Diminished bone formation during diabetic fracture healing is related to premature resorption of cartilage associated with increased osteoclast activity

Abstract

Thesis (DSc) --Boston University, Goldman School of Dental Medicine, 2007 (Department of Oral Biology and Periodontology).Includes bibliographic references: leaves 115-133.Introduction: Diminished bone formation, an increased incidence of non unions and delayed fracture healing have been observed in animal models and in patients with diabetes. Fracture healing is characterized by the formation of a stabilizing callus in which cartilage is formed then resorbed and replaced by bone. To gain insight into how diabetes affects fracture healing, studies were carried out focusing on the impact of diabetes on the transition from cartilage to bone. Methods: A low-dose treatment protocol of streptozotocin in CD-1 mice was used to induce type I diabetes. After mice were hyperglycemic for three weeks, controlled closed simple transverse fractures of the tibia or femur were induced and fixed by intramedullary pins. Histomorphometric analysis of the tibias obtained 12, 16 and 22 days after fracture was performed across the fracture ca11us at O.5 mm proximal and distal increments using computer assisted image analysis. Another group of 16-day samples were examined by micro-computed tomography (micro CT). RNA was isolated from a separate set of animals and the expression of genes that reflect the formation and removal of cartilage and bone was measured by real time PCR. In addition, RNA was used for genetic profiling using microarray and data obtained was analyzed using gene set enrichment analysis (GSEA). Another study was performed on femur fractures and involved an additional group of animals which received insulin treatment through subcutaneous implants. Similar histological analysis was performed lO, 16 and 22 days after fracture. RNA was also isolated from a separate set of animals for real time PCR on genes that regulate cartilage, bone formation and bone remodeling. Results: Molecular analysis of collagen types II and X mRNA expression showed that expression of matrix genes was the same during the initial period of callus formation. Histomorphometric analysis of day 12 fracture calluses showed that callus size and cartilage area were also similar in normoglycemic and diabetic mice. In cotrast, on day 16 ca11us size, cartilage tissue and new bone area were 2.0, 4.4 and 1.5 fold larger respectively in the normoglycemic compared to the diabetic group (P[less than]0.05). Analysis of micro CT images indicated that the bone volume in the normoglycemic animals was 38% 1arger than in diabetics. There were 78% more osteoclasts in the diabetic compared to the normoglycemic group (P=0.05) on day 16 which is consistent with the reduction in cartilage. Real time PCR showed significantly elevated levels of mRNA expression for TNF-[alpha], MCSF and RANKL in the diabetic group. Similarly, mRNAs encoding ADAMTS 4 and 5, major aggrecanases that degrade cartilage, were also elevated in diabetic animals. There was a 4.5 fold increase in apoptotic chondrocytes in the diabetic group compared to the normoglycemic. Gene set enrichment analysis showed that the 16 day diabetic animals exhibited up regulation of gene sets related to matrix degradation, inflammation and apoptosis. Insulin treatment resulted in normalization of the diabetic effect on fracture healing. Conclusion: These results suggest that impaired fracture healing in diabetes is characterized by increased rates of cartilage resorption. This premature loss of cartilage leads to a reduction in callus size and may contribute to decreased bone formation and mechanical strength frequently reported in diabetic fracture healing