Flow cytometric analysis for apoptosis of fibroblasts.

<p>Representative dot plots of the flow cytometric analysis of the Annexin V–FITC/propidium iodide (PI) following induction of apoptosis with H₂O₂. Fibroblasts derived from SM-exposed patients and controls were assessed for spontaneous apoptosis and apoptosis following treatment with 500, 700 and 900 μM of H₂O₂. Spontaneous apoptosis was not significantly different in SM-exposed patients and controls. However, a higher dose-dependent sensitivity to H₂O₂ is observed in patients as compared with controls. Q₁: necrotic cells, Q₂: late apoptotic cells, Q₃: viable cells, and Q₄: early apoptotic cells.</p

Cell migration.

<p>The migration distance of fibroblasts isolated from SM-exposed patients (n = 5) was significantly higher than the distance migrated by controls (n = 4); *p < 0.05. Abbreviations: SM sulfur mustard.</p

Immunocytochemical characterization of fibroblasts.

<p>The cells from both SM-exposed patients and controls were negative for pancytokeratin staining (A and B; 10×) and positive for vimentin (C and D; 10×).</p

Population doubling levels (PDLs).

<p>PDLs of SM-exposed patients and controls were compared at each passage performed at different time points after the start of the primary culture. Each individual culture's PDL was presented by a red dot. PDL of the SM-exposed patients were significantly higher than controls from the day 7 through 28, but did not show any significant difference on the day 35. * p = 0.0025, ** p = 0.0001, *** p < 0.05.</p

Immunocytochemical detection of α-SMA and fibronectin expression.

<p>Both SM-exposed patients and controls expressed α-SMA (A and B, 10×) and fibronectin (C and D, 20×). The number of positive cells was determined by enumeration of 100 cells/sample. Figures E and F present the count of positive cells as mean ± SEM for patients (n = 5) and controls (n = 4). *p < 0.0001. Arrows show positive cells; Abbreviations: α-SMA alpha smooth muscle actin, SM sulfur mustard.</p

3D Protein-Based Bilayer Artificial Skin for the Guided Scarless Healing of Third-Degree Burn Wounds in Vivo

Severe burn injuries can lead to delays in healing and devastating scar formation. Attempts have been made to develop a suitable skin substitute for the scarless healing of such skin wounds. Currently, there is no effective strategy for completely scarless healing after the thermal injuries. In our recent work, we fabricated and evaluated a 3D protein-based artificial skin made from decellularized human amniotic membrane (AM) and electrospun nanofibrous silk fibroin (ESF) in vitro. We also characterized both biophysical and cell culture investigation to establish in vitro performance of the developed bilayer scaffolds. In this report, we evaluate the appropriate utility of this fabricated bilayered artificial skin in vivo with particular emphasis on healing and scar formation due to the biochemical and biomechanical complexity of the skin. For this work, AM and AM/ESF membranes alone or seeded with adipose-tissue-derived mesenchymal stem cells (AT-MSCs) are implanted on full-thickness burn wounds in mice. The healing efficacy and scar formation are evaluated at 7, 14, and 28 days post-implantation in vivo. Our data reveal that ESF accelerates the wound-healing process through the early recruitment of inflammatory cells such as macrophages into the defective site as well as the up-regulation of angiogenic factors from the AT-MSCs and the facilitation of the remodeling phase. In vivo application of the prepared AM/ESF membrane seeded with the AT-MSCs reduces significantly the post-burn scars. The in vivo data suggest that the potential applications of the AM/ESF bilayered artificial skin may be considered a clinical translational product with stem cells to guide the scarless healing of severe burn injuries