Human adipose-derived stromal cells accelerate diabetic wound healing: impact of cell formulation and delivery

Abstract

Human adipose-derived stromal cells (ASCs) have been shown to possess therapeutic potential in a variety of settings, including cutaneous wound healing; however, it is unknown whether the regenerative properties of this cell type can be applied to diabetic ulcers. ASCs collected from elective surgical procedures were used to treat full-thickness dermal wounds in leptin receptor-deficient (db/db) mice. Cells were delivered either as multicellular aggregates or as cell suspensions to determine the impact of cell formulation and delivery methods on biological activity and in vivo therapeutic effect. After treatment with ASCs that were formulated as multicellular aggregates, diabetic wounds experienced a significant increase in the rate of wound closure compared to wounds treated with an equal number of ASCs delivered in suspension. Analysis of culture supernatant and gene arrays indicated that ASCs formulated as three-dimensional aggregates produce significantly more extracellular matrix proteins (e.g., tenascin C, collagen VI alpha3, and fibronectin) and secreted soluble factors (e.g., hepatocyte growth factor, matrix metalloproteinase-2, and matrix metalloproteinase-14) compared to monolayer culture. From these results, it is clear that cell culture, formulation, and delivery method have a large impact on the in vitro and in vivo biology of ASCs.

FIG. 1.

Multicellular aggregates (MAs) formed from human adipose-derived stromal cells (ASCs). Aggregates formed in hanging droplets from solutions of at least 125,000 cells/mL showed very little variability in final diameter, but lower concentrations produced aggregates with much more variability in diameter. MAs also showed more resistance to mechanical dissociation than monolayer-cultured ASCs. Color images available online at www.liebertonline.com/ten.

FIG. 2.

ELISA analysis of wound-healing protein production by ASCs when cultured as MAs compared to monolayer culture. The secretion of several proteins linked to angiogenesis, regeneration, and extracellular matrix remodeling was found to be higher when ASCs were cultured as three-dimensional MAs (black bars) rather than in normal two-dimensional monolayers (white bars; A, B, D). In contrast, secretion of inflammatory proteins such as interleukin 6 (IL-6), interleukin 8, and monocyte chemoattractant protein-1 was increased in monolayer culture relative to MA culture (C).

FIG. 3.

Mass spectrometry analysis of matrix-related protein production by adipose-derived stromal cells (ASCs) when cultured as multicellular aggregates (MAs) compared to monolayer culture. Mass spectrometry data is consistent with gene array data in that increases are seen for each protein (A: MMP14, MMP2 and Tenascin C; B: Collagen and Fibronectin), analyzed when ASCs are cultured as MAs rather than as monolayers. There is no obvious trend for production of these proteins over time from data obtained after three and six days in culture.

FIG. 4.

Comparison of diabetic wound closure over time after treatment with ASCs delivered in suspension or formulated as MAs. Wounds in all groups heal to 90% of original by day 12. During this 12-day open wound interval (gray rectangle), ASCs formed into MAs healed wounds at a significantly faster rate than either ASCs in suspension or vehicle-treated diabetic mice (p < 0.001). Diabetic wounds treated with ASC MAs also healed at a statistically similar rate to wounds inflicted on nondiabetic, vehicle-treated littermates over the same open wound interval. Both vehicle-treated diabetic wounds and cell-suspension-treated wounds healed at a statistically slower rate than nondiabetic controls within the open wound interval (p < 0.001).