Investigating the homing and healing properties of adipose-derived mesenchymal stromal cells using a model of wound repair

Abstract

Adult mesenchymal stromal cells show promise therapeutically due to their multipotent differentiation capacity, immunomodulatory properties, paracrine signalling and ability to migrate to sites of injury. The potential use of adipose-derived stromal cells (ASCs) as a cellular therapy for treating delayed healing, non-healing and chronic wounds, their optimal route of administration, bio-distribution and fate needs to be investigated. Therefore, this study aimed first to establish the location and survival of systemically and locally administered ASCs during wound repair under physiological conditions (model 1) and second, the effect of locally administered ASCs during wound repair under pathological conditions of hyperglycaemia and ischemia (model 2). A dual tracking approach was used to follow firefly luciferase (Fluc) and green fluorescent protein (GFP) expressing ASCs by bioluminescence imaging (BLI) and histological analysis. The immuno-phenotype and differentiation capacity of rat ASCs transduced to express Fluc and GFP were assessed before the cells were used in the wound repair models. In model 1, full-thickness bilateral wounds were created on the dorsal aspect of the hind paws in healthy rats and two treatment modes were assessed: a single dose of 2 x 106 ASCs or NaCl administered systemically into the tail vein or 2 x 105 ASCs injected locally around each wound. Animals were followed by digital photography, BLI and sacrificed for histological analysis. In model 2, ischemia was induced unilaterally by resection of the femoral artery in hyperglycaemic rats before full-thickness bilateral wounds were created and 2 x 105 ASCs or NaCl administered locally around each non-ischemic and ischemic wound. Animals were followed by digital photography and sacrificed for histology and immunohistochemistry (IHC). Haematoxylin/eosin (H/E) staining as well as Masson’s trichrome staining and IHC for alpha-smooth muscle actin (αSMA), ionised calcium binding adaptor molecule 1 (Iba1) and GFP were performed on sample sections. Wound closure time and the contraction/epithelialisation ratio were assessed in both models. Transduced ASCs maintained their immuno-phenotype and differentiation capacity. Under physiological conditions, systemically administered ASCs were filtered out in the lungs, whereas locally administered ASCs survived at the injection site and migrated into the wound bed. Wound closure was accelerated by 5 and 7 days with systemic and local ASC treatment Page | iv respectively compared to control animals, and this was the result of increased epithelialisation. Under pathological conditions, locally administered ASCs significantly enhanced wound closure in non-ischemic and ischemic wounds by 9 days compared to control wounds. Semi-quantitative analysis revealed that ASC treatment led to enhanced cellularity in the wound. No changes in collagen deposition, vascularisation (as determined by αSMA staining) or macrophage infiltration were observed between ASC treated and control groups. However, αSMA staining was detected earlier and remained higher at wound closure in the former without enhancing wound closure by contraction. Despite the limited systemic homing capacity of ASCs, wound healing was improved. Locally injected ASCs migrated from the wound edge into the wound bed where they promoted wound repair. Under pathological conditions, ASCs enhanced wound closure. A significant increase in wound cellularity was observed, possibly through a mechanism of paracrine signalling that recruited more immune regulating and tissue repair cells into the granulation tissue. Administration of ASCs for delayed healing wounds show promise as a cellular treatment for enhancing wound repair.

Key words: Adipose-derived mesenchymal stromal cells (ASCs), in vivo imaging, bioluminescence imaging (BLI), green fluorescent protein (GFP), firefly luciferase (Fluc), re-epithelialisation, contraction, wound healing, wound repair, homing