AN ACELLULAR STABILIZED COLLAGEN MATRIX (ePCM) DRESSING STRENGTHENS ANTI-MICROBIAL DEFENSES IN HUMAN KERATINOCYTES Abstract Results Shomita S. Steiner 1 , Piya Ghatak 1 , Amitava Das 1 , Sashwati Roy 1 , and Chandan K. Sen 1,2 Characterize the physical and biological characteristics of the ePCM dressing and its effect on wound healing Objective Results Conclusions and Future Directions Supported by: Harbor MedTech Inc. ) 1 Comprehensive Wound Center, Davis Heart and Lung Research Institute, 2 Center for Regenerative Medicine and Cell Based Therapies, Columbus, OH Methods A biologically stabilized, acellular, equine pericardial collagen matrix (ePCM) is clinically used as wound dressing. For the care of chronic wounds such as diabetic ulcers, ePCM is used as a single application where the dressing remains embedded in the wound until closure. Preliminary observations support that in patients, ePCM improves wound closure. The mechanism of action of ePCM in wound healing remains unclear. Initial studies from our group characterized human keratinocyte growth, proliferation and differentiation on ePCM in vitro. Our studies indicated that human keratinocytes attached to ePCM and acquired a differentiated phenotype compared to those growing on glass surfaces. This provided first cues suggesting that ePCM may serve as a scaffold for cells within the wound microenvironment. Interestingly, the antimicrobial peptide (AMP) defense system was significantly upregulated in cells adhered to ePCM compared to those on glass. AMPs kill a wide variety of microbes including bacteria. Such upregulation of AMPs in keratinocytes adhered to ePCM could provide effective defenses against bacterial colonization and wound infection. Most bacteria are able to attach and grow on biological surfaces leading to infection. In keeping with this, scanning electron microscopy imaging identified that ePCM by itself was a suitable substrate for robust bacterial growth of the primary wound pathogen Pseudomonas aeruginosa (PA01).This further suggests that effective containment of wound infection in vivo is likely to be contributed by bolstered epithelial antimicrobial defense system caused by ePCM. Our observation demonstrating that an acellular collagen matrix may modify keratinocyte antimicrobial defenses draws attention to the far-reaching influence of biological dressings above and beyond their direct physico-chemical influence on the wound milieu. In vitro studies: • Light microscopy (LM) imaging and atomic microscopic imaging (AFM) of ePCM • Keratinocyte culture on ePCM: Imaging by scanning electron microscopy (SEM) and gene expression studies by qPCR • Bacterial biofilm growth: imaging by SEM Mouse studies: • PVA sponge implantation and analysis of immune cell population recruited at d3 and d7 by flow cytometry. Imaging of ePCM performed by SEM Height Map Surface Topography Microscopy-based characterization of ePCM A B 1mm C ePCM promotes keratinocyte expression of anti-microbial peptides Figure 1. Advanced microscopy based characterization of ePCM. A. Scanning electron microscopic images show structured organization of collagen fibers in the intact dressing. B. 3D light microscopic images show uneven topography of the dressing with randomly oriented collagen bundles. C. Atomic force microscopy (AFM) images show surface topography. stiffness measurements were conducted using wet samples. There was a significant increase in the sample volume once the sample was hydrated and it acquired hydrogel-like material properties. The stiffness measurements are in the range of 1-5 mPa which is higher, than what it has been reported in the literature for collagen gels, but closer to skin tissue. The normal skin modulus distribution peaks at ~322 kPa and ranges from 25.8 kPa to 1.18 mPa. Figure 3. AMP expression is significantly upregulated in human keratinocytes grown on ePCM. Human keratinocytes were plated on glass or ePCM for 24h and RNA was extracted following cell lysis and cDNA was generated. qPCR analysis indicated that compared to control (glass), human keratinocytes grown on ePCM showed upregulation of AMPs – S100A9 and beta-defensin. Data are represented as mean±SEM. *p<0.05, **p<0.005 n=6 ePCM could act as a possible scaffold/catalyst – serving as a homing base for host cells such as epithelial, fibroblast, endothelial and immune cells, creating a gradient of pro-healing cues. Cells potentially drawing nourishment from dressing itself Although ePCM serves as a suitable substrate for bacterial attachment and growth and biofilm formation, it could also act as a decoy, blunting protease mediated invasion and bolstering host pathogen defenses such as AMPs. SEM 3D-LM AFM Min. Value 0.0518 kPa Max. Value 26.9 mPa Elastic Modulus Distribution Modulus (MPa) 0 1 2 3 4 5 6 Count 0 2 4 6 8 10 12 14 ePCM stabilized collagen matrix 0 100 200 300 400 500 600 700 800 Control ePCM 0 100 200 300 400 500 600 700 800 Control ePCM * ** S100A9/-actin rRNA (ct) -defensin/-actin rRNA (ct) Macrophages are recruited early in response to ePCM in vivo Harvest Sponge and Dressing Design - sponge sponge ePCM Design of Experiment d0 sponge implantation d3 d7 harvest wound cells for each time point SEM imaging Flow cytometric analysis A Figure 4. Enhanced early recruitment of immune cells to ePCM implanted in murine wounds. A. ePCM was sandwiched between two polyvinyl alcohol sponges and implanted into wounds created on the back of C56Bl6 mice. The sponges were harvested on days 3 and 7, fixed in glutaraldehyde buffer and processed for SEM imaging. B. Representative SEM images from d3 and d7 are shown. Increased recruitment of immune cells, particularly macrophages (red arrowheads) are seen at the early time point (d3) when usually fewer macrophages are known to be present. C. Flow cytometric analysis of F480 (macrophage specific) positive cells indicated increased macrophage recruitment to the wounds in ePCM treated wounds. Quantitation is shown in the graphs. *p<0.05, n=4. Additionally, significant remodeling of ePCM was noted at both time points. B day 3 day 7 0 10 20 30 40 control ePCM * 0 10 20 30 40 control ePCM % F480 positive cells % F480 positive cells C day 3 day 7 ePCM supports growth of human keratinocytes B Figure 3. Human keratinocyte growth on ePCM alters the integrity of the dressing. A. Human keratinocytes were plated on ePCM or glass for 48h followed by fixation and processing for SEM imaging. Shown are representative images of cells on ePCM and glass substrate. Morphology of the keratinocytes appear different on the biologically relevant substrate compared to glass. B. ePCM was decellularized with 2% SDS and imaged using SEM. Compared to a control (top panel), ePCM exposed to keratinocytes (bottom panel) was found to be significantly remodeled with loss of integrity. ePCM glass ePCM control decellularization post- keratinocyte growth A ePCM supports robust biofilm formation ePCM control biofilmwt PA01 biofilmhi PA01 Figure 2. ePCM is a suitable substrate for bacterial growth. Graded strains of Pseudomonas aeruginosa (PA01) were plated on ePCM for 48h and imaged using SEM. The biofilmwt and biofilmhi strains formed robust biofilms. Shown are representative images of a control and individual PA01 strains grown on ePCM.