Expression And The Role Of The IR And IGF-1R In The Healing Process Of Tooth Extraction Socket In GK Rats

Diabetes is one of the chronic diseases that can cause many serious complications and pose a serious threat to human health. Type 2 diabetes (type 2 diabetes mellitus, T2DM) is by far the most common, affecting 90% of the diabetes population. Diabetes was one of the dental implant contraindications; while good blood glucose control could improve the success rate of dental implants in diabetics. Insulin therapy is a critical part of treatment for diabetics. Professor Liu Hongchen successfully used insulin around the dental implant in diabetics, he proposed that insulin may protect osteoblasts via insulin receptor (IR) and/or insulin-like growth factor 1 receptor. The research team has been confirmed that the effect of insulin on osteoblast proliferation and differentiation, and the expression of IR on osteoblast in vitro.Insulin molecule has docked onto the receptor and effected its action, IR and IGF-IR belongs to the family of tyrosine kinase receptor IGFs, although the IR and the IGF-IR are closely related sharing a high degree of homology, they play a different role in the cell proliferation, differentiation and metabolic process.However, the role of IR and IGF-IR in the healing process of tooth extraction when chitosan-based hydrogel insulin injected is not yet clear. Goto-Kakizaki (GK) rats is a non-obese animal model of spontaneous type 2 diabetes, they are mainly impaired glucose-stimulated insulin secretion. This study utilized the chitosan/β-glycerophosphate hydrogel (chitosan/β-glycerophosphate gels, C/GP) system loaded appropriate physiological concentrations of insulin, injected when the sol to gel in the tooth extraction socket in GK rats. To explore the role of two receptors in the healing process of teeth extraction socket in diabetic rats, expression of two receptors in osteoblasts and new bone formation were observed.Part I Insulin on osteoblasts proliferation and the expression of IR and IGF-IR in osteoblasts Objective:To select an appropriate physiological concentration of insulin, and observe the expression of IR and IGF-1R in the osteoblastsMethods:Isolated, cultured and identified osteoblasts were treated with physiological glucose concentration and high glucose concentration of culture medium prepared with 4 concentrations insulin treated, MTT assay the proliferation of osteoblasts, to determine the appropriate physiological concentration of insulin; identificated expression of IR and IGF-1R of in osteoblasts by immunohistochemistry.Results:Treated insulin in the same concentration, the proliferation of osteoblasts is more active in L-DMEM; 10-6M concentration is the appropriate physiological concentration of insulin for osteoblasts in vitro; IR and IGF-1R expressed in osteoblasts in vitro.Conclusion:10-6 M concentration is the appropriate physiological concentration of insulin for osteoblasts in vitro; IR and IGF-IR expressed in osteoblasts in vitro.PartⅡPreliminary study of chitosan-based hydrogel for the local delivery of insulinObjective:To establish chitosan-based hydrogel for the local delivery of insulinMethods:Concentration and rate of insulin released were assayed by HPLC; to detect the gelation time and the amount of insulin released affected byβ-glycerophosphate concentration and pH value.Results:C/GP gel system was liquid at 25℃, from the sol to gel in 37℃within 8min; the chitosan/β-GP gel systems were not affected after loaded insulin solution; GP content and pH value affect the C/GP gelation time; the final optimal concentration of GP in C/GP system is 5.6%.Conclusion:Chitosan/β-glycerophosphate gel system has been successfully applied to the local administration of insulin.PartⅢLocal administration of insulin gel for tooth extraction sockets in GK rats and expression of IR and IGF-1R in the healing processObjective:To observe the expression of IR and IGF-1R in the healing process of tooth extraction socket in GK rat, and preliminary analyze the relevance of two receptors with new bone in the tooth extraction socket. Methods:Extraction of the rat incisor; local administration of the chitosan-based hydrogel insulin in tooth extraction socket. Randomized:Wistar group, only the extraction group (WN); GK rats,only the extraction group (GN), tooth extraction with chitosan/GP group (G1), tooth extraction of chitosan/GP contained insulin group (G2). After tooth extraction,7 days,14days,21 days and 28days, rats were sacrifced to remove the mandible quickly. To observe IR and IGF-1R in the healing process of tooth extraction socket in GK rat, and analyze the relevance of two receptors with new bone in the tooth extraction socket.Result:Immunohistochemical expression of IR and IGF-1R are in the cytoplasm of the osteoblast, IGF-1R positive cells were located around the new bone trabeculae, IR positive cells around the lacunae; the numbers of IGF-1R positive cells are more than those of IR positive cells. HE staining in G1, G2 group at 28 days there was no C/GP; G2 group was observed by Goldner's trichrome at 28 days after tooth extraction that there are some of the new bone formation and irregular bone matrix, active osteoblasts were also found near those area.During the period of tooth socket from 7d to 28d, WN group showed IR mRNA levels continued to decline, GN group showed IRmRNA level began to decrease after reaching a peak at 14d; IRmRNA level in G1 group were the same trend as the GN group, reached the peak at 21 d and, then dropped to the level which same to 7d when in 28d; G2 group consistent with the WN group performance, decreased in the healing process during 7-28d; IRmRNA level in GN group was the highest (p<0.01), toward GN group, IRmRNA level in G1 and G2 groups were lower than those of GN group but close to WN group; In this process, IGF-1R mRNA in GN and G1 group were lower than other groups; IGF-1R mRNA level in G2 group was significantly higher than other groups at 7d,14d,21d, and reached the peak at 21d, WN group were significantly higher than the GN and G1 group atl4d,21d(p<0.01).Conclusion:Successful injection of the C/GP system contained insulin in the tooth extraction socket in GK rats; local administration of insulin regulates IR mRNA and IGF-1RmRNA level, and new bone formation in G2 group is more active than in GN and G1 group.