No abstract

Large body surface area burns pose significant therapeutic challenges. Clinically, the extent and depth of burn injury may mandate the use of allograft for temporary wound coverage while autografts are serially harvested from the same donor areas. The paucity of donor sites in patients with burns involving large surface areas highlights the need for better skin substitutes that can achieve early and complete coverage and retain normal skin durability with minimal donor requirements. We have isolated autologous stem cells from the adipose layer of surgically debrided burned skin (dsASCs), using a point-of-care stem cell isolation device. These cells, in a collagen—polyethylene glycol fibrin-based bilayer hydrogel, differentiate into an epithelial layer, a vascularized dermal layer, and a hypodermal layer. All-trans-retinoic acid and fenofibrate were used to differentiate dsASCs into epithelial-like cells. Immunocytochemical analysis showed a matrix- and time-dependent change in the expression of stromal, vascular, and epithelial cell markers. These results indicate that stem cells isolated from debrided skin can be used as a single autologous cell source to develop a vascularized skin construct without culture expansion or addition of exogenous growth factors. This technique may provide an alternative approach for cutaneous coverage after extensive burn injuries.

Infection control is necessary for improved burn wound regeneration. In this study contact burn wounds were induced on the dorsum of the rats and were infected with Pseudomonas aeruginosa (10 7 cfu/ml of saline) and left overnight (12–14 hours) to establish the infection. After 12 hours, the wounds were treated with PEGylated fibrin hydrogel containing 50 mgs of silver sulfadiazine (SSD) loaded chitosan microsphere (SSD-CSM-FPEG). On day 9, SSD-CSM-FPEG treated burn wounds further received adipose derived stem cell (5×10 4 ASCs cells/ml) embedded in PEGylated fibrin hydrogel. Wounds were assessed for the healing outcomes such as neovascularization, granulation tissue formation, wound closure and collagen maturation. Analysis of bacterial load in the burn wound biopsies, demonstrated that SSD–CSM-FPEG significantly reduced bacterial infection, while overt infection was still observed in the untreated groups on day 14. Sequential treatment of infected wounds with SSD–CSM-FPEG followed by ASC-FPEGs (SSD-CSM-ASC-FPEG) significantly reduced bacterial colonization (9 log reduction) and pro-inflammatory cytokine (TNF-α) expression. A significant increase in neovascularization markers; NG2 and vWF was also observed. Histological analysis indicated the wounds treated with SSD-CSM-ASC-FPEG increased amount of dermal collagen matrix deposition, a thicker granulation tissue on day 21 and more mature collagen on day 28. This work demonstrates that the sequential treatment of infected burn wounds with SSD-CSM-FPEG followed by ASC-FPEG reduces bacterial infection as well as promotes neo-vascularization with improved matrix remodeling.

The bioengineering of autologous vascular networks is of great importance in wound healing. Adipose-derived stem cells (ASCs) are of interest due to their ability to differentiate toward various cell types, including vascular. We hypothesized that adult human ASCs embedded in a three-dimensional PEG-fibrin (FPEG) gel have the ability to modulate vascularization of a healing wound. Initial in vitro characterization of ASCs isolated from discarded burn skin samples (dsASCs) and embedded in FPEG gels indicated they could express such pericyte/smooth muscle cell markers as α-smooth muscle actin, platelet-derived growth factor receptor-β, NG2 proteoglycan, and angiopoietin-1, suggesting that these cells could potentially be involved in a supportive cell role (i.e., pericyte/mural cell) for blood vessels. Using a rat skin excision model, wounds treated with dsASCs-FPEG gels showed earlier collagen deposition and wound remodeling compared to vehicle FPEG treated wounds. Furthermore, the dsASCs-seeded gels increased the number of vessels in the wound per square millimeter by day 16 (~66.7 vs. ~36.9/mm(2)) in these same studies. dsASCs may support this increase in vascularization through their trophic contribution of vascular endothelial growth factor, as determined by in vitro analysis of mRNA and the protein levels. Immunohistochemistry showed that dsASCs were localized to the surrounding regions of large blood-perfused vessels. Human dsASCs may play a supportive role in the formation of vascular structures in the healing wound through direct mechanisms as well as indirect trophic effects. The merging of autologous grafts or bioengineered composites with the host's vasculature is critical, and the use of autologous dsASCs in these procedures may prove to be therapeutic.

The objective of this study was to demonstrate that stem cells isolated from discarded skin obtained after debridement can be used with collagen and fibrin-based scaffolds to develop a tissue-engineered vascularized dermal equivalent. Discarded tissue samples were collected from severely burned patients undergoing wound debridement. Stem cells were isolated from the adipose tissue layer and their growth and immunophenotype characterized. To develop a skin equivalent, debrided skin adipose stem cells (dsASCs) were added to a collagen-polyethylene glycol (PEG) fibrin-based bilayer hydrogel and analyzed in vitro. The effect of the bilayered hydrogels on wound healing was demonstrated using an excision wound model in athymic rats. The dsASCs isolated from all samples were CD90, CD105, and stromal cell surface protein-1 positive, similar to adipose stem cells isolated from normal human lipoaspirates. Within the bilayer hydrogels, dsASCs proliferated and differentiated, maintained a spindle-shaped morphology in collagen, and developed a tubular microvascular network in the PEGylated fibrin. Rat excision wounds treated with bilayer hydrogels showed less wound contraction and exhibited better dermal matrix deposition and epithelial margin progression than controls. Stem cells can be isolated from the adipose layer of burned skin obtained during debridement. When dsASCs are incorporated within collagen-PEGylated fibrin bilayer hydrogels, they develop stromal and vascular phenotypes through matrix-directed differentiation without use of growth factors. Preliminary in vivo studies indicate that dsASC-bilayer hydrogels contribute significantly to wound healing and provide support for their use as a vascularized dermal substitute for skin regeneration to treat large surface area burns.