Denatured collagen modulates the phenotype of normal and wounded human skin equivalents

Abstract

Epithelial-mesenchymal interactions are known to play an important role in modulating homeostasis and repair. However, it remains unclear how the composition of the extracellular matrix may regulate the ability of dermal fibroblasts to engage in such cross talk. To address this, we studied how fibroblast phenotype was linked to the behavior of normal and wounded human skin equivalents (HSE) by comparing human dermal fibroblasts (HDF) incorporated into the three-dimensional tissues to those extensively cultivated in two-dimensional (2D) monolayer culture on denatured collagen (DC) matrix, native collagen, or tissue culture plastic before incorporation into HSEs. We first established that prolonged passage and growth of HDF on DC increased their migratory potential in a 2D monolayer culture. When HDF variants were grown in HSEs, we found that extended passage on DC and incorporation of DC directly into the collagen gel enhanced proliferation of both HDF and basal keratinocytes in HSEs. By adapting HSEs to study wound reepithelialization, we found that the extended passage of HDF on DC accelerated the rate of wound healing by 38%. Thus, extensive ex vivo expansion on DC was able to modify the phenotype of skin fibroblasts by augmenting their reparative properties in skin-like HSEs.

Figure 1. Prolonged growth of HDF cells on a denatured collagen matrix decreased passage-related morphological changes

(a) HDF-EP cells, passage 8, were maintained for 12 additional passages on the following substrates: (b) tissue culture dishes (HDF-TCP), (c) film of 0.5 mgml⁻¹ denatured collagen (HDF-DC), and (d) film of 0.5 mg ml⁻¹ native collagen (HDF-NC). (e) Cell area represents the mean and SD of N = 20 independent determinants. Bar = 100 µm. ***P<0.001, *P<0.01 (t-test).

Figure 2. Prolonged growth of HDF cells on a denatured collagen matrix decreases the rate of passage-related changes in cell motility

An initial scratch wound was carried out on confluent cultures of (a) HDF-EP cells and on cultures of (b) aged HDF-DC cells or (c) HDF-NC cell. (d–f) Repopulation of the wound surface by (d) HDF-EP cells, and (e) aged HDF-DC cells or (f) HDF-NC cells after 12 hours. (g) Cells that migrated into the wound gap were calculated for each condition and differences were expressed as a percentage of wound closure of the early passaged HDF cell culture (100%). Each point represents the mean and SD of triplicate determinations in two separate experiments. Bar = 100 µm.

Figure 3. Denatured collagen enhances the proliferation of HDF cells incorporated into dermal equivalents

The proliferation rate of dermal equivalents, incorporating either HDF-EP and native collagen (NC), 10% denatured collagen (10% DC) or 25% denatured collagen (25% DC) was measured as the percentage of total cells that were BrdU-positive. Two independent experiments with a minimum of three observations for each condition were analyzed.

Figure 4. Morphologic appearance and proliferation rate of the HDF cells cultivated on collagen gels

(a–c) Collagen gels populated by (a) HDF-EP cells, (b) aged HDF-DC cells, (c) or HDF-NC cells. (d) The proliferation rate was measured by BrdU labeling and expressed as a percentage of total cells. Two independent experiments with a minimum of three observations for each condition were analyzed. Bar = 50 µm.

Figure 5. Proliferation rate of the basal keratinocytes of skin equivalents is influenced by HDF cells populating in the collagen gels

To generate skin equivalents, normal human keratinocytes (NHK) cells were grown on the top of collagen gels populated by (a) HDF-EP cells or (b) aged HDF-DC cells, and (c) HDF-NC cells. An enlargement of each picture shows the morphology of basal keratinocytes. The rate of proliferation of basal keratinocytes was measured by performing BrdU labeling after 14 days of culture. (d) Differences were expressed as a percentage of total basal keratinocytes that were BrdU-positive. Two independent experiments with a minimum of three observations for each condition were analyzed. Bar = 50 µm.

Figure 6. Schematic diagram of 3D wound model of skin equivalent cultures

(1) A wound was created by excising a central portion of human skin equivalent cultures. (2) Keratinocytes from the edge of the wound move across the wound bed to repopulate it. (3) Reepithelialization leads to reestablishment of epithelial integrity as keratinocytes reconstitute a fully stratified epithelium.

Figure 7. HDF-DC cells accelerate the rate of wound reepithelialization of wounded skin-equivalents

(a–d) The morphology of skin equivalent cultures fabricated using (a) HDF-EP cells, (b) HDF-TCP, (c) HDF-NC, or (d) HDF-DC cells 48 hours after wounding. Arrows demarcate the initial wound edges. (e) The degree of reepithelialization was determined by comparing the cultures immediately after wounding with those seen 48 hours later and expressed graphically as the percentage of wound closure. Two independent experiments with a minimum of three observations for each condition were analyzed. Bar = 100 µm.