pGlcNAc Nanofiber Treatment of Cutaneous Wounds Stimulate Increased Tensile Strength and Reduced Scarring via Activation of Akt1

Abstract

Treatment of cutaneous wounds with poly-N-acetyl-glucosamine containing nanofibers (pGlcNAc), a novel polysaccharide material derived from a marine diatom, results in increased wound closure, antibacterial activities and innate immune responses. We have shown that Akt1 plays a central role in the regulation of these activities. Here, we show that pGlcNAc treatment of cutaneous wounds results in a smaller scar that has increased tensile strength and elasticity. pGlcNAc treated wounds exhibit decreased collagen content, increased collagen organization and decreased myofibroblast content. A fibrin gel assay was used to assess the regulation of fibroblast alignment in vitro. In this assay, fibrin lattice is formed with two pins that provide focal points upon which the gel can exert force as the cells align from pole to pole. pGlcNAc stimulation of embedded fibroblasts results in cellular alignment as compared to untreated controls, by a process that is Akt1 dependent. We show that Akt1 is required in vivo for the pGlcNAc-induced increased tensile strength and elasticity. Taken together, our findings suggest that pGlcNAc nanofibers stimulate an Akt1 dependent pathway that results in the proper alignment of fibroblasts, decreased scarring, and increased tensile strength during cutaneous wound healing.

Fig 1. sNAG treatment results in decreased scar size and more organized collagen alignment.

(A) Quantification of scar size from excisional wounds of Wild Type male mice (n = 6) that were either treated with sNAG membrane or left untreated. Scars were measured after 21 days of healing. (B) Masson Trichrome staining of paraffin embedded sections of cutaneous wounds harvested on day 10 post wounding from both WT untreated and sNAG treated mice. The 4x magnification illustrates the entire skin section while the 20x magnification is focused on the regenerating tissue directly under the wounded area. Boxes are included to show the regions of magnification. (C) Quantitative analysis of collagen content in sNAG treated wounds compared to untreated wounds of WT mice using a hydroxyproline assay, n = 10, untreated and n = 9 treated per group (p<0.05).

Fig 2. sNAG treatment results in decreased alpha smooth muscle expression.

(A) Paraffin embedded sections of cutaneous wounds harvested on day 10 post wounding from both sNAG treated and untreated WT mice. Immunofluorescence (20X) was performed using antibodies directed against α-SMA (red), and TOPRO (Blue). White arrows indicate vasculature that is also positively stained with α-SMA antibodies. (B) Quantitation of α-SMA expression from paraffin embedded sections was performed using NIH ImageJ software. (*p<.05) n = 6 for each group.

Fig 3. sNAG treatment causes increased tensile strength and elasticity of wounded skin.

(A) Quantitation of the relative stress wounded skin could withstand from sNAG treated and untreated WT mice. Tissue was harvested 21 days post wounded and subjected to mechanical testing. Measurements are relative to control (CTRL) skin which was derived from unwounded tissue of WT animals. n = 6 for both unwounded CTRL skin and wounded untreated and n = 5 for treated wounds. (*p<.05) (B) Quantitation of skin elasticity from sNAG treated and untreated wounds harvested 21 days post wounding from WT animals. Control skin was derived from unwounded tissue of WT animals (**p<.01). (C) Van Gieson staining of paraffin embedded tissue sections derived from unwounded skin (control), sNAG treated, and untreated wounds of WT animals 10 days post wounding. Blue arrows indicate darkly stained elastin fibers.

Fig 4. sNAG treatment results in increased fibroblast alignment.

(A) Representative images of gel contractions. (B) Representative images of Hematoxylin and Eosin stained sections of paraffin embedded fibrin gels. Fibroblasts were serum starved and either left untreated or stimulated with sNAG (50μg/mL) overnight prior to embedding in fibrin gels. Black circles indicate where minutien pins are located in the fibrin gels to serve as tension points along which the gel contracts.

Fig 5. The sNAG dependent alignment of fibroblasts requires Akt1.

(A) Quantification of contraction of fibrin gels that were embedded with serum starved fibroblasts that were transduced with a SCR control lentivirus, sNAG treated fibroblasts, or fibroblasts that were transduced with a lentivirus directed against Akt1 prior to sNAG stimulation (50μg/mL). (B) Representative images from Hematoxylin and Eosin stained sections of paraffin embedded fibrin gels. Fibroblasts were serum starved and transduced with either Scrambled control or Akt1 lentivirus prior to sNAG stimulation (50μg/mL). (C) Immunofluorescence of both serum starved (untreated) or sNAG treated fibrin embedded fibroblasts using an antibody directed against phospho-Akt (red) and DAPI. (D) Quantitation of the relative stress from sNAG treated and untreated Akt1 null animals n = 3 for both treated and untreated animals. Tissue was harvested 21 days post wounded and subjected to mechanical testing. Measurements are relative to control (CTRL) skin which was derived from unwounded tissue of Akt1-/- animals. (*p<.05)

Fig 6. Model of sNAG-dependent regulation of collagen alignment and tensile strength.

An illustration showing the interaction between pGlcNAC nanofibers and integrins, upstream of Akt1, that leads to increased collagen alignment and tensile strength in cutaneous wound healing.