IMPROVING PERFUSION OF SYNTHETIC SKIN Jordan S. Pober and Jeffrey S. Schechner

CLINICAL PROBLEM Patients with impaired angiogenesis (e.g., the elderly, diabetics) develop non-healing wounds Avascular skin substitutes (Apligraf) may promote healing but fail as long term grafts Graft failure is ischemic, not immunological

HYPOTHESIS Incorporating endothelial cells into synthetic skin will promote perfusion The effect will be most obvious in a setting of impaired angiogenesis

EXPERIMENTAL SYSTEM Synthetic skin prepared from decellularized human dermis seeded with human keratinocytes +/- human endothelial cells Orthotopic skin graft on immunodeficient (C.B-17 SCID/bg) mice +/- rapamycin Assess vascularity by histology

Vascularized Synthetic Skin (orthotopic implant)

Human Neonatal Foreskin Human Adult Cadaver Skin Human Umbilical Vein Human Umbilical Cord Blood Human Adult Peripheral Blood EPC Cultured EC +/- Bcl-2 Transduction EC-Seeded Synthetic Skin Decellularized Dermis Cultured Keratinocytes Simple Synthetic Skin Control SCID Mouse Recipient Moderately Vascularized Skin Graft (Mouse EC-Lined Vessels Only) Poorly Vascularized Skin Graft (Necrosis) Well Vascularized Skin Graft (Mouse and Human EC-Lined Vessels) Well Vascularized Skin Graft (Human EC-Lined Vessels Only) Rapamycin-Treated SCID Mouse Recipient Control SCID Mouse Recipient Rapamycin-Treated SCID Mouse Recipient

AB CD EF Figure 1 Culture of human EC derived from cord blood or adult peripheral blood (a). Colonies of differentiated cells were noted as early as 7-10 days after isolation from CB and days after isolation from AB using the conditions of isolation and culture described in the Materials and Methods(b). Colonies of differentiated EC differentiated from CB-EPC continued to proliferate, establishing a cellular monolayer capable of serial passage and expansion. AB-EC exhibited similar behavior (c,d). Immunofluorescence microscopy of monolayers of cells derived from AB-EPC revealed the presence of VE-cadherin at the cell junctions, and cytoplasmic granules of vWF (e,f), indicative of differentiated EC. Similar cells were derived from CB-EPC. Each cell type was cultured on at least five separate isolations with similar results.

AB C DEF Figure 5 Histologic analysis of microvessels in human skin equivalents harvested 21 days after transplantation. Note the paucity of vessels in grafts constructed without EC-seeding (a), compared with well vascularized grafts seeded with AB-EC (b) or CB-EC (c). In human skin equivalents seeded with CB-EC both mouse EC-lined (d) and human EC-lined (e) vessels were present. The observed microvessels were coated by cells expressing smooth muscle cell-specific alpha actin, indicative of vessel maturation. All images are representative of the results seen in AB-EC or CB-EC-seeded human skin equivalents from at least four separate experiments with each cell type (except actin staining, which was only examined in two experiments).

* ** ###### HUMAN EC-LINED VESSELS MOUSE EC-LINED VESSELS

New Questions Will the inclusion of EC accelerate and/or increase perfusion of synthetic skin? What is the best source of EC? Will Bcl-2 or other transduced genes improve EC performance in synthetic skin? Will endothelialized skin provoke rejection? CAN PERFUSION BE QUANTIFIED?