Topical administration of allogeneic mesenchymal stromal cells seeded in a collagen scaffold augments wound healing and increases angiogenesis in the diabetic rabbit ulcer

Abstract

There is a critical clinical need to develop therapies for nonhealing diabetic foot ulcers. Topically applied mesenchymal stromal cells (MSCs) provide a novel treatment to augment diabetic wound healing. A central pathological factor in nonhealing diabetic ulcers is an impaired blood supply. It was hypothesized that topically applied allogeneic MSCs would improve wound healing by augmenting angiogenesis. Allogeneic nondiabetic bone-marrow derived MSCs were seeded in a collagen scaffold. The cells were applied to a full-thickness cutaneous wound in the alloxan-induced diabetic rabbit ear ulcer model in a dose escalation fashion. Percentage wound closure and angiogenesis at 1 week was assessed using wound tracings and stereology, respectively. The topical application of 1,000,000 MSCs on a collagen scaffold demonstrated increased percentage wound closure when compared with lower doses. The collagen and collagen seeded with MSCs treatments result in increased angiogenesis when compared with untreated wounds. An improvement in wound healing as assessed by percentage wound closure was observed only at the highest cell dose. This cell-based therapy provides a novel therapeutic strategy for increasing wound closure and augmenting angiogenesis, which is a central pathophysiological deficit in the nonhealing diabetic foot ulcer.

FIG. 1.

Example of cross-sectional image of wound stained with hematoxylin and eosin. Scale bar, 1 mm. Six measurements (black arrows) were taken from the cartilage to the wound surface and measured using Cell B software (Olympus), and the average thickness was calculated. The average thickness was used to calculate wound volume, which is used for the calculation of stereological end points. (A high-quality color representation of this figure is available in the online issue.)

FIG. 2.

Scanning electron microscopy images of rabbit MSCs 24 h after seeding on a collagen scaffold. A: Unseeded scaffold. B: Scaffold seeded with 50,000 MSCs. C: Scaffold seeded with 100,000 MSCs. D: Scaffold seeded with 1,000,000 MSCs. The cells were adherent to the scaffold. MSCs were confluent on the scaffold at a dose of 1,000,000.

FIG. 3.

Masson’s trichrome stain of rabbit ear ulcer wounds. A: Fresh wound made on day of sacrifice. B: Untreated wound after 1 week. C: Wounds treated with collagen after 1 week. D: Wounds treated with collagen + 50,000 MSCs after 1 week. E: Wounds treated with collagen + 100,000 MSCs after 1 week. F: Wound treated with collagen + 1.000,000 MSCs after 1 week. Green stain represents collagen. Pink stain represents cytoplasm and epithelium. Purple stain represents cartilage. Original magnification ×2. Scale bar, 1 mm. There appears to be a dose-dependent increase in the epithelialization over the three doses. In the representative images of wounds, there is the subjective appearance of increased new granulation tissue in the wound and a more organized wound healing response in wounds treated with collagen-seeded MSCs in comparison with untreated wounds and wounds treated with collagen alone. (A high-quality color representation of this figure is available in the online issue.)

FIG. 4.

Percentage wound closure of cutaneous ulcers 1 week after treatment with MSCs seeded in a collagen scaffold. Analysis between groups using ANOVA and Fisher pairwise comparison. *P < 0.05. Error bars = SD. MSCs (1,000,000) seeded on a collagen scaffold result in a significantly increased percentage of wound closure as compared with control. There was no observed difference in the observed percentage wound closure between the other treatment groups, i.e., collagen + 50,000 MSCs, collagen + 100,000 MSCs, or collagen alone, when compared with untreated wounds. This result supports the hypothesis that the wound healing effect of MSC and collagen treatment occurs in a dose-dependent fashion. Increased cell doses increase the percentage wound closure and rate of wound healing. (A high-quality color representation of this figure is available in the online issue.)

FIG. 5.

Representative images of neovasculature in control wounds (A) and wounds treated with 1,000,000 MSCs seeded in a collagen scaffold (B). Tissue samples are fixed in paraffin at 5-µm depth and stained with hematoxylin and eosin. Scale bar, 200 µm. An increased blood vessel density and reduced radial diffusion distance are evident in the wounds treated with MSCs and collagen as compared with untreated wounds, as evident in B and A, respectively. (A high-quality color representation of this figure is available in the online issue.)