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. 2019 Feb;33(2):2144-2155.
doi: 10.1096/fj.201800352R. Epub 2018 Sep 27.

Stabilized collagen matrix dressing improves wound macrophage function and epithelialization

Mohamed S El Masry  1   2   3 , Scott Chaffee  2 , Piya Das Ghatak  1   2 , Shomita S Mathew-Steiner  1   2 , Amitava Das  1   2 , Natalia Higuita-Castro  2 , Sashwati Roy  1   2 , Raafat A Anani  3 , Chandan K Sen  1   2
Affiliations

Stabilized collagen matrix dressing improves wound macrophage function and epithelialization

Mohamed S El Masry et al. FASEB J. 2019 Feb.

Abstract

Decellularized matrices of biologic tissue have performed well as wound care dressings. Extracellular matrix-based dressings are subject to rapid degradation by excessive protease activity at the wound environment. Stabilized, acellular, equine pericardial collagen matrix (sPCM) wound care dressing is flexible cross-linked proteolytic enzyme degradation resistant. sPCM was structurally characterized utilizing scanning electron and atomic force microscopy. In murine excisional wounds, sPCM was effective in mounting an acute inflammatory response. Postwound inflammation resolved rapidly, as indicated by elevated levels of IL-10, arginase-1, and VEGF, and lowering of IL-1β and TNF-α. sPCM induced antimicrobial proteins S100A9 and β-defensin-1 in keratinocytes. Adherence of Pseudomonas aeruginosa and Staphylococcus aureus on sPCM pre-exposed to host immune cells in vivo was inhibited. Excisional wounds dressed with sPCM showed complete closure at d 14, while control wounds remained open. sPCM accelerated wound re-epithelialization. sPCM not only accelerated wound closure but also improved the quality of healing by increased collagen deposition and maturation. Thus, sPCM is capable of presenting scaffold functionality during the course of wound healing. In addition to inducing endogenous antimicrobial defense systems, the dressing itself has properties that minimize biofilm formation. It mounts robust inflammation, a process that rapidly resolves, making way for wound healing to advance.-El Masry, M. S., Chaffee, S., Das Ghatak, P., Mathew-Steiner, S. S., Das, A., Higuita-Castro, N., Roy, S., Anani, R. A., Sen, C. K. Stabilized collagen matrix dressing improves wound macrophage function and epithelialization.

Keywords: ECM; antimicrobial peptides; biofilm; cytokines; scaffold.

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Conflict of interest statement

