Abstract
CATEGORY: Basic Sciences/Biologics, Diabetes INTRODUCTION/PURPOSE: Diabetes mellitus is a chronic metabolic disease that interferes with bone formation, increases fracture risk and impairs fracture healing in the foot and ankle. Inflammatory mediators, like fully reduced HMGB1 (fr HMGB1), play a key role fracture healing; however, their activity can be inhibited by Metformin, which is a hypoglycemic drug commonly used for the treatment of diabetes. The effect of Metformin on the bone fracture healing is largely unknown. In this study, we tested the hypothesis that application of inflammatory mediators with metformin improves nonunion bone fracture healing using a diabetic rat model. METHODS: The rats were divided into four groups with 8 rats per group(Fig 1): Group-1 had no additional treatment;Group-2 were injected Metformin (IP, 160 mg/kg) daily;Group-3 were injected frHMGB1 into wound area (250 µg/kg) weekly;Group-4 were injected Metformin (IP, 160 mg/kg) daily and frHMGB1 into wound area (250 µg/kg) weekly. The blood samples were collected for HMGB1 & IL-1β.Tibia samples were also collected and examined by gross inspection, micro-CT images and histological analysis. Data was analyzed by One-way ANOVA followed by Fisher’s Least significance difference test. A p-value less than 0.05 was considered to be significantly different between the groups. RESULTS: Micro-CT images showed completely healed at day-90 post-surgery in frHMGB1 rats while Metformin inhibited frHMGB1 enhanced bone fracture healing as evidenced by the gap in the fractured bone area(Fig 2). Histology analysis indicated that frHMGB1 enhanced fractured bone healing as evidenced by high density of the cells was found in the bone fracture area (Fig. 3). Safranin O and fast green staining along with Masson’s Trichrome staining confirmed the frHMGB1 local injection promoted fractured bone healing (Fig. 4) as evidenced by high quality bone tissue formation with collagen type I (red staining) and collagen type III (blue staining) in the fractured bone wound area. CONCLUSION: Although the treatment with frHMGB1 enhanced nonunion fractured bone healing by promoting cell proliferation, fibroblast migration, and collagen production, some low-quality bone tissues formed at the fractured bone areas due to overgrowth. Metformin slowed the bone fracture healing but regenerated better quality bone tissues by decreasing the inflammatory marker levels, inhibiting fibroblast migration, activating AMPK activity, and reducing collagen III production. These findings highlight the anti-inflammatory actions of Metformin and pro-healing effects when combined with frHMGB1 suggesting the healing potential of non-union fractures in Diabetic Mellitus patients.