Introduction: Skin grafts are one of the most common procedures performed by surgeons. Although graft take rates are generally thought of as very high, this number is difficult to ascertain as graft take largely relies on the recipient wound bed. In cases where the wound bed is poorly vascularized, as in the case of exposed fascia, tendon, or bone, skin grafting is often delayed until the wound bed improves. This subjects patients to multiple procedures and a longer period of time with open wounds that can be painful, cumbersome, and remain a risk for infection. Additionally, wounds that take longer to heal are at increased risk for long-term complications such as scarring and contracture, ultimately affecting quality of life. Here we present a pre-clinical study in which topical nutrients applied to split-thickness skin grafts (STSGs) were examined ex vivo, a poorly vascularized porcine wound bed model was created and normal saline V.A.C. Instill® therapy tested in vivo, as well as a case report of a salvage procedure in which topical saline was administered via the V.A.C. Instill® therapy for a patient with a non-healing lower extremity wound.

Methods: For the ex vivo portion of the pre-clinical study, 12/1000ths inch STSGs were harvested from Duroc and Yorkshire swine post-euthanasia. The skin was then transferred to sterile 50mL tubes with sterile ice cold PBS supplemented with 1x Antibiotic/Antimycotic and 50μg/mL gentamicin and rinsed three times at 30 seconds per rinse. The tissue was then transferred to Dulbecco’s Modified Eagle Medium (DMEM) with low glucose (2g/L) for a soak time of 30 minutes. In a sterile laminar flow hood, 12mm biopsy punches were used to create skin graft discs of equivalent size and transferred into microplate wells with distilled water, physiological PBS, Tyrode’s Buffer, high (4.5g/L) and low (2g/L) glucose DMEM, EpiLife, and William’s E media. Plates were sealed with sterile, breathable sealing film and placed in incubators at 37°C and 5% CO2. Experiments were conducted for 7 days with solutions changed and collected every 24 hours. Collected samples were examined through biochemical analyses conducted by our institution’s laboratory support division including lactic acid production, enzymatic carbonate production, lactate dehydrogenase activity, and glucose quantification to determine consumption rates. Immunohistochemistry was also performed utilizing TUNEL, Ki67, and DAPI assays to provide information regarding the proportion of dying and/or dividing cells within each media. In vivo, twenty full-thickness 5cm-diameter wounds were created on the dorsum of two swine and a dermal substitute placed on each wound. Dermal substitutes of either 0.4mm, 0.8mm, 1.2mm, or 1.6mm thickness were then covered with 12/1000ths inch STSG and V.A.C. Instill® (Kinetic Concepts, Inc. [KCI], San Antonio, TX) placed on top. Therapy either with or without intermittent saline instillation was administered. Installation was performed three times daily with a volume of 300mL and soak time of 15 minutes. Wounds were analyzed for re-epithelialization using SilhouetteStar® (Aranz Medical, Christchurch, New Zealand) at day 7 and day 14. Lastly, a patient who had experienced numerous failures of tissue coverage of a 800cm2 wound on her left knee was treated with the V.A.C. Instill® therapy with normal saline placed over a 12/1000ths inch STSG. Instill therapy consisted of 80mL normal saline followed by a 10 minute soak time every 3.5 hours for 3 days. At day 3, the V.A.C. was removed and the wound dressed with a non-adherent petrolatum dressing.

Results: In both lactic acid and enzymatic carbonate production, DMEM with high glucose and William’s E proved superior to other tested media. Lactate dehydrogenase activity was lowest in the culture wells with William’s E. Although glucose consumption was highest in the DMEM with high glucose, this media also had the most unconsumed glucose at the conclusion of the experiment. William’s E followed as the media with the next highest amount of glucose consumption. Immunohistochemistry showed that wells with the DMEM with high glucose or William’s E had the highest percentage of dividing cells and lowest amount of dying cells. In the porcine model, dermal substitutes of 0.8mm, 1.2mm, and 1.6mm thicknesses inhibited graft take significantly (p<0.01, p=0.02, p<0.01 respectively) for all wounds treated with wound vac alone. Addition of the normal saline instill showed a significant improvement in graft take (p=0.03) over wound vac alone for the wounds treated with the 0.8mm dermal substitute. Wounds covered with 1.2mm and 1.6mm dermal substitute continued to show significantly decreased graft take (p=0.03 and p=0.02, respectively). Wounds with 0.4mm dermal substitute showed similar graft take to control for both the wound vac and wound vac + instill treatments. Clinically, following V.A.C. removal on post-op day 3, the patient was found to have almost complete take of her STSG. There was no exudate, pus, or malodor observed within the wound bed.

Conclusions: While additional studies are ongoing in order to determine the optimal nutrient supplementation to utilize for future experiments, results thus far have shown that different media can have significant effects on the survival of skin grafts ex vivo. Out of the media tested thus far, William’s E performed the best overall when taking into account dividing cells, dying cells, and glucose consumption. In the in vivo portion of the study, we were able to show that dermal substitutes ≥0.8mm create a successful model of a poorly vascularized wound bed. Vac + instill treatment overcame the impedance of a poorly vascularized wound bed only for the 0.8mm dermal substitute thickness. This thickness of dermal substitute creates an ideal poorly vascularized wound bed model from which to conduct further studies incorporating topical nutrients instilled directly onto skin grafts placed onto poorly vascularized wound beds. The case report of the novel use of V.A.C. Instill® treatment over STSG shows promise of utilizing this therapy to improve graft take in the future. A more formal examination of this treatment to conclusively determine its effect on graft take is needed.

References and Resources

1. Greenwood, J., et al., Real-time demonstration of split skin graft inosculation and integra dermal matrix neovascularization using confocal laser scanning microscopy. Eplasty, 2009. 9: p. e33.
2. Maeda, M., et al., The role of serum imbibition for skin grafts. Plast Reconstr Surg, 1999. 104(7): p. 2100-7.