Impact of TNF dysregulation on diabetic fracture repair
Date
2009
DOI
Authors
Alblowi, Jazia A.
Version
OA Version
Citation
Abstract
Introduction: Fracture healing in diabetic individuals and in animal models of diabetes is impaired. Several mechanisms by which diabetes may affect fracture healing were reported. We investigated the effect of diabetes on the catabolic aspect of fracture healing focusing on the transition from cartilage to bone, a midpoint in the fracture healing process.
Methods: CD-1 mice animals were used in this study. Using multiple low-dose streptozotocin diabetes was induced in these animals. Simple closed transverse fractures were then induced in the left femur. Groups of diabetic and normoglycemic mice were treated with TNF inhibitor, Pegsunercept starting from day 10 after fracture. Fracture calluses were collected 10, 16 and 22 days after fracture for histomorphometric analysis. RNA samples were collected from 10, and 16 days groups for gene expression of factors related to cartilage and bone remodeling by real time PCR. Apoptotic, TNF-[alpha], RANKL and CCL4 posistive chondrocytes were investigated by immunhistochemistry. FOXO1 ; a transcriptional factor which was activated by TNF-[alpha] in previous reported studies was also investigated by real time PCR and in chondrocytes by confocal laser scanning microscopy. RNA samples from 16-day group were used for microarray analysis focusing on inflammatory cytokines. An in vitro study was conducted using TNF-[alpha] treated ATDC5 chondrogenic cells to investigate FOXO1 association with TNF-[alpha] and RANKL promoters by chromatin imunoprecipitation assay.
Results: Diabetes was associated with elevated osteoclast numbers and accelerated removal of cartilage as well as pronounced chondrocytes apoptosis (P[less than]0.05). These changes were reflected by smaller callus size on day 16, a stage when cartilage is resorbed and replaced by new bone. No significant changes were noticed between groups on day. When diabetic mice were treated with the TNF-specific inhibitor, pegsunercept, the number of osteoclasts, cartilage loss and apoptotic chondrocytes were significantly reduced (P[less than]0.05). Diabetes also was associated with increased levels of mRNA of TNF-[alpha], RANKL, ADAMTS4 and ADAMTS5 (P[less than]0.05). Immunohistochemical analysis showed that diabetes was associated with increase in TNF-[alpha], RANKL and MIP- 1[beta]/CCL4 positive chondrocytes (P[less than]0.05) each of which was significantly reduced with TNF inhibition in diabetic mice (P[less than]0.05). Diabetes also increased FOXO1 mRNA levels in fracture callus and nuclear accumulation in chondrocytes (P[less than]0.05). Moreover, an association between FOXO1 and the TNF-[alpha] and RANKL promoter was demonstrated by ChIP assay. Microarray analysis revealed an up-regulation of genes related to inflammation including TNF in the diabetic group when cartilage is being replaced by bone on day 16.
Conclusion: These results suggest that diabetes enhanced inflammatory cytokines including TNF-[alpha] and increases osteoclastogenesis, chondrocytes apoptosis and cartilage loss during fracture repair particularly at stage when cartilage is replaced by bone. TNF inhibition reverses these effects demonstrates that they are significantly related to the TNF dysregulation.
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Dissertation (DSc) --Boston University, Henry M. Goldman School of Dental Medicine, 2009 (Department of Periodontology and Oral Biology)
Includes bibliography: leaves 126-143.
Dissertation (DSc) --Boston University, Henry M. Goldman School of Dental Medicine, 2009 (Department of Periodontology and Oral Biology)
Includes bibliography: leaves 126-143.
License
This work is protected by copyright. Downloading is restricted to the BU community. If you are the author of this work and would like to make it publicly available, please contact open-help@bu.edu.