| Diabetes mellitus (DM) has become a globe health problem. The implant failure rate indiabetic patients is often higher than that in normal patients. Diabetic patients usually havehorizontal or vertical bone defect. And bone graft outcome is often compromised. DM isthu s considered as a rela tive contraindication for dental implant prosthesis.Implant failure is one of the comlications of diabetes. It shares the samepathophysiologic mechanism with other diabetic complications. DM may effects implants inmany ways. Glucose may have non enzymatic glycosylation reaction with amino acid andprotein, producing advanced glycation endproduct (AGEs). AGEs may stimulatemonocyte-macrophages and endotheliocyte to release cytokines and inflammatory mediators,which may damage endothelial cells and cause microvascular lesions. And this will degradeosseointegration. AGEs combines with its receptor RAGE on osteoblasts. Binding of agonists like the AGEs to RAGE results in activation of NADPH-oxidases and otherpathways that lead to increased production of reactive oxygen species (ROS)and inhibate the differentiation of osteoblasts. Extracelluar matrix components such ascollagens, laminin and vitronectin maybe damaged by AGEs. And this will affect celladhesion, growth, and matrix accumulation. Insulin may has both direct and indirect effectson bone metabolism. Directly it stimulates osteoblastic matrix synthesis and indirectly itstimulates insulin-like growth factor-I production by the liver. Insulin-like growth factor-1then increases matrix synthesis by two mechanisms: by increasing the number of osteoblastspresent and by upregulating the function of differentiated osteoblasts. Besides, Immunesuppression in diabetes will breakdown collagen and delay wound healing.Adipose-derived stem cells (ASCs) have a higher yield at harvest, easy to obtain, andcan expand more rapidly in vitro as compared with bone marrow mesenchymal stem cells(BMSCs). ASCs could be induced into adipogenic, osteogenic, chondrogenic lineages underappropriate conditions. Thus, ASCs may be a novel and more promising alternative cellsource for bone tissue engineering.Study Objective:The current study was designed to investigate the effect of ASCs on bone formation andosseointegration in diabetic rabbit model. ASCs were obtained from New Zealand rabbitsand indentified by mutilineage differentiation. And ASCs were composite with Bio-Oss andimplanted around implants in the tibia of diabetic rabbits. Bone formation andosseointegration were studied with histological and Micro-CT analysis. Thus, the aim is toimprove the implant success in diabetic patients.Methods and Results:Part1:Methods:For induction of experimental diabetes, rabbits were sedated with intramuscular40mg/kg pentobarbital sodium. Diabetes was induced by injecting Alloxan (100mg/kg) intolateral ear vein. To prevent hypoglycemia, total amount of10ml of5%glucose was injected(iv) at2h,4h, and6h after Alloxan administration. And10%glucose was supplemented for the first24h after Alloxan injection. Fasting blood glucose was measured by a glucometerevery24h after Alloxan administration. The rabbits were treated with insulin (1-4U/kg)when FBG>19.4mmol/L. The rabbits with FBG>13.9mmol/L（250mg/dl） were consideredas diabetic.Results:1. Rabbits got dispirited, decreased activity and loss of appetite. And more drink, morefood, more urine symptoms occured3days after Alloxan adminstration. Two rabbits diedwithin24hours, another two rabbits died with one week. Other rabbits were survived.2. Two rabbits were excluded because fasting blood glucose did not achieve13.9mmol/L. Plasma glucose level raised from5.92（±0.75）mmoL/L to19.51(±5.19) mmoL/L.Body weight decreased0.2-0.5kg. The body weight and plasma glucose difference hasstatistical significance（p<0.05）.Conclusion:Diabetic rabbit could be established by intravenous injection of Alloxan.Alloxan-induced diabetic rabbit is broadly used in diabetic research. Blood glucose shouldbe well maintained to avoid animal death.Part2:Methods:In brief, ASCs were isolated from peritoneal adipose tissue excised from the abdominalregion of male New Zealand rabbit. Adipose tissue was enzymatically digested with0.25%type I collagenase in PBS for60min at37with gentle agitation. Enzyme activity wasneutralized with DMEM. And the infranatant was centrifuged (100g*5min) at roomtemperature. The resulting ASCs were then resuspended and plated in tissue culture dishes.When reached90%confluence, cells were passaged and ASCs at passage3were used in thefollowing study. The ASCs were then coultered with adipogenic and osteogenic medium andconfirmed with Oil Red-O staining and alizarin red staning.Results:1. ASCs from rabbits began to adhere after4h inoculation, first with a small, round andnonuniform cell size. ASCs gradually extended into short or long spindles, and showed atypical fibroblast-like morphology after passage. 