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. 2013 Feb;19(3-4):329-39.
doi: 10.1089/ten.TEA.2011.0738. Epub 2012 Sep 24.

Full-thickness skin wound healing using human placenta-derived extracellular matrix containing bioactive molecules

Ji Suk Choi  1 , Jae Dong Kim, Hyun Soo Yoon, Yong Woo Cho
Affiliations

Full-thickness skin wound healing using human placenta-derived extracellular matrix containing bioactive molecules

Ji Suk Choi et al. Tissue Eng Part A. 2013 Feb.

Abstract

The human placenta, a complex organ, which facilitates exchange between the fetus and the mother, contains abundant extracellular matrix (ECM) components and well-preserved endogenous growth factors. In this study, we designed a new dermal substitute from human placentas for full-thickness wound healing. Highly porous, decellularized ECM sheets were fabricated from human placentas via homogenization, centrifugation, chemical and enzymatic treatments, molding, and freeze-drying. The physical structure and biological composition of human placenta-derived ECM sheets dramatically supported the regeneration of full-thickness wound in vivo. At the early stage, the ECM sheet efficiently absorbed wound exudates and tightly attached to the wound surface. Four weeks after implantation, the wound was completely closed, epidermic cells were well arranged and the bilayer structure of the epidermis and dermis was restored. Moreover, hair follicles and microvessels were newly formed in the ECM sheet-implanted wounds. Overall, the ECM sheet produced a dermal substitute with similar cellular organization to that of normal skin. These results suggest that human placenta-derived ECM sheets provide a microenvironment favorable to the growth and differentiation of cells, and positive modulate the healing of full-thickness wounds.

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Figures

FIG. 1.
FIG. 1.
Preparation of decellularized human placenta-derived ECM. (A, B) ECM was extracted from human placenta and decellularized through physical, chemical, and enzymatic treatment. (C, D) Cell cytoplasm (pink) and nuclei (dark purple spot) in decellularized ECM were stained with H&E. (E, F) Nucleic acids were also stained using DAPI that binds strongly to A-T-rich regions in DNA. Scale bars represent 200 μm. (G) DNA contents in human placenta and ECM. Samples were normalized to the ECM dry weight. Data are shown as mean±standard deviation (n=5) with significance at *p<0.05. ECM, extracellular matrix; H&E, hematoxylin and eosin; DAPI, 4,6-diamidino-2-phenylindole. Color images available online at www.liebertpub.com/tea
FIG. 2.
FIG. 2.
(A) Biochemical analysis of ECM components, including acid/pepsin-soluble collagen, sulfated GAG and soluble elastin. All samples were normalized to ECM or placenta dry weight. Data are shown as mean±standard deviation (n=5) with significance at *, **, ***p<0.05 between the human placenta and ECM. (B) ECM compositions before and after decellularization were identified using Gomori's trichrome staining (collagen, green) and orcinol-new fuchsin staining (elastin, light purple). Scale bars represent 200 μm. GAG, glycosaminoglycans. Color images available online at www.liebertpub.com/tea
FIG. 3.
FIG. 3.
Profiling of bioactive molecules and quantification of wound healing-related growth factors in human placenta and placenta-derived ECM. (A) Cytokines and (B) growth factors were arrayed on glass chip arrays containing 80 different cytokine antibodies and detected by a laser scanner using the Cy3 channel. Bioactive molecules were normalized to positive control. (C) Growth factors that are known to stimulate wound repair were quantified via ELISA. Data are shown as mean±standard deviation (n=5) with significance at *p<0.05 between the human placenta and ECM. BDNF, brain-derived neurotrophic factor; BLC, B-lymphocyte chemoattractant; Ckβ8, chemokine beta 8; EGF, epidermal growth factor; FGF-2, fibroblast growth factor-2; GCP, granulocyte chemotactic protein; GDNF, glial-derived neurotrophic factor; SDF, stromal cell-derived factor; GRO, growth-regulated protein; HGF, hepatocyte growth factor; IGF-1, insulin-like growth factor-1; IGFBP, IGF binding proteins; LIF, leukemia inhibitory factor; IL, interleukin; Lor, Loricrin; MIF, macrophage migration inhibitory factor; MIP, macrophage inflammatory protein; NAP, neutrophil-activating peptide; NT, neurotrophin; PARC, pulmonary and activation-regulated chemokine; PDGF, platelet-derived growth factor; PIGF, placenta growth factor; RANTES, regulated upon activation, normal T-cell expressed, and secreted; SCF, stem cell factor; TIMP, tissue inhibitor of metalloproteinase; TGF-β, transforming growth factor-β; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor.
FIG. 4.
FIG. 4.
Physicomechanical properties of decellularized ECM sheets. (A) The extracted ECM was fabricated into porous sheets. (B) The ECM sheets (13-mm diameter and 1-mm thickness) possessed a highly porous microstructure with a high degree of interconnectivity. Scale bar represents 50 μm. (C) Pore size distribution measured by a porosimeter. (D) Representative stress–strain curves of ECM sheets under tensile loading. Each line curve means each sample (n=5).
FIG. 5.
FIG. 5.
The progression in healing of a full-thickness cutaneous wound treated with a human placenta-derived ECM sheet and without. (A) Wounds were photographed at days 0, 1, 7, 14, and 28. (B) The wound area was expressed as a percentage of the initial wound area at day 0. Data are shown as mean±standard deviation (n=6) with significance at *p<0.05. Color images available online at www.liebertpub.com/tea
FIG. 6.
FIG. 6.
Histological micrographs of wound sections implanted with an ECM sheet and without (n=6) at day 7, 14, and 28 after dermal excision by Gomori's trichrome staining. Wound edges are indicated by red arrows. Insets are the magnified images of the rectangles indicated, and represent the regeneration of the outer layer of the skin. Muscle, keratin, and cytoplasm: red, collagen: green, nuclei: black. Scale bars represent 2 mm (black) and 200 μm (black dotted line), respectively. Color images available online at www.liebertpub.com/tea
FIG. 7.
FIG. 7.
Immunofluorescence staining of the wound sections implanted with an ECM sheet and without. The reconstruction of epithelia was assessed using Ker 15 (Keratin 15, red), Lam 5 (Laminin 5, orange), and Lor (Loricrin, green) on day 14 and 28. Sections were counterstained with DAPI, which stains nuclei blue. Scale bars represent 200 μm. Color images available online at www.liebertpub.com/tea
FIG. 8.
FIG. 8.
The in vivo degradation of an ECM sheet was assessed using antibodies specific for hCol (human collagen type I, red) and rCol (rat collagen type I, green). (A) Day 7, (B) Day 14, (C) Day 28. Sections were counterstained with DAPI, which stains nuclei blue. Scale bars represent 200 μm. Color images available online at www.liebertpub.com/tea
FIG. 9.
FIG. 9.
Distribution and density of newly formed blood vessels were assessed using a CD31 (green) for rat endothelial cells on day 7 and 14. Arrows denote areas of magnified images and show the formation of newly blood vessels. Sections were counterstained with DAPI, which stains nuclei blue. Scale bars represent 1 mm (red) and 200 μm (white). Color images available online at www.liebertpub.com/tea
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