Role of Poly-N-Acetyl-Glucosamine Nanofibers in Cutaneous Wound Healing

Treatment of cutaneous wounds with poly-N-acetyl-glucosamine nanofibers (pGlcNAc), a novel polysaccharide material derived from a marine diatom, results in increases in wound closure, antibacterial activities and innate immune responses. Treatment with nanofibers results in increased defensin, small antimicrobial peptides, expression both in vitro and in vivo. Induction of defensing expression results in bacterial clearance in a cutaneous wound model. We have also shown that Akt1 plays a central role in the regulation of these activities. We show that pGlcNAc treatment of cutaneous wounds in mice results in decreased scar sizes. Additionally, treatment of cutaneous wounds with pGlcNAc results in increased elasticity and a rescue of tensile strength. Masson Trichrome staining suggests that pGlcNAc treated wounds exhibit decreased collagen content as well as increased collagen alignment with collagen fibers oriented similarly to unwounded tissue. Utilizing a fibrin gel assay to analyze the effect of pGlcNAc nanofiber treatment on fibroblast alignment in vitro, pGlcNAc stimulation of embedded fibroblasts results in fibroblasts alignment as compared to untreated controls, by a process that is Akt1 dependent. Our data shows that in Akt1 null animals pGlcNAc treatment does not increase tensile strength or elasticity. Taken together, our findings suggest that pGlcNAc nanofibers stimulate an Akt1 dependent pathway that results in wound closure, the proper alignment of fibroblasts, decreased scarring, and increased tensile strength during cutaneous wound healing

The Role of Poly N Acetyl Glucosamine Nanofibers in Cutaneous Wound Healing

Treatment of cutaneous wounds with poly-N-acetyl-glucosamine nanofibers (pGlcNAc), a novel polysaccharide material derived from a marine diatom, results in increases in wound closure, antibacterial activities and innate immune responses. Treatment with nanofibers results in increased defensin, small antimicrobial peptides, expression both in vitro and in vivo. Induction of defensing expression results in bacterial clearance in a cutaneous wound model. We have also shown that Akt1 plays a central role in the regulation of these activities. We show that pGlcNAc treatment of cutaneous wounds in mice results in decreased scar sizes. Additionally, treatment of cutaneous wounds with pGlcNAc results in increased elasticity and a rescue of tensile strength. Masson Trichrome staining suggests that pGlcNAc treated wounds exhibit decreased collagen content as well as increased collagen alignment with collagen fibers oriented similarly to unwounded tissue. Utilizing a fibrin gel assay to analyze the effect of pGlcNAc nanofiber treatment on fibroblast alignment in vitro, pGlcNAc stimulation of embedded fibroblasts results in fibroblasts alignment as compared to untreated controls, by a process that is Akt1 dependent. Our data shows that in Akt1 null animals pGlcNAc treatment does not increase tensile strength or elasticity. Taken together, our findings suggest that pGlcNAc nanofibers stimulate an Akt1 dependent pathway that results in wound closure, the proper alignment of fibroblasts, decreased scarring, and increased tensile strength during cutaneous wound healing

Anti-Bacterial Effects of Poly-N-Acetyl-Glucosamine Nanofibers in Cutaneous Wound Healing: Requirement for Akt1

