The effects of fibroblast growth factor- 2 blocking on development of chick embryo cranial sutures

Normal growth and morphogenesis of the cranial dome, reflects the balance between cell proliferation in the cranial sutures and osteogenesis in the bone edges. In craniosynostosis, due to early osteogenic differentiation, sutures closure prematurely and craniofacial malformations occur. Nowadays, mutations in fibroblast growth factor (FGF) receptor's genes have been implicated as a major factor in craniosynostotic syndromes. In chicken embryo cranial dome, antibody blocking method is used to disrupt morphogenesis and to manipulate the endogenous FGF-2 ligand. High and low levels of FGF-2 are caused to different reactions. The balance between these different levels is provided normal cranial morphogenesis. In this study, chicken embryos were used as experimental models. The effects of FGF-2 blocking on development of cranial sutures and experimentally formation of the craniosynostosis were investigated. Also morphological, histological and changings that may occur before and after ossification at the protein level was examined. In this process, the relationship between oxidative stress and cell death was investigated. In our study, chicken eggs which obtained from Bornova Veterinary Control and Research Institute were used. Eggs were incubated at 37.5 degrees Celsius in the oven for about 60-80% humidity and four experimental groups were formed. First group was control group and consisted 20 eggs. Second group was included sterile phosphate buffered saline and others (group 3 and 4) were FGF-2 blocking groups. Group 3 and 4 were included 0,1 and 0,01 μg/ml anti-bFGF-2 respectively. Second, 3rd and 4th groups subdivided into 4 groups as E2, E4, E6 and E8. While second group included 5 eggs for each subdivision group (total :20 eggs), 3rd and 4 th groups included 10 eggs for each subdivision groups (total: 80 eggs). In 3rd and 4th group, FGF-2 blocking was done in E2nd, E4th, E6th and E8th days. Anti-basic fibroblast growth factor-2 (anti-bFGF-2) was applied at different stages of development. Fertilized eggs were incubated for 15 days in an incubator and 0.1 and 0.01 µg/ml antibody (in one dose) in PBS was applied under operating microscope around the umbilical cord at 2nd, 4th ,6th and 8th days to each group for blocking. Samples were taken on E15th day after sacrifization and were fixed with Bouin's fixing solution. Tissues were embedded in paraffin according to routine follow-up and 5 µm sectiones were cut. While macroscopic development was examined morphologically, microscopic interactions were evaluated after Hematoxylin-eosin, Mason trichrome, Vonkossa and Alizer Red staining histochemically. Also oxidative stress, proliferation and cell death were examined after immuncytochemically staining with anti-eNOS, anti-PCNA and TUNEL respectively. While comparison of the macroscopic development was done by morphometric measurements, comparison of staining was done by scoring. Graphpad statistical software was used to exposed differences. The comparison was done with the one-way ANOVA test and p value as <0.05 was considered as statistically significant. Embryos which were blocked with antibody on E4th and E8th days evaluated on E15th day with morphologically, histologically and immunocytochemically. Findings revealed the presence of cartilage sovereignty instead of craniosynostosis. In the examples, pathologies such as morphological disturbance, abnormalities or growth retardation were not detected. We observed that, antibody blocking did not affect the systemic structure. It changed the oxidative stress in sutures which is shown by eNOS staining and affected the continuation of proliferation and cell death which were detected by PCNA and apoptosis respectively. These effects were seen most clearly in the E8th embryos and the difference was statistically significant. Our findings showed that, FGF-2 plays an important role in the development of the cranium and blocking of FGF-2 causes to delays in the closure of the sutures. Normally, on the one hand FGF accelerate the normal bone development, on the other cause to delay in suture closure. While our expectation was acceleration of the suture closure by blocking of this factor, opposite effect was observed. The distance between sutures was increased more which was unexpected result. These results suggested that, blocking with antibody can be used for treatment of craniosynostosis. The relationship between craniosynostosis, FGF-2, oxidative stress and apoptosis should be examined in more detail with advanced techniques. So, more important information could be obtained to provide better quality of life to these patients. Keywords: Fibroblast Growth Factor; Chicken embryo; Cranial sutures; craniosynostosis; histology; oxidative stress; apoptosis.