| At present, dental implantation has become an ideal way to restore themissing teeth. Diabetes mellitus (DM) is one of the most commonly encounteredpatients as relative contraindications to dental implant therapy.With prevalenceof DM increasing dramatically worldwide, the number of DM patients,especially type2diabetics（T2DM） requiring for dental implants will growmore and more.Unfortunately, clinical studies revealed that T2DM patients hadhigher implant failure rates than the general population. Besides, T2DM whodesire dental implants are often associated with bone defect in the alveolar ridge.And, the effect of general grafts on bone healing is not good, andbone-implantation osseointegration is inferior after bone grafting. Therefore, thefocal issues are that how to improve bone healing and decrease the failure rateof dental implantation in T2DM patients and manufacture appropriate biomaterials for T2DM patients who are the teeth-missing associating withalveolar bone defect. The aim of the study was to evaluate whether ASCscombined with Bio-Oss anorganic bovine bone has positive effect on healingcalvarial CSD in the T2DM rats model by means of GBR technique and whetherthe mineralization of the newly-formed bone relate to the seeded cells density.Methods and Results:Part Ⅰ:Protocol of type2diabetes mellitus rat models development andpreparation of CSD modelsMethods: A high-fat diet (HFD) with a single low-dose streptozotocin (STZ)intraperitoneal injection was administered to52rats to induce T2DM as theprevious study. After7days, the random plasma glucose levels (PGL) weremeasured via caudal vein using a glucometer.The rats with PGL>16.7mml/Lwere considered T2DM and used in the present study, which were continuouslyfed by the high-fat diet until the end of the study. As well, the PGL and bodyweight were respectively measured before and after feeding the high-fat diet;before and after STZ injection.Then, in the T2DM rat model, a full-thicknesscritical-sized defect (CSD) was created in the parietal bone by means of thesagittal suture as the midline, employing a trephine drill with an outer diameterof5mm. The dura mater and the superior sagittal sinus were carefully protectedin the whole operation.Results: Four rats were excluded from the experiment due to inadequatestandard PGLs following a single low-dose injection of STZ. The remaining48rats presented with characteristics that were analogous to human T2DM, such asobesity, body weight maintaining stable, an increased PGL and development ofhyperglycemia. The mean and standard deviation (SD) of the body weights andPGLs of the rats are shown in Table1. After being fed with the HFD, the body weight was significantly increased (P=0.000), but the PGLs were not significant(P=0.052). There was a significant rise in PGLs following STZ injection(P=0.000), while the body weight of the T2DM rats remained stable (P=0.299).These results indicated that the rat T2DM model was functional for use in thisstudy. Besides, we safely prepared the calvarial CSD in the T2DM model.Thedepth of the CSD is0.06-0.10mm, which can contain bone grafts.Part Ⅱ:Fabrication of ASC-seeded composite scaffoldsMethods:Cells isolated from the bilateral inguinal fat pads by collagenase I digestionwere cultured in vitro.Cells morphology were observed under the microscope.The growth curve was drawn by MTT colorimetry. The surface markers of cellswere analyzed by flow cytometry. Passage3cells were cultured in osteogenic oradipogenic medium and verified with ALP staining, Alizarin red staining or OilRed O staining.Then, to co-culture the composite, the four groups wererandomly treated with different concentrations: control group: Bio-Oss andBio-Gide alone; low-dose group:3×10~5with Bio-Oss and Bio-Gide;Middle-dose group:3×10~6with Bio-Oss and Bio-Gide; High-dose group:3×107with Bio-Oss and Bio-Gide. ASCs were detached and centrifuged to remove thesupernatant, then resuspended50μl the standard medium at a density of3×105,3×10~6,3×10~7cells/graft(0.02g), respectively. The control group only was addedwith the standard medium.The ASC-seeded composite scaffolds were examinedby scanning electron microscope to observe ASCs morphology after co-culturefor4h. Besides, using the same method, the prepared ASCs-seeded compositescaffolds were transported to the operating room.Results: the cells presented fibroblast-like or polygon shape with colony．Thegrowth curve was like ‘S’shape, reflecting strong proliferative ability and active differentiation．