The authors thank Dr. B. Deng (Center for Electron Microscopy and Analysis College of Engineering, The Ohio State University) for her assistance with light microscopy studies on sPCM. This work was supported by U.S. National Institutes of Health Grants GM077185 and GM069589 (National Institute of General Medical Sciences), NR013898 and NR015676 (National Institute of Nursing Research), and DK076566 (National Institute of Diabetes and Digestive and Kidney Diseases). This work was supported, in part, by an unrestricted gift from Harbor MedTech (Irvine, CA, USA). The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Advanced microscopy-based characterization of sPCM. A) SEM images show structured organization of collagen fibers in intact dressing with alternating light and dark bands. B) 3D light microscopic images show uneven topography of dressing with randomly oriented collagen bundles. Scale bar, 1 mm. C) AFM of sPCM immersed in deionized water. Histogram distribution of E obtained for 50 × 50-µm regions scanned under contact mode with minimum and maximum values of 0.56 kPa and 8.52 MPa, respectively. Inset provides detail of highest frequency estimated, E = 35.5 KPa. D) Representative images of height distribution and surface topography of sPCM immersed in distilled water.
Figure 2
Figure 2
Macrophage recruitment. A) Representative SEM images of collected sPCM d 3 after implantation, showing more macrophage recruitment compared to PC. Macrophage is marked with red arrowhead. B) Graphical representation of macrophage (F4/80 is macrophage marker) content in collected sPCM at d 3 and 7, as evident from flow cytometry analysis. Data are presented as means ± sd. ***P < 0.001 (n = 6), *P < 0.05 (n = 4).
Figure 3
Figure 3
sPCM supports efferocytosis and resolves inflammation at wound site. A) Representative images of wound macrophages collected from C57BL/6 mice (F4/80, green) cocultured with apoptotic cells (pHrodo, red). Inset: images showing single macrophage with engulfed apoptotic cells (scale bar, 10 µm). B) Efferocytosis index of d 3 wound macrophage. Efferocytosis index is defined as total number of engulfed apoptotic cells per macrophage present in field of view. Data are presented as means ± sd. ***P < 0.001 (n = 3).
Figure 4
Figure 4
sPCM promoted up-regulation of pro- and anti-inflammatory markers from wound inflammatory cells. Expression of proinflammatory markers at d 3 and 7 (A, C) and anti-inflammatory markers (B, D) were measured using quantitative PCR from d 3 isolated macrophages collected from sPCM postimplantation subcutaneously. Data are presented as means ± sd. *P < 0.05, **P < 0.01, ***P < 0.001 (n = 4).
Figure 5
Figure 5
sPCM promoted rapid resolution of inflammation. Expression of proinflammatory markers from collected tissues at d 7 and 14 (A) and anti-inflammatory markers at d 7 and 14 (B) were measured using ELISA from collected mice wound tissues. Data are presented as means ± sd. *P < 0.05 (n = 5).
Figure 6
Figure 6
sPCM bolstered host immune defenses. A) Representative SEM images of HaCaT cells grown on control surface (glass coverslip) and on sPCM showing distinct morphologic differences. B) HaCaT cells grown on sPCM for 24 h showed up-regulation of AMPs-S100A9 and β-defensin-1. Gene expression data are presented as fold change. Data are presented as means ± sd. *P < 0.05, **P < 0.01 (n = 5).
Figure 7
Figure 7
Biofilm formation was inhibited on sPCM pre-exposed to host immune cells. A) Representative SEM images showing adherence of P. aeruginosa (PA01) on host immune cell primed sPCM compared to PC exposed to same conditions. B) Representative IVIS images of P. aeruginosa (Xen41; base strain PA01), taken at 12 and 24 h, showing less bacterial metabolic activity (red color, more active; blue color, less active) in sPCM compared to PC. C) Representative IVIS images of and S. aureus (SAP231; base strain USA300) taken at 12 and 24 h showing less bacterial metabolic activity in sPCM compared to PC. Average intensity of bacterial activity was quantified and plotted. Data are presented as means ± sd. *P < 0.05, **P < 0.01 (n = 5).
Figure 8
Figure 8
sPCM accelerated wound closure. A) Digital images of C57BL/6 mice at d 0, 7, and 14, showing complete closure of wound at d 14 in sPCM (right) compared to Tegaderm-dressed control (left). B) Representative hematoxylin and eosin–stained images showing full epithelialization and closure of sPCM treated wound at d 14. Wound edges are indicated by red arrows in left panel. Dermis (der) and epidermis (epi) are indicted in magnified images on the right by red dotted lines. C) Plotted graph of wound area percentage over time. Data are presented as means ± sd. *P < 0.05, **P < 0.01, ***P < 0.001 (n = 4).
Figure 9
Figure 9
sPCM accelerated wound re-epithelialization. A) Representative images from formalin-fixed, paraffin-embedded wound tissue biopsy sections at d 14 showing immunostaining with K14. sPCM-treated wounds showed complete re-epithelialization with organization of epithelial layer. Wound edge is indicated by white arrows in left panel. Dermis is indicted by white dotted line in right panel. B) Significantly increased expression of K14 in sPCM compared to Tegaderm-treated wounds was observed and is represented graphically. Data are presented as means ± sd. *P < 0.05 (n = 4).
Figure 10
Figure 10
sPCM enhanced collagen maturation. A) Representative images from formalin-fixed, paraffin-embedded wound tissue biopsy sections at d 14 stained using picrosirius red staining (PRS) showed marked increase of collagen I:III ratio (yellow-orange fibers to green fibers) in sPCM-treated wounds compared to Tegaderm-treated wounds. Collagen content was quantitated and is represented graphically (AU). Data are presented as means ± sd. *P < 0.05 (n = 4). B) Herovici-stained wound tissue showed increased percentage of mature collagen content (mature collagen red fibers to immature blue fibers) in sPCM-treated wounds compared to Tegaderm-treated wounds at d 14 with marked increase of collagen I:III ratio. Wound edge is indicated by red arrows. Collagen content was quantitated and is represented graphically. Data are presented as means ± sd. ***P < 0.001 (n = 6).
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