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A Novel Immune Competent Murine Hypertrophic Scar Contracture Model: A Tool to Elucidate Disease Mechanism and Develop New Therapies
Mohamed M. Ibrahim, MD1, Jennifer Bond, PhD1, Andrew Bergeron, BA1, Kyle J. Miller, BA1, Tosan Ehanire, BA1, Carlos Quiles, MD1, Mark Fisher, MD1, Elizabeth R. Lorden, BS2, Manuel A. Medina, MD1, Angelica Selim, MD3, Bruce Klitzman, PhD1, Kam Leong, PhD2, Howard Levinson, MD1.
1The Division of Plastic and Reconstructive Surgery, Department of Surgery, Duke University School of Medicine, Durham, NC, USA, 2The Department of Biomedical Engineering, Duke University, Durham, NC, USA, 3The Department of Pathology, Duke University School of Medicine, Durham, NC, USA.
Hypertrophic scar contraction (HSc) following burn injury leads to contractures. Contractures are painful and disfiguring. Current HSc therapies are marginally effective. To study disease 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 the dorsum of C57BL/6 mice. Three days post-burn, tissue was excised and wounds were grafted with ear skin. Graft contraction was analyzed by computer planimetry and tissue harvested on post-operative days 3, 7, 9, 14, 28, 70, and 168. Outcomes were compared to human condition to validate the model. To confirm graft survival, green fluorescent protein mice (GFP) were used and histologic analysis was performed to differentiate between ear and back skin. Role of panniculus carnosus (PC) in scar contraction was analyzed by tagging it with titanium clips and X-raying mice. Clip area was measured by ImageJ. Cellularity was assessed with DAPI. 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 qRT-PCR. Elastic moduli of human and murine skin and scar tissue were analyzed using a microstrain analyzer. Samples were strained at a rate of 0.1mm/s until failure. Elastic modulus was stress/strain in the linear portion of the graph.
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 vice-versa and analysis of dermal thickness and hair follicle density in grafts, confirmed graft survival. Interestingly, hair follicles disappeared after grafting and regenerated in ear skin configuration by day 30. Radiological analysis revealed the PC does not contribute to contraction. Microscopic analyses demonstrated that grafts show increase in cellularity. Granulation tissue formed after day 3. Collagen analysis revealed increases in collagen maturation, more immature collagen fibers on day 7 and more mature collagen on day 168. CD31 stain revealed an increase in vascularity compared to normal skin. Macrophages and mast cells were increased. qRT-PCR demonstrated upregulation of TGF-β, ASMA, NMMII, and ROCK2 in HSc. Tensile testing revealed that under low extension rate, human skin and scar tissues are tougher than mouse skin and scar tissues. Both scar tissues displayed increased brittle characteristics compared to uninjured skin.
Through a methodical approach, we have created a validated immune-competent murine HSc model. We unexpectedly found that murine scar contraction occurs independent of PC. We observed that murine graft hair follicles go through a period of dissolution followed by regeneration, indicating our model may also serve to study hair growth. This model will facilitate studying the HSc pathogenesis and accelerate discoveries to prevent HSc.
Characteristics of human and murine hypertrophic scars
|Parameter||Human HSc||Murine HSc|
|Initial injury||Burn||Third-degree burn||Third-degree burn|
|Management||Surgical||Excision and skin graft||Excision and skin graft|
|Gross||Scar contraction||Increased ||Increased |
|Elasticity||Elastic modulus||Increased compared to uninjured human skin||Increased compared to uninjured mouse skin|
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