Treatment of cutaneous wounds with poly-N-acetyl-glucosamine nanofibers (sNAG) results in increased kinetics of wound closure in diabetic animal models, which is due in part to increased expression of several cytokines, growth factors, and innate immune activation. Defensins are also important for wound healing and anti-microbial activities. Therefore, we tested whether sNAG nanofibers induce defensin expression resulting in bacterial clearance.The role of sNAG in defensin expression was examined using immunofluoresence microscopy, pharmacological inhibition, and shRNA knockdown in vitro. The ability of sNAG treatment to induce defensin expression and bacterial clearance in WT and AKT1-/- mice was carried out using immunofluoresent microscopy and tissue gram staining. Neutralization, using an antibody directed against β-defensin 3, was utilized to determine if the antimicrobial properties of sNAG are dependent on the induction of defensin expression.sNAG treatment causes increased expression of both α- and β-type defensins in endothelial cells and β-type defensins in keratinocytes. Pharmacological inhibition and shRNA knockdown implicates Akt1 in sNAG-dependent defensin expression in vitro, an activity also shown in an in vivo wound healing model. Importantly, sNAG treatment results in increased kinetics of wound closure in wild type animals. sNAG treatment decreases bacterial infection of cutaneous wounds infected with Staphylococcus aureus in wild type control animals but not in similarly treated Akt1 null animals. Furthermore, sNAG treatment of S. aureus infected wounds show an increased expression of β-defensin 3 which is required for sNAG-dependent bacterial clearance. Our findings suggest that Akt1 is involved in the regulation of defensin expression and the innate immune response important for bacterial clearance. Moreover, these findings support the use of sNAG nanofibers as a novel method for enhancing wound closure while simultaneously decreasing wound infection

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

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

sNAG treatment reduces bacterial infection in an Akt1 dependent manner.

<p>(<b>A</b>) Tissue gram staining of paraffin embedded <i>S. aureus</i> infected wounds from WT and Akt1null mice <b>(n = 3)</b>. Infected wounds were either untreated or treated with sNAG membrane and wound beds were harvested on day 3 and day 5 for analysis. Dark purple staining indicates the presence of gram positive bacteria in the wound bed. Black arrows indicate examples of gram positive staining. Note the accumulation of positive staining in untreated WT that is lacking in WT animals treated with sNAG. Scale bars = 50 µm. (<b>B</b>) CFUs derived from day 5 post wounding were quantitated from <i>S. aureus</i> infected wounds using both treated and untreated WT <b>(n = 3)</b> and Akt1 mice <b>(n = 3)</b>. Wild type mice that were sNAG treated show a significant (p<.01) decrease in bacteria load in the wound beds as compared to Akt1 null animals. All experiments were repeated three independent times and the p values are shown. (<b>C</b>) CFU quantitated from infected wounds at day 3 post wounding in a similar fashion described in (B). sNAG treatment of infected wounds shows a significant decrease in CFU of both WT and Akt1 null animals on day 3, but the WT animals show an approximate 10 fold difference compared to a 2 fold difference in Akt1 animals. (<b>D</b>) Quantitation of CFUs in <i>S. aureus</i> cultures that were either untreated or treated with various amounts of sNAG nanofibers. Each experiment was performed three independent times and p values are shown. (<b>E</b>) Tissue gram staining of <i>S. aureus</i> infected wounds harvested on day 3 post wound from WT mice <b>(n = 3)</b> that were treated with or without β-defensin 3 peptide. Note the decrease in gram positive staining in infected wounds that were treated with β-defensin 3 peptide. (<b>F</b>) Quantitation of CFUs from <i>S. aureus</i> infected WT mice <b>(n = 3)</b> treated with or without β-defensin 3 peptide. Infected wounds that were treated with peptide show a significant decrease (p<.05) in CFU. Scale bars = 50 µm. Each experiment was performed three independent times and p values are shown.</p

sNAG induced defensin expression <i>in vivo</i> requires Akt1.

<p>(<b>A</b>) Paraffin embedded sections of cutaneous wounds harvested on day 3 post wounding from both WT (n = 3) and Akt1 mice. Wounds were either untreated or treated with sNAG membrane. Immunofluorescence was performed using antibodies directed against β-defensin 3 (green), Involucrin (Red), and Topro (Blue). (<b>B</b>) Paraffin embedded section from WT treated with sNAG harvested on day 3. Immunofluorescence was performed using antibodies directed against β-defensin 3 (green), Involucrin (Red), and Topro (Blue). This lower magnification (20×) is included to better illustrate the epidermal layers expressing β-defensin 3. Scale bars = 50 µm. (<b>C</b>) Quantitation of β-defensin 3 expression from paraffin embedded sections was performed using NIH ImageJ software. Experiments were repeated three independent times and p values are shown.</p

sNAG treatment results in expression and secretion of defensins <i>in vitro</i>.

