Effect Of Insulin On The Biological Activity Of Osteoblasts Obtained From Diabetic Rats' Mandibles In Vitro

Diabetic patients have a low rate of osseointegration,and hydroxyapatite coating is usually used to improve the osseointegration,however,hydroxyapatite coatings are not stable,they are inclined to cause infection and tend to delaminate from the implant surface.Both patients with type 1 and advanced stage of type 2 diabetes mellitus need insulin treatment,and a new design proposed for medicine administration via dental implant may be a possible resolvement for the above problems.If the design be realized,it may take place of coating for osseointegration and patients could get dental therapy as well as system treatment at the same time.Osteoblasts of jaw bone play a key role in the formation of osseointegration.With the development and application of bone cell culture technique,osteoblasts have been found to be able to remember the character of their donor resource.But now there is few report about the osteoblasts obtained from diabetic donor.The present paper is to study the effect of insulin on the biological activity of osteoblasts obtained from diabetic rats' mandibles,and to provide feasibility evidence for the local application of insulin via jaw bone.Part 1 Primary culture and identification of osteoblasts obtained from diabetic rats'mandiblesObjective:To explore the feasibility of the culture of osteoblasts got from alloxan-induced diabetic rats,and to supply cells for further experiments.Methods:16 SD rats were assigned to two groups:diabetic group(10)induced by alloxan(200mg/kg)peritoneal injection and control group(6)injected physiologic saline.Rats were killed and their mandibles were harvested 10 days after the establishment.And primary bone cells were obtained from association of enzyme digestion and tissue explants in culture media containing 5.5 or 25mM glucose according to postprandial blood sugar examined before and after diabetic model establishment.The cells' growth situation was observed under inverted phase contrast microscope.The second passage cells were identified by alkaline phosphatase Ca-Co stain,typeⅠcollagen special stain and calcium nodules alizarin bordeaux stain.Cells' ultrastructure was observed by transmission electron microscope(TEM).Results:Cells of diabetic group cultured in 5.5mM glucose concentration media had typical osteoblasts' appearance under light microscope and positive identification stain.TEM results showed that there were few golgiosome but more lysosomes in diabetic osteoblasts' endochylema,which is different from the control group.Cells in 25mM glucose concentration media,with no obvious osteoblasts' morphology,had weakly positive identification and could not grow in multilayer to form calcium nodule.Conclusions:A large amount of osteoblasts can be obtained from alloxan-induced diabetic rats' mandibles when cultured in 5.5mM glucose media,which supply cells for further experiments.Part 2 Biological activity of osteoblasts obtained from diabetic rats' mandiblesObjective:To study the biological activity of osteoblasts obtained from diabetic rats' mandibles.Methods:Comparison of cell biological activity was made between the diabetic and control group using MTT,PNPP,BGP content radioimmunoassay and alizarin bordeaux stain of calcified nodules.The expression of ALP,ColⅠ,Runx2 was measured by RT-PCR.The expressions of insulin receptorα(IRα)and glucose transporter-1(GLUT-1)were measured by RT-PCR,Westemblot and immunohistochemistry stain.Results:1.MTT value of diabetic group was significantly higher than the control group (P＜0.05),yet ALP(3d,7d,14d,21d)and BGP(28d)value were on the contrary (P＜0.01).The ALP,ColⅠmRNA expression of diabetic group were lower than control group(P＜0.05),yet Runx2 were on the contrary(P＜0.01).2.The expressions of IR a and GLUT-1 of diabetic group were higher than control group(P＜0.05).Conclusion:Osteoblasts obtained from diabetic rats' mandibles have defects in their differentiation ability,leading to deregulated proliferation and a minimal capacity of these cells to develop into fully differentiated mineralizing osteoblasts. Meanwhile,these cells keep the adaptation changes to hyperglycemia and hypoinsulinemia,which may contribute to their dysfuntion.Part 3 Effect of insulin on the biological activity of osteoblasts obtained from diabetic rats' mandiblesObjective:To study the effect of insulin on the differentiation and glucose uptake of osteoblasts obtained from diabetic rats' mandibles.Methods:1.Osteoblasts were divided into 5 groups(N,DM,DM+Inse-5,DM+Inse-6,DM+Inse-7)according to their origin(control group or diabetic group)and insulin concentration(0,10^-5,10^-6,10^-7M insulin)in culture media.Comparison of cell differentiation ability was made among the five groups using MTT,PNPP, BGP content radioimmunoassay and alizarin bordeaux stain of calcified nodules. RT-PCR was used for the measure of ColⅠA1 and Runx2 mRNA expression.2.Osteoblasts were divided into 4 groups(N,N+Ins,DM,DM+Ins)according to their origin(control group or diabetic group)and insulin concentration(0,10^-6M insulin)in culture media.Comparison of glucose uptake was made among the four groups by measuring fluorescence of intracellular 2-NBDGResults:1.N group had the highest MTT OD value after the first 24h(P＜0.01),DM group the lowest.In the following 48 hours their proliferation rate from the high to low was DM+Inse-6＞DM+Inse-5,DM+Inse-7＞N＞DM in order.2.From 3 to 21 days,the highest ALP value among diabetic groups turned to be DM+Inse-7,DM+Inse-6,DM+Inse-5 by turns.3.N group had the highest general BGP value among the five groups(P＜0.05). The BGP secretion peak of N group appeared in the first week,the lowest in the fourth week.Diabetic group had the opposite tendency.4.There was large area red stain of calcium deposition in N group.In DM group there was only red stain of calcium nodules odds and ends,and DM plus insulin groups had enlarged red stain area.5.DM plus insulin groups expressed significantly more ColⅠA1 and less Runx2 mRNA than DM group.6.The uptake of 2-NBDG was N+Ins＞DM+Ins＞DM＞N by turn at 20minutes, and DM＞N+Ins＞N and DM+Ins at 40 minutes.Conclusion:Insulin may correct the differentiation defects of osteoblasts obtained from diabetic rats,and inhibit their too high glucose uptake.