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. 2015 Feb;193(2):953-962.
doi: 10.1016/j.jss.2014.08.045. Epub 2014 Sep 1.

Cell recruitment by amnion chorion grafts promotes neovascularization

Zeshaan N Maan #  1 , Robert C Rennert #  1 , Thomas J Koob  2 , Michael Januszyk  1 , William W Li  3 , Geoffrey C Gurtner  1
Affiliations

Cell recruitment by amnion chorion grafts promotes neovascularization

Zeshaan N Maan et al. J Surg Res. 2015 Feb.

Abstract

Background: Nonhealing wounds are a significant health burden. Stem and progenitor cells can accelerate wound repair and regeneration. Human amniotic membrane has demonstrated efficacy in promoting wound healing, though the underlying mechanisms remain unknown. A dehydrated human amnion chorion membrane (dHACM) was tested for its ability to recruit hematopoietic progenitor cells to a surgically implanted graft in a murine model of cutaneous ischemia.

Methods: dHACM was subcutaneously implanted under elevated skin (ischemic stimulus) in either wild-type mice or mice surgically parabiosed to green fluorescent protein (GFP) + reporter mice. A control acellular dermal matrix, elevated skin without an implant, and normal unwounded skin were used as controls. Wound tissue was harvested and processed for histology and flow cytometric analysis.

Results: Implanted dHACMs recruited significantly more progenitor cells compared with controls (*P < 0.05) and displayed in vivo SDF-1 expression with incorporation of CD34 + progenitor cells within the matrix. Parabiosis modeling confirmed the circulatory origin of recruited cells, which coexpressed progenitor cell markers and were localized to foci of neovascularization within implanted matrices.

Conclusions: In summary, dHACM effectively recruits circulating progenitor cells, likely because of stromal derived factor 1 (SDF-1) expression. The recruited cells express markers of "stemness" and localize to sites of neovascularization, providing a partial mechanism for the clinical efficacy of human amniotic membrane in the treatment of chronic wounds.

Keywords: Amniotic membrane; Chronic wounds; Hematopoietic progenitor cell; Neovascularization; Progenitor cell recruitment; SDF-1; dHACM.

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Figures

Fig. 1
Fig. 1
Fluorescence-activate cell sorting (FACS) analysis of HPCs in the implants and overlying skin. (A) A Lin-/CD45+/c-Kit+/Sca-1+ gating scheme was used to identify HPCs. (B) The relative number of HPCs in specimens of healthy skin, sham surgery sites, and control ADM are reduced compared with dHACM. ** indicates P ≤ 0.05 for dHACM compared with healthy skin and sham implant. ++ indicates P ≤ 0.05 for dHACM compared with control ADM. (Color version of the figure is available online.)
Fig. 2
Fig. 2
Immunohistochemistry for CD34 expression in dHACM and control groups. (A and B) Increased CD34 positive progenitor cells were visualized in the peri-implant space and skin overlying dHACM compared with controls. (C and D) Intraimplant CD34 + progenitor cell engraftment was increased in the dHACM group compared with control. ** indicates P ≤ 0.05 for dHACM compared with healthy skin and sham implant. ++ indicates P ≤ 0.05 for dHACM compared with control ADM. Scale bar: 50 μm. (Color version of the figure is available online.)
Fig. 3
Fig. 3
Flow cytometric analysis of GFP + cells in healthy skin, sham operated skin, control ADM, and dHACM after murine parabiosis. (A) Representative flow cytometry plots from day 7 samples identifying GFP + cells. (B) Flow cytometric quantification of GFP + cells showing increased recruitment by dHACM from day 3–day 14. ** indicates P ≤ 0.05 for dHACM compared with healthy skin and sham implant. ++ indicates P ≤ 0.05 for dHACM compared with control ADM. (Color version of the figure is available online.)
Fig. 4
Fig. 4
Immunohistochemistry of SDF-1 expression. (A) SDF-1 expression colocalizes with GFP expression in the dHACM group and (B) is relatively increased compared with controls. Scale bar: 50 μm. (Color version of the figure is available online.)
Fig. 5
Fig. 5
Immunohistochemistry of CD90 expression demonstrating coexpression with GFP and 4’,6-diamidino-2-phenylindole (DAPI) nuclear stain within dHACM implants. White arrows demonstrate CD90+, GFP + recruited cells. Scale bar: 25 μm. (Color version of the figure is available online.)
Fig. 6
Fig. 6
Immunohistochemical localization of recruited GFP + cells to sites of neovascularization. (A) GFP + cells are recruited to perivascular sites of CD31 expression within the dHACM; (B) a process that is relatively increased compared with control groups. Scale bar: 50 μm. (Color version of the figure is available online.)
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