Diabetic model
After injection of STZ, the animals presented significant high level of glucose in blood and in urine (Fig. 1a and b, p < 0.01). The consumption of food and drinking water was significantly increased while the body weight and growth rate were significantly reduced in the diabetic animals (Fig. 1c-e), compared with the non-diabetic animals (all p < 0.01).
Radiological Evaluation
X-ray showed that, at the second and the fourth weeks after fracture, the calluses of the fractured femurs in the diabetic animals were much weaker than their non-diabetic counterparts, by presenting predominant fibrous calluses with few calcified calluses (Fig. 2). At the eighth post-fracture week, the non-diabetic group had almost finished fracture remodeling while diabetic one had not (Fig. 2). Thus, the results of radiological examines at the end of the fourth week were employed in comparative and quantitative analyses. The size of callus was presented as the ratio of the callus diameter to the femoral diameter. Based on the analysis, the significantly smaller size of calluses was observed in diabetic animals, compared to that of the non-diabetic group (diabetic vs non-diabetic, 1.64 ± 0.14 vs 2.14 ± 0.18, n = 8, p < 0.05). The calcification ratio of diabetic calluses was also significantly lower than that of non-diabetic calluses (diabetic vs non-diabetic, 53.40 ± 6.49% vs 60.61 ± 2.13%, p < 0.05).
Mechanical Evaluation
The mechanical endurance of diabetic callus was dramatically reduced than that of non-diabetic callus at both the second and the fourth post-fracture weeks. But only the difference was found statistically significant (p < 0.05, Table 1), between the groups at the end of the fourth week.
Table 1
Mechanical endurance of the fractured bones (kPa)
| 2nd week | 4th week |
Groups | normal (n = 5) | DM (n = 4) | normal (n = 5) | DM (n = 6) |
| 92.40 | 30.19 | 170.88 | 211.92 |
| 110.50 | 28.72 | 260.58 | 212.80 |
| 12.90 | 48.66 | 230.39 | 108.45 |
| 61.55 | 87.93 | 255.30 | 148.61 |
| 130.73 | | 170.30 | 151.60 |
| | | | 137.73 |
Mean | 81.62 | 48.88 | 217.49 | 161.85* |
SD | 41.20 | 23.88 | 39.63 | 38.33 |
*, p < 0.05, compared with the ‘normal’ at the end of 4th week after fracture. |
Histological And Immunohistochemistry Assay
It can be clearly seen that the fracture ends were surrounded by a spindle red-stained callus. Calcification started from the four angles formed by periosteum and cortex (Fig. 3a). The (red-stained) callus was significantly smaller in the diabetic group at the second and the fourth post-fracture weeks (Fig. 3b and c vs Fig. e and f). At the eighth week after the fracture, the fracture in the non-diabetic control was well healed and showed typical double-track sign as intact bones (Fig. 3d). However, the cortex of the fracture bones in diabetic animals was still under remodeling (Fig. 3g), which was consistent with the results of radiological evaluation (Fig. 2).
The immunohistochemistry assay revealed that BMP-6 and BMP-9 were normally expressed in skeleton muscles, periosteum, marrow and woven bone inside the callus. Positive spots were also found scattered in osteocytes and extracellular matrix in cortex (Fig. 4a and b showing expression of BMP-6). Fewer BMP-6 immunoreactive materials were detected in the cortex of the bones of the diabetic animals (f, g and h), compared with the non-diabetic ones (4c d and e).
Changes Of Bmp-6 And Bmp-9
The results of western blot assay showed multiple bands of immunoreactive positive for BMP-6. Four of them located between 49 kDa and 90 kDa. One was between 35 kDa and 49 kDa and another band was aligned with the marker of 35 kDa (Fig. 5a and d). The optical density values of all bands of different molecular weights were quantified and compared among groups. Although the pattern of band layout was not strictly identical among each lane (sample), four most-frequently showed bands, including the one with a molecular weight of 35 kDa, were compared and analyzed. As the exact molecular weight of the other three bands was unknown (not aligning with any molecular marker), here they were presented as BMP-6-1, -2 and − 3 (in the order of molecular weights from high to low, as shown in Fig. 6a and d). Only the 35 kDa one was stably upregulated in fractured femurs at both the second and the fourth post-fracture weeks. However, only one band for BMP-9 was found, but not related to fracture (Fig. 5b, c, e, f and Fig. 7).
Fracture And Bmp-6
In comparison with the non-fractured femurs, the expression of the BMP-6 (35 kDa) was significantly up-regulated in fractured femurs of both the diabetic (Fig. 5a, c, d and f; Fig. 6g and h) and the non-diabetic rats (Fig. 6a-h). For the time-expression differences, greater amount of expression of BMP-6 (35 kDa) was found at the end of the second week in both of the fractured and the non-fractured femurs of both of the diabetic and the non-diabetic animals, when compared to those at the fourth post-fracture week (Fig. 6g and h; Fig. 7, all p < 0.05). The findings indicate the participation of BMP-6 (35 kDa) in the early time after fracture.
Diabetes And Bmp-6
As the molecular weights of the other proteins showing immunoreactive positivity to the BMP-6 antibody were greater than the 35 kDa, if the BMP-6 at 35 kDa was the functioning form, the others (with greater molecular weights) could be the precursor and mid-products. Therefore, as the BMP-6 at 35 kDa was significantly up-regulated in the fractured femurs, the volume of precursor and mid-products may be getting lower because of the shift of the precursor and the mid-products to the smaller functioning BMP-6 (35 kDa).
Our finding indicates that the transforming procedure from the molecules of greater molecular weights to the BMP-6 at 35 kDa was inhibited in diabetic animals. Therefore, it may suggest that diabetes down-regulate the BMP-6 at 35 kDa through slowing down its maturing procedure. Obviously, the underlying mechanism is worth to be investigated.
In conclusion, downregulation of BMP-6 at 35 kDa may correlate with the impaired fracture healing in diabetes. The finding may suggest a potential target for the intervention of the pathological mechanism underlying the impairment of fracture healing in diabetes.
Ratio Of Bmp-6 Molecules Of Different Molecular Weights
The ratio of the BMP-6 in forms of different molecular weights to the BMP-6 in 35 kDa were analyzed in this study to figure out the mechanism underlying the downregulation of the BMP-6 (35 kDa). The results showed significantly higher ratios in the BMP-6-1/BMP-6 (35 kDa) in diabetic ones (normal fracture vs diabetic fracture, 1.19 ± 0.44 vs 4.89 ± 1.69, p < 0.01) and BMP-6-3/BMP-6(35 kDa) (normal fracture vs diabetic fracture, 0.47 ± 012 vs 1.47 ± 0.43, p < 0.01). The results indicate the impairment of the maturation of BMP-6 (35 kDa) in fractured bones of diabetics (Table 2).
Table 2
Ratio of optical density of the BMP-6 bands (M ± SD)
Groups | BMP6-1/35 kDa | BMP6-2/35 kDa | BMP6-3/35 kDa |
Normal fracture | 1.19 ± 0.44 | 0.55 ± 0.41 | 0.47 ± 0.12 |
Normal intact | 3.85 ± 2.02& | 2.77 ± 1.84& | 3.50 ± 2.59& |
Diabetic fracture | 4.89 ± 1.69* | 0.59 ± 0.25 | 1.47 ± 0.43* |
Diabetic intact | 5.17 ± 1.78 | 1.08 ± 0.51 | 4.61 ± 2.28# |
&, p < 0,01, compared with ‘Normal fracture’; *, p < 0.01, compared with ‘normal fracture’; #, p < 0.05, compared with ‘Diabetic fracture’. |