Flow cytometry showed that ASCs expressed CD29、CD90, butnot CD34、CD45. The ALP staining, Alizarin red staining and Oil Red Ostaining showed positive reaction. After4h co-culture, the ASCs were viewedadhered and extended on Bio-Oss and Bio-Gide membranes surfaces.Furthermore, the increasing number of adhered ASCs was correlated with anincrease in the seeding cell density.Part Ⅲ: The effect of ASCs/Bio-Oss composite on bone healing in the T2DMrat calvarial CSDMethods: The48T2DM rats were randomly divided into4groups (n=12). TheCSDs were perapared according to the method in PartⅠ. The four groups withCSDs were randomly filled with the prepared different composite scaffolds asfollowing: control group: Bio-Oss and Bio-Gide alone; low-dose group:3×10~5with Bio-Oss and Bio-Gide; Middle-dose group:3×10~6with Bio-Oss andBio-Gide; High-dose group:3×10~7with Bio-Oss and Bio-Gide. The Bio-Ossgranules of the composite scaffolds were placed over the dura mater and furthercovered with the Bio-Gide membrane. The PGL was monitored pre-operationand post-operation every2weeks, respectively. At4and8weekspost-implantation, the rats were sacrificed using overdose anesthetics. Calvarialspecimens were harvested, and analysed with micro-CT (μCT),histomorphology and Energy Disperse Spectroscopy (EDS).Results:(1) Gross appearance: At4weeks, new bone formation in the control goup waslesser than in the experiental groups, and the range of the CSDs was notapparently changed in control group. At8weeks, the appearance of the controlgroup was similar to that of4weeks. However, Newly-formed bone wassignificantly increased post-implantation8W compared with4weeks and the size of the CSD was apparently decreased in the experimental groups.(2) Three dimensional images of μCT showed that newly-formed bone was notonly observed at the periphery of CSDs, but also at the center in experimentalgroups. And, more new bone appeared in the ASC treated groups than in thecontrol group, being consistent with the gross appearance. The statistic datesrevealed there was a dose-dependent response on BV/TV of new bone.Combined with means and the interaction between ASC seeding dose and timefacts in the statistic results, BV/TV was significantly increased in high-doseASCs treated group than any other groups at4weeks（multiple comparison ofgroups P<0.05）. Whereas, we found Tb.Th was highest and Tb.Sp was lowest inmiddle-dose group at8weeks, thanks to Tb.Th (P=0.136) and Tb.Sp (P=0.052)had no statistic significance between middle-and high-dose ASCs treatedgroups. Apart from these, BS/BV and Tb.N values achieve the highest inmiddle-dose group at8weeks, but the two facts of ASCs seeding dose and timehad no interaction for BS/BV and Tb.N. Shortly, the most of results show thatnew bone formation has improved significantly in middle-dose ASCs treatedgroup at8weeks postimplantation in this study.(3) The Ca/P ratio analyzed by EDS and INCA software was higher in thecontrol group compared with the low-dose ASC group (P=0.000). The Ca/P ratioin the low-dose group was lower than the middle-and high-dose ASC groups,respectively (P=0.012, P=0.000), but the differences between the control,middle-dose and high-dose groups (control versus middle, P=0.068; controlversus high, P=0.712; middle versus high, P=0.117) were not statisticallysignificant. The Ca/P ratio (1.40±0.19) was the highest in the middle-dose ASCgroup at8weeks in this study when associated with the mean.(4) Minimal new bone formation was observed in the defect area in control group at4weeks post-implantation, and new bone didn’t obviously increased at8weeks. The representative samples of the experimental groups at4weeksrevealed more new bone at the periphery of the CSDs and immature osseousislands away from the periphery, where trabecular bone can be observed. And,newly formed osseous islands didn’t bridge the remodeled Bio-Oss granules.Furthermore, at8weeks post-implantation, new bone formation of the calvarialdefect sites was significantly enhanced, and there is a fusion or bridge betweennew bone creeping from the periphery to the center of the CSD along theendocranial surface and partial remodeled Bio-Oss granules.Conclusions:(1) The T2DM rat model was induced by HFD combined with low-dose STZ inthis study, which would analogously mimic the natural history and metaboliccharacteristics of human T2DM. And, we successfully prepared the calvarialCSD in the T2DM rat model.(2) The cells were substantiated as ASCs, being of precise osteogenicpotentiality. After co-culture with Bio-Oss, the ASCs can adhere and extend onBio-Oss and Bio-Gide membranes surfaces.(3) Allogenic ASCs also enhanced new bone formation for T2DM rat calvarialdefect healing, when used in association with Bio-Oss scaffolds. Our resultssuggested that middle-dose ASCs (3×10~6/0.02g graft) chosen in the study wasthe most effective dose for improving new bone formation and degree ofmineralization in a T2DM rat calvarial defect model. Due to a short evaluationperiod (8weeks), we cannot claim that8weeks is the optimal duration for bonehealing. Alonger observation period in vivo is necessary. |
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