Novel Progenitor Cell Therapies Augment Ischemic Tissue Healing: Preclinical Studies

Introduction: Diabetic wounds, radiation injury, bone fractures and distraction osteogenesis are characterized by an ischemic microenvironment that limits healing. We hypothesize that progenitor cells (PCs), derived from lipoaspirate or bone marrow, can be utilized to enhance neovascularization, alleviate ischemia, and augment healing. Methods: Diabetic wounds: Murine stented wounds were randomized into experimental arms: AMD3100 (CXCR4 partial agonist and bone marrow PC mobilizing agent) treated, topical lineage-negative PC treated (lineage-negative PCs were isolated from murine bone marrow by magnetic cell sorting, suspended in collagen), and controls. Radiation injury: Murine dorsal skin was isolated and irradiated with 45 Gy. Four weeks following radiation, mice were grafted with lipoaspirate or sham-grafted with saline. Diabetic and radiation injury healing were assessed photometrically, and vascularity was assessed by CD31 immunofluorescence. Fractures: 3-mm defects were created on murine parietal bone. AMD3100 or sterile saline was injected intraperitoneally daily post-operatively, and bony regeneration was assessed using micro-CT. CD31 and osteocalcin staining were performed to assess for vascular density and osteoblast density. Distraction osteogenesis: Rats were subjected to mandibular distraction. From the beginning of the consolidation phase, animals received daily injections of AMD3100 or sterile saline. Mandibles were harvested and bony regeneration was assessed using micro-CT and 3-point strength testing. CD31 and osteocalcin staining were performed to assess for vascular density and osteoblast density. Results: Diabetic wounds: AMD3100 significantly improved wound healing compared to controls at 7, 14, and 21 day time points (p=0.05, p<0.01, and p=0.02). The improvement in wound healing was associated with increased circulating PCs as well as increased wound vascularity at day 21 (155.3±16.1 vs. 431.8±19.3, p<0.01). Wounds treated with lineage-negative cells demonstrated a significantly decreased time to closure (14 days) vs. controls (28 days, p=0.004), and a significant improvement in percentage closure at 14 days compared with controls (p< 0.01). Radiation injury: The processed lipoaspirate used for fat grafting contained 2.5±.2x105 PCs/g. Chronic ulceration and fibrotic skin thickening became stable 4 weeks post-irradiation. Alopecia, skin color/texture, and ulceration were improved and vascular density increased in fat-grafted mice. Fractures: AMD3100-treatment improved bony regeneration post-op week 8 (34.8±11.5% vs. 50.3±11.5%, p=0.017) and week 12 (36.0±5.7% vs. 61.8±11.4%, p<0.0001). AMD3100-treatment significantly increased the vascular and osteoblast density. Distraction osteogenesis: AMD3100-treated animals had substantially improved bony regeneration (max. load 100 N vs. 60 Np<0.05). Immunohistochemistry demonstrated that AMD3100 treatment increased vascular density (10.76±0.74% vs. 6.59±1.21%, p=0.043). Osteoblast density was substantially increased in AMD3100 treated animals. Conclusions: We demonstrate methods for internal mobilization and external delivery of progenitor cells to enhance neovascularization and augment murine diabetic wound healing, fracture healing, radiation injury repair and distraction osteogenesis. Clinically applied progenitor cell mobilization and topical therapies could decrease patient morbidity and improve outcomes.