Topical application of laminin-332 to diabetic mouse wounds

Abstract

Background: Keratinocyte migration is essential for wound healing and diabetic wound keratinocytes migrate poorly. Keratinocyte migration and anchorage appears to be mediated by laminin-332 (LM-332). Impaired diabetic wound healing may be due to defective LM-332 mediated keratinocyte migration.

Objective: To evaluate LM-332 expression in diabetic (db/db) and control (db/-) mice and to test LM-332 wound healing effects when applied to mouse wounds.

Methods: LM-332 expression in mouse wounds was evaluated using immunohistochemistry. LM-332 wound healing effects were evaluated by directly applying soluble LM-332, a LM-332 biomaterial, or a control to mouse wounds. Percent wound closure and histology score, based on healing extent, were measured.

Results: Precursor LM-332 expression was markedly reduced in db/db when compared to db/- mice. In vitro, soluble LM-332 and LM-332 biomaterial demonstrated significant keratinocyte adhesion. In vivo, soluble LM-332 treated wounds had the highest histology score, but significant differences were not found between wound treatments (p>0.05). No differences in percentage wound closure between treatment and control wounds were found (p>0.05).

Conclusion: The db/db wounds express less precursor LM-332 when compared to db/-. However, LM-332 application did not improve db/db wound healing. LM-332 purified from keratinocytes was primarily physiologically cleaved LM-332 and may not regulate keratinocyte migration. Application of precursor LM-332 rather than cleaved LM-332 may be necessary to improve wound healing, but this isoform is not currently available in quantities sufficient for testing.

Figure 1

LM-332 trimer with α3, β3 and γ2 subunits. The α chain of precursor LM-332 is cleaved at the G4/5 globular domains of the carboxy terminus to form cleaved LM-332. C2-5 antibody binds to the amino terminus of the α chain.

Figure 2

Detection of LM-332 using immunofluorescence of whole mount wounds. Wounds treated with soluble biotinylated LM-332 and saline control at 1 and 24 hours. Arrowheads indicate wound margin. In soluble biotinylated LM-332 treated wounds, bright orange staining (fluorescent emission of streptavidin-Cy5) can be seen in the wound bed at both 1 and 24 hours after application, while immunofluorescence is absent in control wounds.

Figure 3

Wounds treatments were soluble or biomaterials. Soluble wound treatments included LM-332 and phosphate buffered saline (PBS) as a control. Treatment solutions (0.1 ml) were infused beneath the Tegaderm™ using a syringe and 30-gauge needle. LM-332 biomaterial or C2-5 biomaterial as a control, was also delivered to wounds.

Figure 4

Immunohistochemistry showing presence of precursor LM-332. Six millimeter punch wounds created on db/- (normal littermates) (Figures 4A, C and E) and db/db mice (Figures 4B, D and F) were harvested post-wounding at days 3 (Figures 4A and B), 7 (Figures 3C and D) and 10. Precursor LM-332 is present along the migrating epithelial wound tongue at 3 days for both the db/- and db/db mouse but is markedly reduced in the 7 day and 10 day db/db mouse wound (Figure 4D and F, respectively) as compared to their normal littermates (Figures 4C and E).

Figure 5

Adhesion assay of human foreskin keratinocytes (HFKs) comparing Tegaderm™, Tegaderm™ coated with C2-5 antibody (C2-5 biomaterial) and Tegaderm™ coated first with C2-5 followed by LM-332 (LM-332 biomaterial). LM-332 biomaterial demonstrated adhesion of a relatively high number of HFKs, while few cells adhered to the control biomaterials (Tegaderm™ alone and C2-5 biomaterial).

Figure 6

Graph comparing % wound closed of LM-332, PBS, LM-332 biomaterial and C2-5 control biomaterial treated wounds at days 0, 7, 14 and at day 14 after removal of Tegaderm™ and hematoxylin staining. Wounds enlarged after excision, had limited % wound closed at day 7 and were not yet closed at day 14. No statistically significant differences in % wound closed were found at either day 7 or 14 between wounds treated with soluble LM-332 or PBS or wounds treated a LM-332 or C2-5 biomaterial (p=0.10 at day 7, p=0.52 at day 14).

Figure 7

Tegaderm™ covered wound at day 14 compared to the same wound with Tegaderm™ removed and stained with hematoxylin. Removal of Tegaderm™ and staining with hematoxylin aids in evaluation of the % wound closed.

Figure 8

Wound histology scores. Tissue sections were scored from 1 to 12, with 1 representing no healing and 12 representing complete closure. When individual comparisons were made between wound treatments, no significant differences were found between histology score based on wound treatment.