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 h as been recognized that a permanent skin replacement must contain a functio nal complex structure consisting of epidermis integrated with dermis. Altho ugh a practical solution for the replacement of missing epidermis exists th rough the culture expansion of the autologous epidermis, a practical soluti on for permanently replacing missing dermis has not been achieved. While it is generally recognized that the insoluble matrix components-largely colla gen 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 a nd cryopreserved human dermis. Through the use;of 2 different colorimetric assays and cellulose acetate electrophoresis, we found the following: 1) th e principal dermal GAGs are those of the heparin family; 2) dermatan sulfat e is the second most predominant GAG component; 3) chondroitin-6-sulfate is found at concentrations of 2 orders of magnitude less than the heparins; a nd 4) hyaluronan and keratan sulfate were both found as only minor constitu ents. When the GAG composition of fresh skin was compared with that of cryo preserved skin, no significant differences were observed. This study also e xamined the time course of GAG leaching during the preparation of deconstru cted human dermis, which is human dermis reduced to the native insoluble ma trix components by exhaustive saline soaking. We found that GAG leaching wa s 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 resul ts of the leaching study generate important considerations for short-term s kin storage and long-term skin banking. Because GAG leaching commences imme diately, appropriate precautions must be taken to minimize the potential fu nctional compromise of cryopreserved human dermis.