The effect of nano-scale topography on keratinocyte phenotype and wound healing following burn injury

Abstract

Topographic modulation of tissue response is an important consideration in the design and manufacture of a biomaterial. In developing new tissue therapies for skin, all levels of architecture, including the nanoscale need to be considered. Here we show that keratinocyte phenotype is affected by nanoscale changes in topography with cell morphology, proliferation, and migration influenced by the pore size in anodic aluminum oxide membranes. A membrane with a pore size of 300 nm, which enhanced cell phenotype in vitro, was used as a dressing to cover a partial thickness burn injury in the pig. Wounds dressed with the membrane showed evidence of advanced healing with significantly less organizing granulation tissue and more mature epidermal layers than control wounds dressed with a standard burns dressing. The results demonstrate the importance of nanoscale topography in modulating keratinocyte phenotype and skin wound healing.

FIG. 1.

Scanning electron microscope (SEM) images of prepared anodic aluminium oxide (AAO) membranes. (A) Membrane prepared in sulfuric acid at 24 V, pore size 41 nm. (B) Membrane prepared in oxalic acid at 30 V, pore size 58 nm. (C) Membrane prepared in oxalic acid at 60 V, pore size 114 nm. (D) Membrane prepared in phosphoric acid at 130 V, pore size 300 nm.

FIG. 2.

SEM images of keratinocytes grown on: (A, B) Flat glass control surface (scale bars [SB]: 2 μm, 1 μm). (C, D) 41 nm membrane (SB: 100 μm, 1 μm). (E, F) 58 nm membrane (SB: 100 μm, 1 μm). (G, H) 114 nm membrane (SB: 200 μm, 200 nm). (I, J) 300 nm membrane (SB: 100 μm. 1 μm). Cells produced many filopodia to explore the nanoporous substrate and had a mostly rounded morphology on the 41 nm, 58 nm, and 114 nm membranes. However, on the control and 300 nm membrane, cells had a more flat and spread morphology.

FIG. 3.

Keratinocyte proliferation and migration on the prepared AAO membranes. (A) There was no significant difference between the control and 300 nm membrane, with both supporting the fastest cell proliferation. There was significantly less proliferation on all other membranes. Values are the mean absorbance of three replicates±SE (*p<0.05 as significant). (B) Cell migration was significantly faster across the 300 nm membrane compared to the control and the other membranes. There was no difference in the rate of change for migration for the 41 and 58 nm membranes compared to the control. Values are the mean of three replicates±SE (*p<0.05 as significant). Color images available online at www.liebertonline.com/tea

FIG. 4.

Gross morphology of wound healing. (A, C, E) Digital photographs of a control treated wound at time of burn injury and day 21 and day 70 post burn respectively. (B, D, F) Matched membrane treated wound at corresponding time-points. Color images available online at www.liebertonline.com/tea

FIG. 5.

(A) Average re-epithelialization (% of original wound area) for control and membrane treated wounds (data points are mean±SE). There was no significant difference between treatments (p>0.05). (B) Total aluminum content in blood plasma collected pre-injury and days 7 and 14 post burn. There was no difference in aluminum concentration after contact with the nanoporous membrane. Color images available online at www.liebertonline.com/tea

FIG. 6.

Effect of 300 nm nanoporous membrane dressing on porcine wounds. (A) There was no significant difference in the gross clinical assessments of the final scars between control and membrane treated wounds. (B) Total skin thickness. Regardless of the day euthanized (day 56 or day 70) there was no significant difference in the total skin thickness between control and membrane treated wounds. (C) Epidermal thickness. For animals euthanized day 56 post burn there was no significant difference between treatments, but for animals euthanized day 70 post burn, membrane treated wounds had a significantly thinner epidermis (*paired t-test; p=0.0380). The epidermal thickness decreased over time. (D) Organizing granulation tissue. There was significantly less organizing granulation tissue organizing granulation tissue (OGT) in membrane treated wounds compared to control wounds at an earlier time-point (day 42; *paired t-test, p=0.0283). There was no significant difference between treatments at day 56 or day 70. The amount of OGT decreased over time. For each of the histological analyses, the data presented is the paired data of control and test wounds from each individual pig (n=4 euthanized at day 56; n=4 euthanized at day 70. Each data point is averaged from the 6 sections collected and analyzed from both anteroposterior and dorsoventral axes through the wound). The broken line (········ ) represents the thickness of normal skin. Color images available online at www.liebertonline.com/tea