Diabetic model
Data reported here were collected from the twenty-two animals presented significant high level of glucose in blood and in urine (Fig. 1a and b, p < 0.01) and lived through the observation period. The consumption of food and drinking water was significantly increased while the body weight and growth rate were significantly reduced in the diabetic animals, compared with the non-diabetic controls (Fig. 1c-e, all p < 0.01).
Radiological evaluation
The results 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 (non-diabetic vs diabetic, 2.14 ± 0.18 vs 1.64 ± 0.14, n = 8, p < 0.05). The calcification ratio of diabetic calluses was also significantly lower than that of non-diabetic calluses (non-diabetic vs diabetic, 60.61 ± 2.13% vs 53.40 ± 6.49%, n = 8, p < 0.05).
Mechanical evaluation
The mechanical endurance of diabetic callus was markedly 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, between the non-diabetic and diabetic groups at the end of the fourth week of fracture (non-diabetic vs diabetic: 217.49 ± 39.63 kPa vs 161.85 ± 38.33 kPa, p < 0.05, Table 1).
|
2nd week
|
4th week
|
|
Groups
|
Non-DM (n=5)
|
DM (n=4)
|
Non-DM (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 ‘non-DM’ at the end of 4th week after fracture.
Table 1
Mechanical endurance of the fractured bones (kPa)
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 expressed in skeleton muscles, periosteum, marrow and woven bone inside the callus. The immunoreactive 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 to 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 analyzed. Although the pattern of band layout was not strictly identical among each lane (sample), four most-frequently presented bands, including the three located between 90 and 45 kDa and 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 of the 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 - f). Only the 35 kDa one was stably upregulated in fractured femurs at both the second and the fourth post-fracture weeks (Fig. 5a, c, d and f, Fig. 6g and h). However, only one band of immunoreactive to BMP-9 was found, but no relationship to fracture was detected (Fig. 5b, c, e, f and Fig. 7).
Fracture and BMP-6
In comparison with the intact 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-dependent expression differences, greater amount of expression of BMP-6 (35 kDa) was found at the end of the second week after fracture in both of the fractured and the non-fractured femurs of the diabetic (n = 5) and the non-diabetic animals (n = 5), when compared to those at the end of the fourth post-fracture week (diabetic group, n = 6; non-diabetic group, n = 6; Fig 6g and h; Fig. 7, all p < 0.05). The findings indicate the participation of BMP-6 (35 kDa) in the healing of the fracture from the early time after fracture.
Diabetes and BMP-6
However, the values of the BMP-6 (35 kDa) in both of the fractured and the non-fractured femurs from diabetic rats were much lower than their non-diabetic counterparts. Precisely, compared with the non-diabetic rats, the BMP-6 (35 kDa) in fractured and non-fractured femurs of the diabetic ones reduced by 25.95% (n = 5) and 19.15% (n = 5) respectively, at the end of the second post-fracture week, but by 34.01% (n = 6) and 18.83% (n = 6) respectively, at the end of the fourth week (all p < 0.05, Fig. 6 g, h and 7). Therefore, it may indicate that the BMP-6 (35 kDa) participated in fracture healing, which was significantly inhibited by diabetes (by up to 34.01%).
Ratio of BMP-6 molecules of different molecular weights
The ratio of the immunoreactive BMP-6 in forms of different molecular weights to the BMP-6 in 35kDa was analyzed from the data collected from the animals of diabetic and non-diabetic group, at the end of the fourth week after femur fracture, to figure out the mechanism underlying the downregulation of the BMP-6 (35kDa). The results showed significantly higher ratios in the BMP-6-1/BMP-6 (35kDa) in diabetic ones (normal fracture vs diabetic fracture, 1.19 ± 0.44 vs 4.89 ± 1.69, n = 6, p < 0.01) and BMP-6-3/BMP-6 (35kDa) (normal fracture vs diabetic fracture, 0.47 ± 012 vs 1.47 ± 0.43, n = 6, p < 0.01). The results indicate the impairment of the maturation of BMP-6 (35kDa) in fractured bones of diabetic animals (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’. |