Methods: An experimental mouse model of fracture-healing was used to test the impact of diabetes on MSCs in areas of bone formation. Type-1 diabetes was induced by treatment of mice with multiple low-dose streptozotocin. To determine whether the results were specifically due to diabetes, a diabetic group was treated with slow-release insulin. To test whether diabetes-enhanced inflammation affects MSCs, normoglycemic and diabetic mice were treated with tumor necrosis factor (TNF) inhibitor, pegsunercept. Closed fracture of the femur was induced, and areas of bone formation were identified in H&E-stained paraffin sections. The percentage number of MSCs was assessed by immunofluorescence using antibody to CD271. Double-immunofluorescence was used to identify double-positive TUNEL+/CD271+ apoptotic MSCs and Ki67+/CD271 double-positive proliferating MSCs. Statistical analysis was prepared by one-way analysis of variance.
Results: Diabetic mice had a 40% reduction in CD271+ MSCs in areas of bone formation (p<0.05), which was reversed by insulin-treatment. MSCs had a 3.2-fold increase in apoptosis in diabetic mice compared to normoglycemic control mice (p<0.05), and insulin-treatment decreased MSC apoptosis in diabetic mice so that it was similar to control mice (p>0.05). When inflammation was inhibited by treatment of a TNF blocker, there was a 1.6-fold increase in the number of MSCs (p<0.05). Pegsunercept treatment increased the number of MSCs by reducing apoptotic MSCs by 3.6-fold (p>0.05) and increasing proliferating MSCs by 2.2-fold (p>0.05).
Conclusions: Diabetes significantly reduced the number of MSCs in areas of bone formation, which is due in part to diabetes-enhanced TNF levels. This occurs through a mechanism that involves both enhanced apoptosis and reduced proliferation of MSCs.