THE MACROSCOPIC, HISTOLOGIC, AND IMMUNOMODULATORY EFFECTS OF INTRAVENOUS ALLOGENEIC EQUINE UMBILICAL CORD BLOOD-DERIVED MULTIPOTENT MESENCHYMAL STROMAL CELL THERAPY ON EXPERIMENTALLY CREATED LIMB WOUNDS IN THE HORSE

Abstract

BACKGROUND: Limb wound on horses are often slow to heal and are prone to developing exuberant granulation tissue (EGT) and close primarily through epithelialization, which results in a cosmetically inferior and less durable repair. In contrast, wounds on the body heal rapidly and primarily through contraction and rarely develop EGT. Intravenous (IV) multipotent mesenchymal stromal cells (MSCs) are promising. They home and engraft to cutaneous wounds and promote healing in laboratory animals, but this had not been demonstrated in the horse. Furthermore, the clinical safety of administering >1.00 x 108 allogeneic (allo) MSCs IV to a horse has not been determined. PILOT PROJECT: A proof-of-principle pilot project was performed with two horses that were administered 1.02 x 108 fluorescently labelled allo-cord blood-derived MSCs (CB-MSCs) following surgical wound creation on the forelimb and thorax. Results confirmed preferential homing and engraftment to wounds with persistence of CB-MSCs at 33 days following wound creation, without clinically adverse reactions to the infusion. SECONDARY PROJECT: A minor secondary project was developed from the pilot project, where the mRNA of the inflammation-associated proteins β-arrestin-2 (βarr2), CXC ligand (CXCL) 8, CXC receptor (CXCR) 2, CXCL10, and CXCR3 was compared in limb and thoracic wounds. Results suggest that there are differences in expression between βarr2 and CXCL8/CXCR2 in limb and thoracic wounds. MAJOR PROJECT: Materials and Methods: Wound were surgically created on the forelimbs of treatment and control horses. 1.51 – 2.46 x 108 allo-CB-MSCs were administered to treatment horses 12 hours after wound creation and were monitored for clinically adverse reactions during infusion. Control horses were administered diluted cellular suspension medium (HTS-FRS) only. Biopsies of the wounds were collected on days 0, 1, 2, 7, 14, and 28 from treatment and control horses. The biopsies tissue was dived – a portion was snap frozen and analyzed using multiplex mRNA assays for proinflammatory (TNFα, CXCL8), anti-inflammatory (IL-4, IL-8), inflammation resolving INFγ, CXCL10), profibrotic (TGFβ1, TGFβ2), and anti-fibrotic (TGFβ3) cytokines, and the other portion was evaluated histologically and given a combined repair and inflammation score. The wounds were photographed on days 7, 14, and 28 and evaluated for wound size and total percentage of wound closure by contraction and epithelialization using planimetric analysis. Day of wound closure was recorded when the granulation tissue was covered with epithelium. Rate of wound closure, percentage of contraction, percentage of epithelialization, and mean fold change of cytokines was evaluated using generalized estimating equations for an overall treatment effect over all days (P≤0.2) and pairwise comparison for treatment effect at individual days (P≤0.05). Results: 3/6 (50%) treatment horses and 1/6 (17%) control horses experiences clinically adverse responses during injection. Day to wound closure was not significantly improved (treatment 26+/- 4 days, control 27 +/- 3 days; two sample T-test, P=0.702), although overall wound size (GEE; P=0.145) was decreased, characterized by overall increased contraction (GEE; P=0.145) and decreased epithelization (GEE: P=0.015) with significantly less epithelization on day 14 (GEE, PWC; P=0.0173). Histologic repair score was not improved and virtually identical between groups, thus no static analysis was performed. There was overall decrease in proinflammatory (GEE; P=0.032), anti-inflammatory (GEE; P=0.022), inflammation resolving (GEE; P=0.033) and profibrotic (GEE; P=0.191) cytokines with significantly less proinflammatory cytokines on day 2 (GEE; P=0.0003). There was no difference in antifibrotic cytokines. CONCLUSIONS: Administered >1.51 x 108 IV allo-CB-MSCs suspended in HTS-FRS can induce infusion reactions in recipients which may be caused by antigenic stimulation or the suspension medium. IV allo-CB-MSC therapy did not improve time to wound closure or histologic repair scores, although overall wound size was smaller due to increased contraction and decreased epithelization. IV allo-CB-MSCs decreased expression of all cytokines except for antifibrotic cytokines. Although minor improvements in wound healing were measured, the advantages of IV allo-CB-MSC therapy likely do not justify the risks of infusion reactions. Investigation into other methods of MSC delivery and therapies are warranted.