Different degrees of reductions in bone mass of cancellous bone and cortical bone in mice with type 1 diabetes and related mechanism

Abstract:

Objective：To investigate the different degrees of reductions in bone mass of cancellous bone and cortical bone in mice with type 1 diabetes mellitus（T1DM） and related mechanism. Methods：A total of 30 C57BL/6J mice aged 8 weeks were randomly divided into wild-type（WT） group and T1DM group. The mice in the T1DM group were given intraperitoneal injection of strep－tozotocin to establish a model of T1DM，and those in the WT group were given injection of the same dose of normal saline. Micro-CT，X-ray radiology，HE staining，and bone morphometry were used to observe the changes in bone mass and osteogenic activity. Immunohistochemistry and real-time PCR were used to measure the mRNA and protein expression of the factors involved in the Wnt/β-catenin pathway，including low-density lipoprotein receptor-related protein 6（LRP-6），β-catenin，lymphoid enhancer factor（LEF-1），T cell factor（TCF），and osteoblast-related transcription factors，including alkaline phosphatase（ALP），osteocalcin（OCN），osterix（OSX），and runt-related transcription factor 2（Runx2）. Results：According to the results of morphological examination，the T1DM group had the greatest reduction in bone mass of the distal femur and a slight reduction in bone mass of the middle femur. Micro-CT analysis revealed that compared with the WT group，the T1DM group had significant reductions in the following indices of cancellous bone：bone volume per total volume（0.16±0.01 vs. 1.27±0.08，P<0.01），trabecular thickness（0.04±0.01 vs. 0.09±0.01，P<0.01），and trabecular number（1.90±0.27 vs. 4.53±0.45，P<0.01）；as for the corresponding indices of cortical bone，compared with the WT group，the T1DM group had significant reductions in bone area per total area（0.35±0.05 vs. 0.40±0.02，P<0.05） and cortical thickness（0.17±0.01 vs. 0.18±0.01，P<0.05）. Bone mor－phometry showed that compared with the WT group，the T1DM group had significant reductions in the following indices of cancellous bone：osteoblast surface per bone surface（12.15±0.46 vs. 21.69±0.37，P=0.000），mineralizing surface per bone surface（30.19±1.02 vs. 38.02±0.70，P=0.000），mineral apposition rate（0.74±0.06 vs. 1.13±0.10，P=0.000），and bone formation rate（0.25±0.02 vs. 0.48±0.04，P=0.000）；as for the corresponding indices of cortical bone，the T1DM group also had significant reductions in these indices compared with the WT group（osteoblast surface per bone surface：12.80±0.13 vs. 16.41±0.29，P=0.000；mineralizing surface per bone surface：36.16±1.22 vs. 42.21±0.54，P=0.000；mineral apposition rate：0.67±0.34 vs. 0.83±0.03，P=0.000；bone formation rate：0.27±0.02 vs. 0.36±0.04，P=0.000）. RT-PCR showed that compared with the WT group，the T1DM group had significantly lower expression of the Wnt/β-catenin pathway factors LRP-6，β-catenin，TCF，and LEF-1 and osteogenic differentiation indices ALP，OCN，OSX，and Runx2 in cancellous bone（0.18±0.13 vs. 1.00±0.13，0.23±0.20 vs. 1.00±0.15，0.29±0.23 vs. 1.00±0.23，0.22±0.17 vs. 1.00±0.12，0.52±0.10 vs. 1.00±0.00，0.52±0.11 vs. 1.00±0.10，0.42±0.01 vs. 1.00±0.10，0.34±0.12 vs. 1.00±0.08，all P<0.01） and cortical bone（0.74±0.65 vs. 1.00±0.07，0.63±0.45 vs. 1.00±0.09，0.75±0.58 vs. 1.00±0.08，0.70±0.63 vs. 1.00±0.05，0.67±0.19 vs. 1.00±0.23，0.73±0.08 vs. 1.00±0.23，0.63±0.08 vs. 1.00±0.39，0.62±0.08 vs. 1.00±0.11，all P<0.05）. Immunohistochemistry showed a similar trend as RT-PCR. Conclusion：In T1DM mice，the reduction in cancellous bone mass is greater than that in cortical bone mass，which might be caused by the greater downregulation of the Wnt/β-catenin signaling pathway in cancellous bone than in cortical bone.