A bioreactor for studying negative pressure wound therapy on skin grafts

Abstract

Negative pressure wound therapy (NPWT) has become the prevailing standard of care for treating complex soft tissue wounds and is now being considered for use in alternative applications including improving skin graft take. While it is generally agreed that negative pressure leads to improved wound healing, universal consensus on its optimal application is not supported in the literature. We describe the design and validation of a bioreactor to determine the prospective benefits of NPWT on skin grafts and engineered skin substitutes (ESS). Clinically relevant pressures were applied, and the native human skin was able to withstand greater negative pressures than the engineered substitutes. Both skin types were cultured under static, flow-only, and -75 mm Hg conditions for 3 days. While it remained intact, there was damage to the epidermal-dermal junction in the ESS after application of negative pressure. The normal skin remained viable under all culture conditions. The engineered skin underwent apoptosis in the flow-only group; however, the application of negative pressure reduced apoptosis. Vascular endothelial growth factor levels were significantly higher in the normal flow-only group, 152.0 ± 75.1 pg/mg protein, than the other culture conditions, 81.6 ± 35.5 pg/mg for the static and 103.6 ± pg/mg for the negative pressure conditions. The engineered skin had a similar trend but the differences were not significant. This bioreactor design can be used to evaluate the impacts of NPWT on the anatomy and physiology of skin to improve outcomes in wounds after grafting with normal or engineered skin.

Keywords: NPWT; bioreactor; skin graft; skin substitute.

FIGURE 1

Custom‐made NPWT bioreactor. A, An overview of one flow loop. (a) The peristaltic pump is used to re‐circulate the culture medium through the chamber of the bioreactor (b), pulling the medium from the medium reservoir (d). The wound vac (c) is applied to the top of the sample in the bioreactor. B, Each sample is placed in an individual bioreactor chamber (a) through which the culture medium (b) can flow below the sample. A perforated stainless‐steel grate (c, solid line) allows for medium exchange and a wicking material (d, hashed) is placed on top to help with medium distribution. The skin or ESS (e, solid line) is placed on top of the wicking material (c). A medical sponge (g) is placed on top of the skin or ESS as would be done clinically. A suction port (i) is placed on top of the sponge and sterile adhesive dressing (h) is applied. A vacuum line or a sterile filter is attached to the vacuum cup for the NPWT or flow‐only conditions, respectively. C, The assembled bioreactor in the incubator. There are four separate channels with individual flow loops and culture medium. The peristaltic pump and the wound vac are outside of the incubator. ESS, engineered skin substitutes; NPWT, negative pressure wound therapy

FIGURE 2

Masson's Trichrome staining. The collagen looks denser in both the flow‐only and negative pressure wound therapy samples compared with static for both skin types. While the epidermis is still intact, the NPWT does appear to cause some damage to the ESS but not to the NHS (indicated by arrow). Scale bar = 50 μm. ESS, engineered skin substitutes; NHS, normal healthy skin

FIGURE 3

DNA and VEGF contents in grafts. A, The DNA content did not significantly change with any of the culture conditions; however, it was significantly higher in the ESS than in the NHS. B, VEGF content changes showed a similar pattern between the ESS and the NHS due to treatment; however, only the NHS had significantly more VEGF after flow alone than static culture. The VEGF content was significantly higher in the ESS than in the NHS. n = 12; *P < .05 compared with NHS of the same treatment; ^# P < .05 compared with static control of same skin type. ESS, engineered skin substitutes; NHS, normal healthy skin

FIGURE 4

TUNEL staining. Red staining indicates DNA nicks, blue DNA counterstaining was used for comparison. Some red autofluorescence can be seen, especially in the epidermis. The NHS remained highly viable in all culture conditions. The ESS showed significant DNA damage in the Flow‐only culture; however, this was reduced with the application of negative pressure. The static controls had minimal DNA damage. Scale bar = 100 μm. ESS, engineered skin substitutes; NHS, normal healthy skin; TUNEL, transferase dUTP nick end labelling;