Stimulatory Effect of Autologous Adipose Tissue-Derived Stromal Cells in an Atelocollagen Matrix on Wound Healing in Diabetic db/db Mice

Abstract

We aimed to evaluate the effectiveness of the application of an atelocollagen matrix containing autologous adipose tissue-derived stromal cells (ASCs) on wound healing in diabetic (db/db) mice. Cultured ASCs from db/db mice and from db/+ mice secreted identical amounts of growth factors, cytokines, and type I collagen. ASCs from db/db mice proliferated at the same rate as those from db/+ mice. When DiI-labeled ASCs were applied to full-thickness round skin wounds on the backs of diabetic db/db mice, histological observation at 2 weeks showed that red fluorescent-labeled tissues were formed in the epidermis, dermis, and capillaries. Twelve db/db mice were treated with either matrix alone or matrix containing ASCs and then sacrificed at 1 or 2 weeks. A histological examination demonstrated significantly advanced granulation tissue formation, capillary formation, and epithelialization in those wounds treated with atelocollagen matrix containing ASCs, compared with wounds treated with matrix alone.

Figure 1

After 4 days of culture, ASCs from db/db (°) and db/+ (▲) mice were detached with trypsin-EDTA solution and cultured for 3 days with identical growth rates.

Figure 2

Photographs are representative of 6 wounds treated with ACM alone or with ACM containing ASCs immunostained with anti-Type I collagen.

Figure 3

Growth factors and cytokines present in cell culture supernatants were analyzed on day 4 in ASCs from db/+ (white bars) and db/db (black bars) mice. Data represent the mean ± SD of duplicate samples from 3 culture supernatants.

Figure 4

Labeling efficiency of Vybrant DiI-labeled ASCs isolated from db/db mice was about 25–30% (a). ACMs containing Dil-labeled ASCs were then prepared and applied to the wounds of db/db mice (b).

Figure 5

CD31-immunostained (a) and DiI-labeled (b) tissue on the transplant areas of db/db mice at 2 weeks. The transplanted DiI-labeled ASCs were mainly incorporated into regenerated granulation and epithelial tissues. Some microvessels were double-labeled with DiI and CD 31 (c). Each photograph is representative of 6 experiments.

Figure 6

ACM containing ASCs or ACM alone was applied to each wound by suture. The silicon membranes were removed at 1 week; the skin surrounding the wounds was removed at 2 weeks and sectioned for histological examination. Sections were made perpendicular to the wound surface. Histological examination of wound repair was performed at 2 weeks after the initial wounding. Photographs are representative of 6 wounds treated with ACM alone and 6 wounds treated with ACM containing ASCs. Tissue was stained with hematoxylin-eosin. Short arrows show blood vessels containing erythrocytes. Long arrows show the thickness of the granulation tissue.