Diabetes-impaired wound healing is improved by matrix therapy with heparan sulfate glycosaminoglycan mimetic OTR4120 in rats

Abstract

Wound healing in diabetes is frequently impaired, and its treatment remains a challenge. We tested a therapeutic strategy of potentiating intrinsic tissue regeneration by restoring the wound cellular environment using a heparan sulfate glycosaminoglycan mimetic, OTR4120. The effect of OTR4120 on healing of diabetic ulcers was investigated. Experimental diabetes was induced by intraperitoneal injection of streptozotocin. Seven weeks after induction of diabetes, rats were ulcerated by clamping a pair of magnet disks on the dorsal skin for 16 h. After magnet removal, OTR4120 was administered via an intramuscular injection weekly for up to 4 weeks. To examine the effect of OTR4120 treatment on wound heal-ing, the degree of ulceration, inflammation, angiogenesis, and collagen synthesis were evaluated. We found that OTR4120 treatment significantly reduced the degree of ulceration and the time of healing. These effects were associated with reduced neutrophil infiltration and macrophage accumulation and enhanced angiogenesis. OTR4120 treatment also increased the collagen content with an increase of collagen type I biosynthesis and reduction of collagen type III biosynthesis. Moreover, restoration of the ulcer biomechanical strength was significantly enhanced after OTR4120 treatment. This study shows that matrix therapy with OTR4120 improves diabetes-impaired wound healing.

FIG. 1.

Body weight measurement during diabetes induction (i.e., days 1–49 after STZ injection), ulceration, and ulcer healing (i.e., days 49–134). Data are presented as means ± SEM. *P < 0.05 indicates significant difference in weight restoration after STZ injection between OTR4120-treated rats and control rats.

FIG. 2.

Macroscopic evaluation of the ulcer healing time in OTR4120-treated and control rats. A: Representative photographs of ulcers in OTR4120-treated rats on days 14 (a), 23 (b), and 42 (c) and in control rats on days 14 (d), 23 (e), and 42 (f). B: Percentage of complete closed ulcers. *P < 0.05 indicates significant difference between the OTR4120-treated rats and control rats. (A high-quality color representation of this figure is available in the online issue.)

FIG. 3.

Effect of OTR4120 treatment on reducing neutrophil infiltration assessed by MPO activity assay (A) and hematoxylin and eosin (H&E)–stained histology (B and C). Representative H&E staining section on day 7 after compression release of a control ulcer (B) shows intense infiltration of neutrophils (arrows) (original magnification ×1.25; scale bar = 4 mm) in the ulcer area, and an OTR4120-treated ulcer (C) shows less intense infiltration of neutrophils (arrows) (original magnification ×1.25; scale bar = 4 mm). B and C, top left insets: Normal skin tissue, located 2 mm from the ulcer margin of a control ulcer (B1) and an OTR4120-treated ulcer (C1) (original magnification ×10; scale bar = 500 µm). B and C, top right insets: Neutrophil infiltration at higher magnification of a control ulcer (B2) and an OTR4120-treated ulcer (C2) (original magnification ×40; scale bar = 100 µm). Data are presented as means ± SEM. *P < 0.05 and **P < 0.01 indicate significant differences between the OTR4120-treated groups and control groups. (A high-quality color representation of this figure is available in the online issue.)

FIG. 4.

Effect of OTR4120 treatment on inflammation reduction assessed by monocyte/macrophage marker CD68 immunohistochemistry. A and B: Representative CD68-staining sections of OTR4120-treated ulcers (A1–A3) and control ulcers (B1–B3) on days 7, 14, and 84 after compression release, respectively. Original magnification ×40. Original inset magnification ×200. C: Graphic visualization of scores of CD68 staining at indicated time points. Data are presented as means ± SEM. **P < 0.01 indicates significant difference between the OTR4120-treated groups and control groups. (A high-quality color representation of this figure is available in the online issue.)

FIG. 5.

Effect of OTR4120 treatment on angiogenesis assessed by the endothelial cell marker CD34 immunohistochemistry. A and B: Representative CD34-staining sections of OTR4120-treated wounds (A1 and A2) and control wounds (B1 and B2) on days 14 and 42 after compression release, respectively. Original magnification ×40. Original inset magnification ×200. C: Graphic visualization of scores of CD34 staining at indicated time points. Data are presented as means ± SEM. **P < 0.01 indicates significant difference between the OTR4120-treated groups and control groups. (A high-quality color representation of this figure is available in the online issue.)

FIG. 6.

Effect of OTR4120 treatment on VEGF, TGF-β1, and iNOS protein contents as assessed by Western blot analysis. Representative bands of VEGF (A), TGF-β1 (B), and iNOS (C) on days 3, 14, and 84 after compression release, respectively. Quantification of the VEGF (D), TGF-β1 (E), and iNOS bands (F). Data are presented as means ± SEM. *P < 0.05 and **P < 0.01 indicate significant differences between the OTR4120-treated rats and control rats.

FIG. 7.

Ratio of the wound-breaking strength of ulcer tissue compared with normal skin tissue on days 18, 42, and 84, respectively. Data are presented as means ± SEM. ***P < 0.001, and *P < 0.05 indicate significant differences between treated and control groups.

FIG. 8.

Biosynthesis of collagen type I and type III in ulcer tissue in control and OTR4120-treated rats. Ulcer tissue samples were ex vivo labeled with [³H]hydroxyproline and digested by pepsin. Pepsin-soluble collagen types I and III were determined by SDS-PAGE. A: The percentage of synthesized collagen type I to total synthesized collagen. B: The percentage of synthesized collagen type III to total synthesized collagen. C: The ratio of collagen type I/collagen type III. Data are presented as means ± SEM. *P < 0.05 indicates significant difference between treated and control groups.