Xenotransplantation Using Lyophilized Acellular Porcine Cornea with Cells Grown in vivo and Stimulated with Substance-P

Abstract

Corneal allograft transplantation has a high success rate, but the clinical use of corneal allografts is limited by an insufficient number of human donor corneas (Alldredge & Krachmer, 1981; Sedlakova & Filipec, 2007). Artificial substitutes may serve as an alternative to donor allograft use, but widely accepted substitutes are not currently available (Chen et al., 2001; Griffith et al., 1999; Trinkaus-Randall et al., 1988). Porcine corneas may serve as a reasonable alternative due to the ease of use and the potential for genetic engineering (Auchincloss, 1988; Insler & Lopez, 1991; Ross et al, 1993). In porcine organ xenografts, overcoming xenoantigene expression is of central importance to avoid graft rejection. Gal1α3Gals1-4GlcNAc (α-gal) on porcine tissues is one of the best known antigens involved in xenograft rejection (Amano et al., 2003; Collins et al., 1995; Good et al., 1992). We previously demonstrated that lyophilization of porcine corneas could eliminate the α-gal antigen by removing antigen-expressing cells, and lyophilized acellular porcine corneas (APCs) survived longer than fresh porcine corneas in pig-to-rat model (Lee et al., 2010). Though decellularization using lyophilization appear favorable in reducing graft rejection, but the early inflammation frequently encountered is another issue which requires resolution. The lyophilization process based on glycerol and surgical manipulation might be responsible for early inflammation, and the delayed healing caused by an acellular substrate graft is another concern. Recently, several studies have reported that corneal transparency is highly dependent on corneal cells as well as the extracellular matrix (Meek et al., 2003; Mourant et al., 2000). Thus, adequate recellularization of acellular substitute might not only extend graft survival, but enhance optical transparency. Accordingly, we hypothesized that repopulation of lyophilized APCs with cell grown in vivo before transplantation can enhance the survival of the graft by reducing the damage caused by inflammation or apoptotic environment. To address this issue, lyophilized APCs were transplanted under the limbus of rabbit corneas in advance for repopulation with cells in vivo, then optical transparency and histologic findings were compared with controls over the follow-up period. Furthermore, lamellar keratoplasties were performed to evaluate the usefulness of lyophilized APCs with cells grown in vivo in a rabbit model.