Development of the Mechanical Properties of Engineered Skin Substitutes After Grafting to Full-Thickness Wounds

Permanent wound closure is critical to the survival of patients with massive burns, traumatic injuries, or congenital conditions that require the replacement of significant portions of their skin [1,2]. It is estimated that as many as 500,000 burns are treated in the U.S. every year with approximately 20,000 of these resulting in hospitalization [3]. Advances in burn care have significantly reduced mortality rates for massive burns. From 2003–2012, pediatric burn injuries covering 50% of their total body surface area (TBSA) or greater had an average of 29.4% mortality [3]. These survival rates are achieved by providing a regimen of critical care that is costly and requires lengthy hospitalizations. Furthermore, in those instances where TBSA is high and donor sites for skin autograft are not readily available, the need for an alternative skin substitute may become definitive. Several tissue-engineering strategies have been developed to improve permanent wound closure for patients with massive burns or other injuries that require the replacement of significant portions of their skin [1,4,5]. These strategies generally involve the use of degradable polymer scaffolds, cells, or some combination of the two and have provided significant benefits to patients by providing a life-saving barrier that promotes rapid healing and stable wound closure [6,7]. However, many skin substitutes suffer from poor mechanical properties and can be easily damaged during handling or by tensile or shear forces in situ after transplantation. Even after remodeling and integrating with the host tissue, the repaired tissue often does not recover the mechanical properties of native skin and the extracellular matrix (ECM) is never restored to its uninjured condition [8–10]. Previous studies from this laboratory have reported an autologous engineered skin substitute (ESS) for excised, full-thickness burns that provides permanent wound closure [6,11,12]. For this model of ESS, keratinocytes from the epidermis and fibroblasts from the dermis are isolated from a biopsy of a patient's remaining viable skin. The cells are then expanded in vitro until sufficient numbers are obtained and then inoculated into a collagen-glycosaminoglycan scaffold (CGS). After inoculation, the ESS is incubated in vitro for one to two weeks to allow the formation of an epidermal-dermal construct that may be grafted onto the patient. Over time, the ESS graft remodels, becomes soft and pliable, and restores the essential physiologic barrier to fluid loss and infection. Despite the success of this model, much remains to be learned regarding the relationships between the degradation of the collagen scaffold and synthesis of extracellular matrix during the remodeling process and how these factors integrate across scales ranging from nanometers to centimeters to generate the mechanical and physiologic properties of the engineered skin. To evaluate these properties, the ESS containing human cells were tested mechanically at two weeks of incubation in vitro and at six weeks after grafting to a full-thickness wound in athymic mice. Additional context for changes in the remodeling and mechanical properties of the ESS was obtained by comparing ESS in vitro to an acellular CGS, or to a dermal skin substitute (DSS) consisting of CGS populated with hF and by comparing grafted ESS to murine autograft or uninjured murine skin.