<p>(<b>A</b>) RTPCR analysis of serum starved (SS) primary endothelial cells treated with sNAG (50 µg/ml) for the times indicated and assessed for expression of β-defensin 3 and α-defensin 1. (<b>B</b>) Immunofluorescent labeling of endothelial cells either serum starved (untreated) or treated with sNAG nanofibers (10 µg/ml for 5 hrs). Antibodies are directed against α-defensin 5 (Green, FITC), β-defensin 3 (Red, Texas Red). Nuclei are stained with TOPRO-3 (Blue). Lower right hand corner represents triple overlay. (<b>C</b>) Immunofluorescent labeling of keratinocytes (HaCat) that are either serum starved (untreated) or treated with sNAG nanofibers (10 µg/ml for 5 hours). Antibodies are directed against α-defensin 5 (Green, FITC), β-defensin 3 (Red). Nuclei are stained with TOPRO-3 (Blue).</p

sNAG induced defensin expression is dependent on Akt1.

<p>(<b>A</b>) Quantitative RT-PCR analyses using primers directed against α-defensin 1 from total RNA isolated from serum starved endothelial cells treated with or without sNAG for 3 hours, with or without pretreatment with PD098059 (50 µM), wortmannin (100 nm). Quantitation is relative to the S26 rprotein subunit. (<b>B</b>) Quantitation of β-defensin 3 expression from total RNA isolated from serum starved endothelial cells treated with or without sNAG for 3 hours, with or without PD98059 (50 µm), wortmannin (100 nm) and shown as relative to S26. (<b>C</b>) Western Blot analysis of phospho-Akt in serum starved endothelial cells (SS) stimulated with sNAG for the times indicated. Line indicates where lanes have been removed (<b>D</b>) Quantitative RT-PCR analyses of serum starved endothelial cells infected with a scrambled control (SCR) or Akt1 shRNA lentiviruses, treated with or without sNAG and assessed for α-defensin 4 expression. Quantitation is shown relative to S26. (<b>E</b>) Quantitation of β-defensin 3 expression from total RNA isolated from serum starved endothelial cells infected with a scrambled control (SCR) or Akt1 shRNA lentiviruses, treated with or without sNAG. Quantitation is shown relative to S26. All experiments were done in at least triplicate and repeated at least three independent times and p values are shown.</p

Antibodies against β-defensin 3 impedes antibacterial effects of sNAG treatment.

<p>(<b>A</b>) Tissue gram staining of paraffin embedded <i>S. aureus</i> infected wounds treated with sNAG from WT mice <b>(n = 3)</b> that were harvested on Day 3. sNAG treated wounds were treated with either β-defensin 3 antibody or isotype control goat IgG antibody prior to sNAG treatment. Representative images show increased accumulation gram positive staining (black arrows) in the wound beds of mice treated with an antibody directed against β-defensin 3. Scale bar = 20 µm. (<b>B</b>) Quantitation of CFUs from <i>S. aureus</i> infected WT mice treated either β-defensin 3 antibody <b>(n = 3)</b> or control IgG antibody <b>(n = 3)</b> prior to sNAG treatment. β-defensin 3 application significantly increasedd (p<.05) CFU.</p

Rapid induction of defensin expression by sNAG treatment of <i>S. aureus</i> infected wounds.

<p>(<b>A</b>) Paraffin embedded tissue sections from <i>S. aureus</i> infected wounds, harvested on day 3, were subjected to immunofluorescence using antibodies directed against β-defensin 3 (green), Involucrin (red) to mark the keratinocyte layer, and Topro (blue) from both sNAG treated WT <b>(n = 3)</b> and untreated WT mice <b>(n = 3)</b>. Non specific staining of keratin is indicated by the no primary control which was stained with secondary antibody only. Scale bar = 50 µm. (<b>B</b>) Quantitation of β-defensin 3 expression from paraffin embedded sections using NIH ImageJ software. S. aureus infected wounds that were treated with sNAG show a significant increase (p<.05) in β-defensin 3 staining. Experiments were repeated three independent times and p values are shown.</p