Fig 1.
In the canonical pathway, keratinocytes that acquire DNA damage, usually through ultraviolet light (UVB), respond by upregulating tumor protein 53 (p53). p53 in turn is a transcription factor for proopiomelanocortin (POMC), thus upregulating its expression. POMC is proteolytically cleaved to its products, alpha melanocyte stimulating hormone (α-MSH) and adrenocorticotropin hormone (ACTH), which are secreted by keratinocytes and bind the melanocortin 1 (MC1R) on melanocyte membranes. This ligand-receptor binding propagates the signal transduction in melanocytes via adenylcyclase, protein kinase A (PKA), and cyclic AMP response element binding protein (CREB) to ultimately upregulate MiTF expression. When MiTF is phosphorylated through other mechanisms, it is translocated to the nucleus where it acts as a transcription factor for tyrosinase (TYR), tyrosinase related protein 1 (TYRP1), and dopachrome tautomerase (DCT), which are all enzymes required to convert tyrosine to eumelanin. Eumelanin is then packaged and transferred back to keratinocytes via protease-activated receptor 2 (PAR2) to protect against further keratinocyte nuclear damage.
Fig 2.
Heterogeneous dyschromia with hyper- and hypo-pigmentation develops after full thickness excision wounding in porcine HTS.
Examples of HTS from 3 pigs (A). SCC non-invasive skin probe was used to measure melanin in the different pigmentation phenotypes (B) (mean ± SEM, n = 8 scars). **p<0.01, ****p<0.0001.
Table 1.
Porcine demographics, injury details, and acute burn and hypertrophic scar management.
Fig 3.
H&E staining reveals structural architecture of hyper- and hypo-pigmented porcine HTS compared to normal skin.
Punch biopsies of distinct regions of hyper- (A), hypo- (B), and normally-pigmented (C) scar and skin were taken and were FFPE and H&E stained. (Scale bar = 100 μm for 10X, top and 20 μm for 40X, bottom).
Fig 4.
Fontana-Masson staining reveals melanin deposition of hyper- and hypo-pigmented porcine HTS compared to normal skin.
Punch biopsies of distinct regions of hyper- (A), hypo- (B), and normally-pigmented (C) scar and skin were taken and were FFPE and Fontana-Masson stained. (Scale bar = 100 μm for 10X, top and 20 μm for 40X, bottom).
Fig 5.
Regions of hyper- and hypo-pigmentation in pig HTS contain melanocytes in equal numbers.
Epidermal sheets from regions of hyper- or hypo-pigmentation were stained for melanocyte marker, S100β by en face staining. S100β (red), DAPI (blue). Scale Bar = 50 μm at 10X (left) or 10 μm at 40X (right) (A). Melanocytes were counted in each region of pigmentation. Differences are not significant (B, top). Melanocyte dendrites were counted in each region of pigmentation (B, bottom). Images are from Pig #1 from Table 1. (mean ± SEM, n = 10 scars, ***p<0.001).
Fig 6.
Heterogeneous dyschromia with hyper- and hypo-pigmentation develops after burn injury in patient HTS.
Examples of HTS from Subjects 1–5 from Table 2 (A). SCC non-invasive skin probe was used to measure melanin in the different pigmentation phenotypes (B) (mean ± SEM, n = 5 scars). **p<0.01, ****p<0.0001.
Table 2.
Subject demographics, injury details, and acute burn and hypertrophic scar management.
Fig 7.
H&E staining reveals structural architecture of hyper- and hypo-pigmented patient HTS compared to normal skin.
Punch biopsies of distinct regions of hyper- (A), hypo- (B), and normally-pigmented (C) scar and skin were taken and were FFPE and H&E stained. (Scale bar = 100 μm for 10X, top and 20 μm for 40X, bottom).
Fig 8.
Fontana-Masson staining reveals melanin deposition of hyper- and hypo-pigmented patient HTS compared to normal skin.
Punch biopsies of distinct regions of hyper- (A), hypo- (B), and normally-pigmented (C) scar and skin were taken and were FFPE and Fontana-Masson stained. (Scale bar = 100 μm for 10X, top and 20 μm for 40X, bottom).
Fig 9.
Regions of hyper- and hypo-pigmentation in patient HTS contain melanocytes in equal numbers.
Epidermal sheets from regions of hyper- or hypo-pigmentation were stained for melanocyte marker, S100β by en face staining. S100β (red), DAPI (blue). Scale Bar = 50 μm at 10X (left) or 10 μm at 40X (right) (A). Melanocytes were counted in each region of pigmentation (B, top). Melanocyte dendrites were counted in each region of pigmentation (B, bottom). Images are from Patient #1 from Table 2. (Scale bar = 50 μm for 10X, top and 20 μm for 40X, bottom) (mean ± SEM, n = 8 scars, ***p<0.001).
Fig 10.
Heterogeneous dyschromia with hyper- and hypo-pigmentation develops after burn injury in patient HTS.
Examples of HTS from Subjects 9–11 from Table 2 (A). SCC non-invasive skin probe was used to measure melanin in the different pigmentation phenotypes (B) (mean ± SEM, n = 3 scars). *p<0.05, **p<0.01.
Fig 11.
Staining reveals structural architecture and melanin deposition in hyper- and hypo-pigmented patient HTS compared to normal skin.
Punch biopsies of distinct regions of hyper- (I), hypo- (II), and normally-pigmented (III) scar and skin were taken and were FFPE and H&E stained (A). The same biopsies were Fontana-Masson stained (B). Images are from Subject #6 in Table 2. (Scale bar = 100 μm for 10X, and 20 μm for 40X).
Fig 12.
Melanocytes were cultured from biopsies regardless of pigmentation phenotype.
The sample biopsies were treated with dispase to isolate epidermal cells which were seeded in culture. Images were taken under bright field microscopy at a fixed light intensity after 3 days in culture. Images are from Subject #9 (A) and 12 (B) in Table 2.
Fig 13.
Melanocytes isolated from hypo-pigmented scar can be stimulated to produce melanin in media containing α-MSH.
Punch biopsies of distinct regions of hyper- and hypo-pigmented scar were taken during a pre-planned surgical excision and were treated with dispase to isolate epidermal cells which were seeded in culture in media containing α-MSH. Images were taken under bright field microscopy at a fixed light intensity for 77 days Hypopigmented melanocytes over time are shown (B).
Fig 14.
Melanocytes isolated from hypo-pigmented scar can be stimulated to produce melanin through the up-regulation of TYR, TYRP1, and DCT gene expression by exogenous treatment with NDP α-MSH.
Punch biopsies of distinct regions of hypopigmented scar were taken and treated with dispase to isolate epidermal cells which were seeded in culture in media that did not contain α-MSH. After 3 days in culture, cells were treated with 10 μM NDP α-MSH for 72 hours. Images were taken under bright field microscopy at a fixed light intensity after 72 hours. RNA was isolated and qRT-PCR was performed for TYR, TYRP1, and DCT. GAPDH was used as a housekeeping control (mean ± SEM, n = 6) (B).