Objective To observe the effects of cobalt chloride (CoCl2)-simulated hypoxia on VEGF and TGF-β1 expression and to provide theoretical basis for deci phering the molecular mechanism of cl inical distraction osteogenesis. Methods The mandibular osteoblasts were obtained from newborn Wistar rats within 24 hours and cultured and purified through modified enzymatic digestion. The morphological and histological changes of cells were evaluated by the HE staining,the histochemical staining for ALP, the collagen I immunohistochemistry staining and the calcified nodules staining, and the growth curves were drawn. The best cells of the 3rd-passage rats were treated with CoCl2, and then immunofluorescence was used to detect the expressions of VEGF and TGF-β1 at 0, 3, 6, 9, 12 and 24 hours after culture. Results The HE staining demonstrated that the cellular forms were diverse, triangular, polygonal, circular and scaly and so on. The prominence varied in length and extended outwards. The nucleus was clearly discernible. The cytoplasma was rich and pink, with the nucleus royal purple. Sometimes 2 cell nuclei were seen. At the crowded place, cellular form was not clear, the dividing l ine was indistinct, and just the great-circle nuclear cells could be seen. The ALP immunohistochemistry staining demonstrated that the cell butcher nature appeared black pellets, the cell nucleus outl ine was unclear, and at the cell compact district, massive mascul ine cells could be seen clearly. The collagen I immunohistochemistry staining demonstrated that mascul ine cells were seen evenly, cytoplasma appeared yellowish brown especially around the nucleus. However, yellowish brown pellets were not seen in negative cells. The osteoblast calcium tubercle staining demonstrated that the cells gathered in the opaque region with the shape of tubercle after15 days of culture. After al izarin red staining, the reddish orange pigmentation appeared. At various time points, weak VEGF fluorescence was seen in the cells in the control group under the laser confocal microscope. As the hypoxia time prolonged, VEGF fluorescence of cells in the experimental group intensified, and reached the peak 9 hours after peration, and then dropped to the normal level. At various time points, TGF-β1 fluorescence was found in both groups under the laser confocal microscope, and fluorescence intensity in the control group was sl ightly ber than that in the VEGF control group. In the experimental group, TGF-β1 expression had short-term increase 3 hours after hypoxia, and reduced gradually with the prolonging of hypoxia time. Conclusion The method of culturing osteoblast from Wistar rats mandibular is practicable. The cells can be used for further studies. Moderate hypoxia can affect bone synthesis and turnover in distraction osteogenesis and up-regulate the expressions of VEGF and TGF-β1.
Objective To observe the result of reconstructing quadriceps femoris function in the paraplegia rats by using the 7th cervical nerve root (C7) transposition with autologous and allogeneic neural transplantation. Methods Twenty16-week-old SPF male Wistar rats were adopted to prepare frozen sciatic nerve. Thirty-six Wistar rats were divided into 2 groups (group A and group B, n=18). The left paraplegia model was establ ished with left spinal cord hemisection by the micro scissors under the operation microscope. After the model establ ishment, the homolateral autologous sciatic nerve was bridged with the femoral nerve root by the translocation of C7 in group A, while the allogeneic sciatic nerve was bridged with the femoral nerve root by the translocation of C7 in group B. At 16 weeks and 24 weeks after operation, 9 rats in each group were selected for the neuroelectric-physiological test and then the histomorphology of the nerves was observed under the microscope and the electron microscope. The fresh weight recovery rate of quadriceps femoris was calculated. Results At 16 and 24 weeks after operation, the nerve action-evoked potential (NAP) was (1.14 ± 0.07) mV and (1.21 ± 0.07) mV in group A, and (0.87 ± 0.06) mV and (0.99 ± 0.05) mV in group B; the nerve conduction velocity (NCV) was (17.34 ± 2.15) m/s and (19.00 ± 3.02) m/s in group A, and (11.23 ± 1.45) m/s and (12.54 ± 1.59) m/s in group B, respectively, indicating significant differences (P lt; 0.05) between 2 groups. At 16 and 24 weeks after operation, HE staining and Bielschowsky staining showed that group A had a large number of nerve fiber regeneration, with a regular arrange of axons; while group B had l ittle nerve fiber regeneration with a scattered arrange of axons. At 24 weeks after operation, images in TEM showed a large number of regeneration myel inated nerve fibers and a small number of unmyel inated nerve fibers through the transplanted nerve in two groups. At 16 weeks after operation, the number of myel inated nerve fibers in group A and group B was (438 ± 79) and (196 ± 31) / vision, the areas of myel inated nerve fiberswere (5 596.00 ± 583.94) and (4 022.63 ± 615.75) μm2 / vision; after 24 weeks, the number of myel inated nerve fibers in groups A and B were (642 ± 64) and (321 ± 75)/vision, the areas of myel inated nerve fibers were (6 689.50 ± 1 142.10) and ( 4 733.00 ± 982.22) μm2/vision, indicating significant differences between two groups (P lt; 0.05). There was no statistically significant difference (P gt; 0.05) in the wet weight recovery rate of quadriceps between group A and group B at 16 weeks (87.96% ± 4.93% vs. 86.47% ± 7.47%) and at 24 weeks after operation (90.10% ± 4.22% vs. 87.66% ± 3.14%). Conclusion C7 transposition combined with autograft and allograft of sciatic nerve can reconstruct the partial function of the quadriceps femoris in paraplegia rats. The effect of graft is better than that of graft obviously.