Effects of cryopreservation and deconstruction on the dermal glycosaminoglycan content of human skin.

Since the concept of a fabricated skin replacement was first proposed, it has been recognized that a permanent skin replacement must contain a functional complex structure consisting of epidermis integrated with dermis. Although a practical solution for the replacement of missing epidermis exists through the culture expansion of the autologous epidermis, a practical solution for permanently replacing missing dermis has not been achieved. While it is generally recognized that the insoluble matrix components--largely collagen and elastin--are essential, the role of other matrix components such as glycosaminoglycans (GAGs) and proteoglycans remains undefined. This article describes both the qualitative and quantitative GAG composition of fresh and cryopreserved human dermis. Through the use of 2 different colorimetric assays and cellulose acetate electrophoresis, we found the following: 1) the principal dermal GAGs are those of the heparin family; 2) dermatan sulfate is the second most predominant GAG component; 3) chondroitin-6-sulfate is found at concentrations of 2 orders of magnitude less than the heparins; and 4) hyaluronan and keratan sulfate were both found as only minor constituents. When the GAG composition of fresh skin was compared with that of cryopreserved skin, no significant differences were observed. This study also examined the time course of GAG leaching during the preparation of deconstructed human dermis, which is human dermis reduced to the native insoluble matrix components by exhaustive saline soaking. We found that GAG leaching was readily detectable even within the first day. Sixty percent of total GAG leaching occurred by day 7. These investigations establish a benchmark for the reproduction of GAGs in synthetic dermal constructs. Further, the results of the leaching study generate important considerations for short-term skin storage and long-term skin banking. Because GAG leaching commences immediately, appropriate precautions must be taken to minimize the potential functional compromise of cryopreserved human dermis.

A reliable and cost-effective in vitro assay of skin viability for skin banks and burn centers.

The tetrazolium salt, MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), was used to evaluate the viability of fresh native human skin and cryopreserved human skin under a wide variety of conditions. Viability of fresh and cryopreserved skin was determined with our modification of the MTT assay, and compared with the well-described tetrazolium reductase assay. The MTT assay provides a precise and reproducible index of viability for both fresh and cryopreserved skin. In comparison, the tetrazolium reductase assay (1) is subject to higher levels of variability and, (2) underestimates the viability of fresh native skin. The precision, simplicity, and economy of the MTT assay support its utility in the routine assessment of skin viability in skin banks, burn centers, and skin biology research units.

Nuclear magnetic resonance spectroscopy of skin: predictive correlates for clinical application.

The first application of phosphorous 31 (31P) and proton (1H) nuclear magnetic resonance (NMR) spectroscopy to the analysis of the metabolic profiles of skin flaps in a rat model and of human skin grafts is presented. Resonances of adenosine triphosphate (ATP), phosphocreatine (PCr), and inorganic phosphate (Pi) were identified in 31P nuclear magnetic resonance spectra. Resonances of phosphocreatine, creatine (Cr), and lactate (Lac) were identified in 1H nuclear magnetic resonance spectra. The most significant finding was the substantial presence of phosphocreatine as the major high-energy phosphometabolite in mammalian skin, a finding which heretofore has not been widely recognized. An energy shuttle between phosphocreatine and ATP is operative in skin to buffer the fall in ATP during ischemic (anaerobic) insult. Inability to replenish exhausted phosphocreatine reserves predictively correlates with eventual flap necrosis. We have defined and analyzed temporal fluxes in the phosphocreatine-creatine and phosphocreatine plus creatine-lactate ratios by proton nuclear magnetic resonance. Both are sensitive, accurate, and unambiguous early prognostic indices of eventual flap outcome. These findings support the concept that the fate of a flap may be established as early as 3 hours after elevation and have laid the groundwork for development and application of noninvasive in vivo nuclear magnetic resonance spectroscopy to the study of skin flaps in animals and humans.