Stijn Dickens

Integration of Blood Outgrowth Endothelial Cells in Dermal Fibroblast Sheets Promotes Full Thickness Wound Healing

Vascularization is the cornerstone of wound healing. We introduced human blood outgrowth endothelial cells (hBOEC) in a self‐assembled human dermal fibroblast sheet (hDFS), intended as a tissue‐engineered dermal substitute with inherent vascular potential. hBOEC were functionally and molecularly different from early endothelial progenitor cells and human umbilical vein endothelial cells (HUVEC). hBOEC alone, unlike HUVEC, efficiently revascularized and re‐oxygenated the wound bed, both by active incorporation into new vessels and by trophic stimulation of host angiogenesis in a dose‐dependent manner. Furthermore, hBOEC alone, but not HUVEC, accelerated epithelial coverage and matrix organization of the wound bed. In addition, integration of hBOEC in hDFS not only further improved vascularization, epithelial coverage and matrix organization but also prevented excessive wound contraction. In vitro analyses with hBOEC, fibroblasts and keratinocytes revealed that these effects were both due to growth factor crosstalk and to short cutting hypoxia. Among multiple growth factors secreted by hBOEC, placental growth factor mediated at least in part the beneficial effects on keratinocyte migration and proliferation. Overall, this combined tissue engineering approach paves the way for clinical development of a fully autologous vascularized dermal substitute for patients with large skin defects that do not heal properly. STEM CELLS 2010;28:1165–1177

Regulable vascular endothelial growth factor165 overexpression by ex vivo expanded keratinocyte cultures promotes matrix formation, angiogenesis, and healing in porcine full-thickness wounds.

The intricate wound repair process involves the interplay of numerous cells and proteins. Using a porcine full-thickness wound (FTW) healing model, we hypothesized that the ex vivo gene transfer of vascular endothelial growth factor (VEGF)-transfected basal keratinocyte (KC) cell suspensions may generate cross-talk and induce matrix formation, angiogenesis, and accelerated healing. Moreover, to regulate overexpression of isoform 165 of VEGF and its effect on healing, we introduced a tetracycline (TC)-inducible gene switch in the expression plasmid. Autologous basal KCs were cultivated from the porcine donor and transfected using cationic liposomes. A dose-response curve was established to determine optimal activation of the gene switch by TC. In vivo, FTWs were treated with VEGF-transfected KCs and controls. Wound fluids were collected daily and examined using enzyme-linked immunosorbent assay. Biopsies were evaluated using hematoxylin and eosin and immunostaining for fibronectin, CD144, and lectin BS-1. In vitro, highest regulable VEGF165-expression was obtained with 1 microg/mL of TCs. In vivo, after induction of the gene switch by adding 1 microg/mL of TCs to the FTW, we obtained upregulated VEGF165 levels and enhanced fibronectin deposition and found more endothelial cell tubular formations and higher rates of reepithelialization than in controls. This ex vivo gene transfer model may serve as a platform for vascular induction in full-thickness tissue repair.

A Plasma-Based Biomatrix Mixed with Endothelial Progenitor Cells and Keratinocytes Promotes Matrix Formation, Angiogenesis, and Reepithelialization in Full-Thickness Wounds

In search of an autologous vascularized skin substitute, we treated full-thickness wounds (FTWs) with autologous platelet-rich plasma gel (APG) in which we embedded endothelial progenitor cells (EPCs) and basal cell keratinocytes (KCs). We cultivated autologous KCs in low-serum conditions and expanded autologous EPCs from venous blood. FTWs (n = 55) were created on the backs of four pigs, covered with wound chambers, and randomly assigned to the following treatments: (1) APG, (2) APG + KCs, (3) APG + EPCs, (4) APG + KCs + EPCs, and (5) saline. All wounds were biopsied to measure neovascularization (lectin Bandeiraea Simplicifolia-1 (BS-1), alpha smooth muscle actin [alphaSMA], and membrane type 1 matrix metalloproteinase (MT1-MMP)), matrix deposition (fibronectin, collagen type I/III, and alphavbeta3), and reepithelialization. Wound fluids were analyzed for protein expression. All APG-treated wounds showed more vascular structures (p < 0.001), and the addition of EPCs further improved neovascularization, as confirmed by higher lectin, alphaSMA, and MT1-MMP. APG groups had higher collagen I/III (p < 0.05), alphavbeta3, and fibronectin content (p < 0.001), and they exhibited higher concentrations of platelet-derived growth factor subunit bb, basic fibroblast growth factor, hepatocyte growth factor, insulin growth factor-1, transforming growth factor-beta1 and -beta3, matrix metalloproteinase-1 and -z9, and tissue-inhibiting matrix metalloproteinase-1 and -2. Applying APG + KCs resulted in the highest reepithelialization rates (p < 0.001). No differences were found for wound contraction by planimetry. In this porcine FTW model, APG acts as a supportive biomatrix that, along with the embedded cells, improves extracellular matrix organization, promotes angiogenesis, and accelerates reepithelialization.