < 0.05) between grafted ESS and ungrafted murine skin. f indicates significant difference (p < 0.05) between autograft and skin.