J Korean Med Sci.  2011 Jun;26(6):726-733. 10.3346/jkms.2011.26.6.726.

Mesenchymal Stem Cells Improve Wound Healing In Vivo via Early Activation of Matrix Metalloproteinase-9 and Vascular Endothelial Growth Factor

• Kim CH
• Lee JH
• Won JH
• Cho MK

• KMID: 1785956
• DOI: http://doi.org/10.3346/jkms.2011.26.6.726

Abstract

We investigated the effects of mesenchymal stem cells (MSCs) on wound healing using a three-dimensional (3D) collagen gel scaffold. Three circular full-thickness skin defects were created on the back of Sprague-Dawley rats. One site was covered with a 3D collagen gel containing 2 x 10(6) MSCs (MSCs+/3D collagen+). Another site was replaced with a 3D collagen gel without MSCs and the third site was left empty. The wound size was significantly reduced in the MSCs+/3D collagen+ sites. MSCs+/3D collagen+ sites exhibited the most neovascularization. FISH showed that Y-chromosome possessing cells were found within the dermis of MSCs+/3D collagen+ sites. Gelatin zymography revealed that the most intense expression of MMP-9 was detected early in the MSCs+/3D collagen+ sites. Our results indicate that MSCs upregulate the early expression of MMP-9 which induces the early mobilization of VEGF. Thus, MSCs appear to accelerate significantly wound healing via early activation of MMP-9 and VEGF.

Keyword

Mesenchymal Stem Cells; Wound Healing

MeSH Terms

Animals
Collagen/pharmacology
Female
Immunohistochemistry
Matrix Metalloproteinase 9/*metabolism
Mesenchymal Stem Cell Transplantation
Mesenchymal Stem Cells/*cytology
Neovascularization, Physiologic
Rats
Rats, Sprague-Dawley
Skin/pathology
Vascular Endothelial Growth Factor A/*metabolism
*Wound Healing

Figure

• 

  Fig. 1 Characterization of MSCs in vitro. Differentiation of MSCs. (A) Cultured in appropriate differentiate media, MSCs differentiated osteocytes. (B) Chondrocytes in pellet culture which were positive for toluidine blue. (C) Adipocytes were demonstrated by the accumulation of lipid vesicles with oil red O staining (original magnification, × 40).

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  Fig. 2 Flow cytometer analysis of rat mesenchymal stem cells. MSCs were analyzed by flow cytometer after staining with FIFC- or PE-conjugated antibodies against indicated cell surface proteins. MSCs did not produce CD11b, CD45, CD106 antigens, but produced CD44H, CD90 antigens.

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  Fig. 3 Effects of MSCs. The wound size was significantly smaller in the MSCs+/3D collagen+ site at day 7.

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  Fig. 4 Wound size measurement. MSCs+/3D collagen+ sites were significantly decreased (0.48 ± 0.01 cm2: *P < 0.05), which were significantly smaller than the MSCs-/3D collagen+ sites (0.92 ± 0.01 cm2) or MSCs-/3D collagen- sites (0.66 ± 0.02 cm2).

• 

  Fig. 5 Immunohistochemistry. VEGF was not detected in the normal rat skin tissue. The MSCs+/3D collagen+ site was showed the most expression of VEGF than MSCs-/3D collagen+ site and MSCs-/3D collagen- site (A, Normal rat skin; B, MSCs-/3D collagen+ site; C, MSCs-/3D collagen- site; D, MSCs+/3D collagen+ site; A to D, × 400 magnification).

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  Fig. 6 Fluorescence in situ hybridization. (A) Positive control. Probe (Whole chromosome Painting Probe): Rat X/Y (FITC/Cy3) Probe. (B) Y-chromosome positive cells were identified within the dermis of MSCs+/3D collagen+ site at day 7. Y-chromosomes were probed with spectrum red (Cy3), X-chromosomes with spectrum green (FITC).

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  Fig. 7 Gelatin zymography. The level of MMP-9 (92-kDa/gelatinase B) was detect early in MSCs+/3D collagen+ site at day 3. MMP-2 (72-kDa/gelatinase A) was expressed in all experimental sites (HT1080, positive control: Human fibrosarcoma cell line).

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