A novel immune competent murine hypertrophic scar contracture model: A tool to elucidate disease mechanism and develop new therapies

Abstract

Hypertrophic scar (HSc) contraction following burn injury causes contractures. Contractures are painful and disfiguring. Current therapies are marginally effective. To study pathogenesis and develop new therapies, a murine model is needed. We have created a validated immune-competent murine HSc model. A third-degree burn was created on dorsum of C57BL/6 mice. Three days postburn, tissue was excised and grafted with ear skin. Graft contraction was analyzed and tissue harvested on different time points. Outcomes were compared with human condition to validate the model. To confirm graft survival, green fluorescent protein (GFP) mice were used, and histologic analysis was performed to differentiate between ear and back skin. Role of panniculus carnosus in contraction was analyzed. Cellularity was assessed with 4′,6-diamidino-2-phenylindole. Collagen maturation was assessed with Picro-sirius red. Mast cells were stained with Toluidine blue. Macrophages were detected with F4/80 immune. Vascularity was assessed with CD31 immune. RNA for contractile proteins was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Elastic moduli of skin and scar tissue were analyzed using a microstrain analyzer. Grafts contracted to ∼45% of their original size by day 14 and maintained their size. Grafting of GFP mouse skin onto wild-type mice, and analysis of dermal thickness and hair follicle density, confirmed graft survival. Interestingly, hair follicles disappeared after grafting and regenerated in ear skin configuration by day 30. Radiological analysis revealed that panniculus carnosus doesn't contribute to contraction. Microscopic analyses showed that grafts show increase in cellularity. Granulation tissue formed after day 3. Collagen analysis revealed increases in collagen maturation over time. CD31 stain revealed increased vascularity. Macrophages and mast cells were increased. qRT-PCR showed up-regulation of transforming growth factor beta, alpha smooth muscle actin, and rho-associated protein kinase 2 in HSc. Tensile testing revealed that human skin and scar tissues are tougher than mouse skin and scar tissues.

Mitigation of hypertrophic scar contraction via an elastomeric biodegradable scaffold

“…To improve cellescaffold interactions, we covalently attached bovine collagen to the scaffold prior to in vivo implantation. To ensure adequate time for HSc stabilization in skin-grafted murine wounds, we followed all treatment groups out for 30 days following implantation [24]. While wound healing studies are often conducted over 14 days, this extended timeline allowed us to gain an early determination of the effects of scaffold degradation on scar contraction.…”

“…Mice were housed under protocols approved by the Institutional Animal Care and Use Committee of Duke University. Surgical methods were performed as described in our previous work; in all instances, skin graft refers to the use of a split thickness skin graft fashioned from the skin tissue of two donor mouse ears [24]. In brief, a third degree burn was created on the dorsum of the donor mouse, the burn was left for 3d, and a 14 mm diameter circle over the burn site was excised for recipient skin grafting.…”

“…Routine staining was carried out as previously described [24]. Primary antibodies used included: F4/80 (1:1500, eBioscience, San Diego, CA, USA) and anti-CD31 (1:50, Abcam).…”

“…Human and murine skin and scar tissues were tested as previously described [24]. In breif, scar and uninjured skin samples were gathered from human and murine donors.…”

“…We first studied the behavior of human dermal fibroblasts on this scaffold using the fibroblast populated collagen lattice (FPCL) assay (22), which has been used to model HSc contraction [16,22,23]. This was followed by in vivo evaluation using a murine HSc contraction model recently established in our laboratory; our model is the only immune-competent murine model that mirrors the human condition in terms of causality [24]. Together our in vitro and in vivo data suggest that slowly degrading electrospun scaffolds prevent myofibroblast activation associated with HSc, and provide the necessary longevity to prevent HSc contraction in vivo.…”