| Objective To explore the peptidergic nerve development in the developing bone and its relationships with angiogenesis and bone mesenchymal cell differentiation. Furthermore, the importance of innervation to the bone development via immunochemical neurectomy will be observed. Besides the researches about the osteogenesis on the stage of development, the regeneration aspect of bone tissue will be also investigated. In vitro the proliferation and differentiation of bone marrow stem cell (BMSc) after the treatment of neurotrophic factors (NTFs) will be evaluated and the possibility of applying NTFs in the bone tissue engineering will be discussed. Finally, the effects of NTFs on the fracture healing will be also observed.Methods1. The lower extremities of SD embryo and new-bom SD rats were taken, fixed in formalin and packed by paraffin wax. The developmental stages of bone were determined by HE stain. The representative specimen were picked out and within the same layer the sections were taken immunohistochemical stain respective to anti-NPY, VIP, VEGF and ALP. The location and intensity of stain were compared by microscopic observation.2. 60 SD new-bom rats (20 3-day-old ones and 40 2-week-old male ones) were randomly allocated into treatment and control groups after weight by weight. Anti-NGF, 6-OHDA and 192 were injected subcutaneously tothe three treatment groups respectively in which contains 10 rats each.The rats' weights were recorded everyday. After two weeks treatment, the rats were killed and the sciatic nerves and the whole femurs were isolated. The femurs were investigated by both gross measurement and biomechanics investigation, and the development of sciatic nerves was taken pathological observation.3. The BMSc of SD rats were cultured and identified by morphological observation and immunohistochemical stain of ALP. The cells of control group were cultured with non-serum DMEM/F12, and the BDNF or NGF of different concentration was added in the culture media of treatment group respectly. The proliferation of BMSc was evaluated by MTT method and the differentiation was compared by detecting the ALP activity after breaking cells with ultrasound.4. 42 adult male SD rats were randomly allocated into treatment group and control group after weight by weight. All the rats were prepared by fixing the tibia shaft fracture with intrameduHary pins. BDNF and NGF were injected subcutaneously every other day to the treatment groups respectively in which contains 12 rats and 9 rats each and saline to the control groups simultaneously. The tibia samples were taken at the second, third, fourth and fifth week respectively for gross observation, measurement, biomechanical test and scanning electron microscopy.Results1. During the earliest stage of osteogenesis, there was no sign of angiogenesis while the VIP begins to express. That suggests the original bone tissue may have the independent nerve control before angiogenesis. And the VIP expression parallels the intensity of ossification activity according to ALP expression. At the first ossification center, the location and pattern of NPY expression are similar to those of VEGF. The expression similarity of timing and location between NPY and VEGF suggests there would be certain relationship between NPY-energetic nerve and angiogenesis.2. As a whole, the living condition and weights of all the treatment groups animals were not much changed compared with control groups (P>0.05).1) After anti-NGF treatment, the sciatic nerve pathological stain showed the axon number of treatment group was slightly less man that of control group (P>0.05). In anti-NGF treatment group, the diameter of femur was larger than that of control group (P<0.05), but the max stress and stress curve slope were not different with the latter (P>0.05).2) After 6-OHDA treatment, the sciatic nerve pathological stain didn't find obvious difference in the axon density and arrangement could be observed (P>0.05). The difference of femur diameters... |
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