| Objective: To observe the expression and distribution of nerve growth factor(NGF) and basic fibroblast growth factor(bFGF) in rat dental pulp after direct pulp capping with vitapex by immunohistochemical techniques; To evaluate the effects and significance of NGF and bFGF in dental pulp wound healing; To explore some better therapies and means for the clinical treatment of dental disease.Methods:35healthy male Wistar rats with no caries and periodontal disease were selected, the weight220-250g each. All rats were provided by the Experimental Animal Center of Hebei Medical University. The rats were randomly divided into7groups. These groups were the control group, Group with immediate pulp capping, the3-d, the5-d, the7-d, the14-d and the28-d group. The rats were anesthetized with100g/L chloral hydrate (3ml/Kg).In supine position, bilateral surgery areas of the rats were disinfected using1%iodine and5%ethanol. The experimental group of teeth were randomly selected from one side of the upper first molar, Drill and grind down the first molar meiso-occlusal with a1/4round bur until the bottom of the cavity appeared pink. We perforated the pulp through the remaining thickness of dentin by pressing the tip of sharp probe which diameter was0.05mm. The cavities were rinsed with physiolonical serum and gently dried with a cotton pellet before being capped with vitapex. To protect the wound site from oral contaminants, we filled the cavities with a glass ionomer cement. Rats were killed under anesthesia by perfusion through the heart with the4%paraformaldehyde.The jaws were dissected free, the teeth were exposed by removal from the buccal bone plate and were carefully elevated from the socket. The apical of each root was carefully removed and the tooth was then placed in fixative solution, and kept at4℃for48hrs, then demineralized in 10%EDTA for4wks. The glass ionomer cement were removed, Demineralized molar were dehydrated with graded ethanol and vitrification by dimethylbenzene, embedded in paraffin, then serial5μm sections were cut for hematoxylin-eosin staining and iummunohistochemistry.NGF and bFGF were stained under procedures in accordance with the SP kit, respectively, using PBS as a negative control instead of primary antibody. Results of staining were analyzed with the image analysis system and the percentage of positive cells and staining intensity of the two factors were recorded. The final results were obtained by multiplication of the two values. All statistical analysis were performed using SPSS for Windows, version13.0. Kruskal-Wallis H test was used to analyze different values among seven groups, Wilcoxon test was introduced for multiple-comparison. As a=0.05, if P≤0.05, there is a difference statistically.Result:1The experiment change of bFGFIn the normal control group, bFGF were strongly positive expression mainly in the odontoblasts, fibroblast and vascular endothelial cells. There is no significant histological difference between the instant group and the control group. In the group of3days, expression of odontoblasts were decreased. After5days later, expression of bFGF began to increase, and it was strongly expressed mainly in vascular endothelial cells and fibroblasts within the pulp. In experimental group of14ds and28ds, it was weakly positive or positive expression. The results of the Kruskal-Wallis test showed there was a significant defference among the seven groups(H=24.425, P<0.05).The results of Wilcoxon test indicated that there was no difference statistically in expression level among normal group,0-d,14-d and28-d group; and among3-d,14-d,28-d group(P>0.05). There was no difference statistically in expression level between5-d and7-d group,(P>0.05). The expression level in normal group was different from that in3-d group,5-d group and7-d group (P<0.05). The expression level in0-d group was different from that in3-d group,5-d group and7-d group (P<0.05). The expression level in3-d group was different from that in5-d group and7-d group (P<0.05). The expression level in5-d group was different from that in14-d group and28-d group (P<0.05). The expression level in7-d group was different from that in14-d group and28-d group (P<0.05).2The experiment change of NGFIn the normal group, NGF showed negative expression, the expression of instant group was weakly positive.3-day,5-day group, strong positive expression, mainly part of the dental pulp cells around the vessels and nerve fibers in the odontoblasts. Down regulated after7days, showing positive expression of group14d and group28d, mostly negative or weakly positive expression. The results of the Kruskal-Wallis test showed there was a significant difference among the seven groups(H=25.431, P<0.05).The results of Wilcoxon test indicated that there was no difference statistically in expression level among normal group,0-day,14-day and28-day group; and among normal group,0-d,7-d group(P>0.05). There was no difference statistically in expression level between3-day and5-day group,7-day and14-d group (P>0.05). The expression level in normal group was different from that in3-d group and5-d group (P<0.05). The expression level in0-d group was different from that in3-d group and5-d group (P<0.05). The expression level in3-day group was different from that in7-d group,14-d group and28-d group (P<0.05). The expression level in5-d group was different from that in14-d group and28-d group (P<0.05). The expression level in7-d group was different from that in28-d group (P<0.05).Conclusion:1The expression level of bFGF can be altered by the different time point of direct pulp capping, it promote the formation of reparative dentine by regulating angiogenesis and differentiation of odontoblast-like cell. And it may play an important role in the process of dental pulp wound healing.2NGF may play an important role in the reaction and nerve restoration after dental pulp injury as its expression varied in different stages. |
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