2. ASCs proliferation rate decreased obviously after adipogenic differention. Afterfeeding ASCs with adipogenic inducing media for5days, small oil droplates were present inthe cytoplasm. And the oil droplates got bigger with continue differention. Adipogenicdifferention of rabbit ASCs was confirmed by Oil Red-O staining.3. After osteogenetic differention for3days, dark red mineralized bone matrix wasconfirmed by alizarin red staining, indicating that rabbit ASCs had differenrd intoosteoblasts.Conclusion:ASCs were isolated from peritoneal adipose tissue excised from the abdominal region ofmale New Zealand rabbit. The ASCs were then coultered with adipogenic and osteogenicmedium and confirmed with Oil Red-O staining and alizarin red staning. Adipose-derivedstem cells (ASCs) have a higher yield at harvest, easy to obtain, and can expand morerapidly in vitro as compared with bone marrow mesenchymal stem cells (BMSCs). Thus,ASCs may be a novel and more promising alternative cell source for bone tissueengineering.Part3:Methods:Diabetic rabbits were sedated with intramuscular40mg/kg pentobarbital sodium. Thetibia of the rabbit was shaved and disinfected with a povidone-iodine solution. In eachanimal, an incision was made down to the surface of the tibia. An MAO treated pure titaniumimplant (3.3*6mm) was insert into the tibia of the rabbit under physiological saline irrigation.Four more holes were then drilled around the implant to induce bleeding from the marrowspaces. A pre-formed titanium dome was placed to cover the implant. Randomly, one sidewas chosen as the experimental side. In the ASCs group, the titanium dome filled with themixture of ASCs and Bio-Oss was tightly fitted with two miniscrews. While in the controlgroup, the titanium dome was filled with the mixture of physiological saline and Bio-Oss.Each reflectd skin flap was then repositioned and sutured with interrupted sutures. Boneformation and osseointegration was studied by general observation, histological andMicro-CT analysis at4,8, and12weeks after operation, respectively. Results:1. Two New Zealand rabbits were excluded because of infection. The left12rabbitsrecovered without postoperative sign of infection and healed uneventfully. Samplepreparation was made at4,8,12weeks, respectively.2. Micro-CT measurements show that new bone formed around the implants withoutdifference between the ASCs group and the control group at4weeks after operation. Whilenew bone formation was significantly higher than that in the control group at8and12weeksafter operation, with an higher BV/TV value.（p<0.05）.3. Ponceau staining shows that new bone formed both in the ASCs group and controlgroup without significant difference4weeks after operation. Newly formed bone in thecontrol group was still thin at8weeks after operation, while bone substitute had graduallybeen replaced by newly formed bone in the ASCs group. And better osseointegration wasachieved in the ASCs group at12weeks than the control group（p<0.05）.Conclusion:More bone formation and better osseointegration achieved when ASCs applied. ASCsmight improve the new bone formation and osseointegration in diabetic rabbits.Summary:1. Stable diabetic rabbit animal model could be established with intravenous injectionof Alloxan. Plasma glucose should be well mentained to reduce the death rate.2. ASCs could be isolated from adipose tissue from rabbits. And ASCs possess theablity of self-renewal, proliferation and muti-differention.3. More bone formation and better osseointegration achieved when ASCs applied.ASCs might improve the new bone formation and osseointegration in diabetic rabbits. Boneengineering with ASCs coule be an ideal treatment for the diabetes with missing teeth. |
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