Reconstructed human keloid models show heterogeneity within keloid scars

Abstract

Keloid scars are often described as having an actively growing peripheral margin with a regressing centre. The aim of this study was to examine the possible heterogeneity within keloids and the involvement of different regions within and around keloid scars in the pathogenesis, using an in vitro keloid scar model. In vitro skin models were constructed from keratinocytes and fibroblasts from normal skin and different regions within and around keloid scars: periphery, centre, and (adjacent) surrounding-normal-skin regions. Additionally, fibroblasts were isolated from the superficial-central and deep-central regions of the keloid and combined with central keratinocytes. All keloid regions showed increased contraction compared to normal skin models, particularly in central regions. Myofibroblasts were present in all keloid regions but were more abundant in models containing central-deep keloid fibroblasts. Secretion of anti-fibrotic HGF and extracellular matrix collagen IV gene expression was reduced in the central deep keloid compared to normal skin. No significant differences between peripheral and central regions within keloids were observed for inflammatory cytokine CCL20, CCL27, CXCL8, IL-6 and IL-18 secretion. Parameters for surrounding-normal-skin showed similarities to both non-lesional normal skin and keloids. In conclusion, a simple but elegant method of culturing keloid-derived keratinocytes and fibroblasts in an organotypic 3D scar model was developed, for the dual purpose of studying the underlying pathology and ultimately testing new therapeutics. In this study, these tissue engineered scar models show that the central keloid region shows a more aggressive keloid scar phenotype than the periphery and that the surrounding-normal-skin also shares certain abnormalities characteristic for keloids.

Keywords: Cytokine; Extracellular matrix; In vitro; Keloid; Keloid center; Keloid periphery.

Fig. 1

Construction of skin models. a, b Keloids are dissected into: peripheral region (P), the central superficial (Cs) and central deep regions (Cd); the normal skin directly adjacent to the keloid periphery (sN: surrounding normal skin). Keratinocytes and fibroblasts are isolated from each region (circles indicate where biopsies were taken for cell isolation) and combined to form a peripheral keloid (P-Kscar) model, central superficial keloid (Cs-Kscar) model, central deep keloid (Cd-Kscar) model, surrounding normal skin (sNskin) model (c). Skin models are constructed by first seeding fibroblasts into Matriderm® (d). After 3 weeks, keratinocytes are added and the skin models are then cultured air-exposed for an additional 2 weeks

Fig. 2

Increased contraction, epidermal and dermal thickness in central keloid regions. Absolute surface area of the skin models was measured in duplicate in n = 8 normal skin (Nskin), n = 8 peripheral keloid (P-Kscar), n = 7 central superficial keloid (Cs-Kscar), n = 7 central deep keloid (Cd-Kscar), n = 5 surrounding normal skin (sNskin). a shows macroscopic views of SE at the start and end of culturing; b shows the number of viable epidermal cell layers in the SE; c shows the dermal thickness measured in µm; d shows contraction measured as a reduction in end surface area after 5 weeks of culturing. *p < 0.05, if 0.05 > p < 0.08 then the exact p value is listed in the graph

Fig. 3

Increased α-SMA staining in central keloid regions. Upper panel shows representative pictures of HE, α-SMA and vimentin stainings performed on one of the duplicate skin models in n = 8 normal skin (Nskin), n = 8 peripheral keloid (P-Kscar), n = 7 central superficial keloid (Cs-Kscar), n = 7 central deep keloid (Cd-Kscar), n = 5 surrounding normal skin (sNskin). Magnification 200×, scale bar 100 µm. Lower panel shows the results of immunohistochemical stainings of epidermal markers (Ki67, keratin 10), dermal cellular markers (vimentin, α-SMA) in the skin models. Ki67 is expressed as mean ± SEM; SPB: suprabasal expression; SB: stratum basale; PAN: panepidermal (both SB and SPB); +/−: minimal expression; +: normal expression; ++: increased expression; +++: strongly increased expression; –: absent

Fig. 4

Differential dermal expression of ECM related genes. Dermal expression of COL4A2, MMP3 and HAS1 was determined in one of the duplicate skin models in n = 4 normal skin (Nskin), n = 5 peripheral keloid (P-Kscar), n = 4 central superficial keloid (Cs-Kscar), n = 4 central deep keloid (Cd-Kscar), n = 4 surrounding normal skin (sNskin). The scatter plots show the individual data points with the median, with *p < 0.05

Fig. 5

secretion of wound healing mediators. Wound healing mediator secretion of HGF, CCL27, CXCL8, CCL20, IL-6, IL-18, CCL5, CXCL1, VEGF, and CCL2 was determined in duplicate in n = 8 normal skin (Nskin), n = 8 peripheral keloid (P-Kscar), n = 7 central superficial keloid (Cs-Kscar), n = 7 central deep keloid (Cd-Kscar), n = 5 surrounding normal skin (sNskin). Graphs show mean ± SEM, with *p < 0.05